pyrimidinones and merocyanine-dye

M540 bodies are membrane-surrounded round bodies occurring in semen of sub-fertile men. They appear variable in size and density, virtually devoided of chromatinic material and especially frequent in oligoasthenoteratozoospermic patients. Up to now, data collected by our group suggest that they may be apoptotic bodies that somehow escaped from testicular/epididymal phagocytosis. Indeed, they promptly stain with Merocyanine 540, a probe detecting the changes occurring in the membrane of apoptotic cells. In addition, they exhibit many of the apoptotic markers occurring in testicular apoptosis, including caspases activity, Fas receptor, p53 and DNA fragmentation (the latter detected by TUNEL assay). Due to the similarity in size and density between head sperm and M540 bodies, traditional protocols of sample preparation fail to yield sperm population completely free of M540 bodies, except for swim-up selection. In flow cytometry, it is possible to distinguish sperm from bodies by labelling samples with nuclear probes, because the latter fail to stain M540 bodies. Occurrence of M540 bodies in semen has been revealed only recently, at least in quantitative studies, and ,hence, many flow cytometric studies have not accounted for them. We summarise two studies (one on sperm ubiquitination and another on sperm DNA fragmentation) in which flow cytometric analyses were conducted both including and excluding M540 bodies from the sperm population. We found that in both cases M540 bodies largely affected the results. The study on sperm ubiquitination reveals that the direct correlation between sperm ubiquitination and good semen parameters is unmasked only after exclusion of M540 bodies from the analysis. The study on sperm DNA fragmentation shows that the amount of DNA damage in sub-fertile patients is more dramatic than expected from past investigations that included M540 bodies in the analysis.

Topics: Apoptosis; Biomarkers; Cell Separation; DNA Fragmentation; Flow Cytometry; Fluorescent Dyes; Humans; Infertility, Male; Male; Pyrimidinones; Semen; Spermatozoa; Staining and Labeling; Ubiquitin

The reactive nature of species derived from oxygen, such as singlet oxygen and hydrogen peroxide, has been exploited in the clinical setting for targeting bacteria, viruses, and tumor cells by photodynamic excitation of a variety of chromophores. This modality, termed photodynamic therapy (PDT), is currently being used to treat some forms of cancer. However, the applicability of conventional PDT is limited due to the absolute dependence on simultaneous exposure of the target to the photoactive compound and light. In 1990, we demonstrated that the need for simultaneous exposure of the biological target to light and photosensitizer could be circumvented by prior exposure (activation) of the sensitizer molecule to light and its subsequent use as any other anti-cancer or anti-viral drug. By dint of the nature of the protocol, this process was termed preactivation. Since then, the generation of biologically active molecules in vitro by preactivation has been validated using a variety of chromophores, such as merocyanine 540, Photofrin II, and naphthalimide. Here we briefly review the role of reactive oxygen species in the photodynamic effect, and provide an explanation for the mechanism of preactivation. We propose that photo-oxidation not only provides a novel means for the generation of biologically active molecules, but could also explain, at least in part the mechanism of conventional PDT. It is likely that the light-dependent breakdown of the chromophore to generate novel active compounds, in addition to reactive oxygen species, also contributes to the photodynamic damage observed on simultaneous exposure of the chromophore and target tissue to light during PDT.-Pervaiz, S. Reactive oxygen-dependent production of novel photochemotherapeutic agents.

Topics: Animals; Caspases; Humans; Neoplasms; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Reactive Oxygen Species

The pros and cons of purging of either bone marrow or peripheral blood stem cell preparations for autologous transplantation for cancer has been debated strongly over the past decade. Recent data implicating the role of minimal residual disease in autografted marrow in cancer relapse have renewed interest in this question. There is a considerable body of literature supporting the possibility that photosensitizer molecules in combination with light might provide a therapeutic window permitting selective elimination of malignant stem cells while sparing those of normal lineage. Molecules of this class are known to be taken up more actively by most malignant cells, and intracellular concentrations are critical in their cytotoxic effect when they are activated by light at an appropriate wavelength. The present paper reviews the observations made over the past decade on a variety of photosensitizers and their effects on hemopoietic progenitors.

Topics: Bone Marrow Purging; Bone Marrow Transplantation; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Hematoporphyrin Derivative; Humans; Indoles; Light; Neoplasms; Neoplastic Stem Cells; Organometallic Compounds; Photosensitizing Agents; Porphyrins; Pyrimidinones

Recent preclinical and clinical investigations indicate that phototherapy and photochemotherapy may have applications that go far beyond their "traditional" roles in the treatment of skin disorders, selected solid tumors, and neonatal hyperbilirubinemia. Bone marrow transplantation is one area that may benefit substantially from these new developments. This review focuses on new applications of phototherapy and photochemotherapy that pertain to the inactivation of tumor cells in autologous bone marrow grafts, the prevention and treatment of acute and chronic graft-versus-host disease, the prevention of transfusion-induced allosensitization and graft rejection, and the inactivation of pathogenic viruses and parasites in bone marrow grafts and blood products.

Topics: Animals; Antiviral Agents; Blood; Bone Marrow Purging; Bone Marrow Transplantation; Graft Rejection; Graft vs Host Disease; Humans; Immunization; Infection Control; Mice; Neoplasms; Neoplastic Stem Cells; Photochemotherapy; Photosensitizing Agents; Phototherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transfusion Reaction

Topics: Antiviral Agents; Blood Banks; Bone Marrow Transplantation; Disinfection; Humans; Light; Molecular Structure; Pyrimidinones; Radiation-Sensitizing Agents; Transfusion Reaction

A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.

Topics: Animals; Antiviral Agents; Blood Proteins; Bone Marrow Purging; Cell Membrane; Erythrocyte Membrane; Humans; Liposomes; Mice; Oxygen; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Rats; Singlet Oxygen; Virus Physiological Phenomena; Virus Replication; Viruses

Topics: Animals; Carbocyanines; Carotenoids; Energy Metabolism; In Vitro Techniques; Isoxazoles; Kinetics; Membrane Potentials; Models, Molecular; Molecular Probes; Pyrimidinones

Topics: Animals; Cells; Coloring Agents; Humans; Leukemia; Neoplasms; Photolysis; Pyrimidinones; Radiation-Sensitizing Agents

Topics: Adolescent; Adult; Animals; Bleomycin; Bone Marrow; Bone Marrow Transplantation; Cell Separation; Child; Child, Preschool; Clinical Trials as Topic; Cyclophosphamide; Drug Evaluation; Etoposide; Graft vs Host Disease; Hematopoietic Stem Cells; Humans; Leukemia; Leukemia, Lymphoid; Lymphoma; Lysophosphatidylcholines; Methylprednisolone; Neoplasms, Experimental; Phospholipid Ethers; Pyrimidinones; Transplantation, Autologous; Transplantation, Isogeneic

Topics: Acute Disease; Animals; Bone Marrow; Bone Marrow Transplantation; Cyclophosphamide; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Leukemia; Lymphoma; Mechlorethamine; Neoplastic Stem Cells; Photosensitivity Disorders; Podophyllotoxin; Pyrimidinones; Rats; Transplantation, Autologous

To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa.. Case-control, prospective study.. Academic centers.. Fertile and subfertile subjects.. Semen samples from subfertile and fertile men, 11 per group, were analyzed for basic semen parameters and early (annexin-V binding) and late (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with M540 to assess the presence of M540 apoptotic bodies.. Presence of M540 apoptotic bodies.. Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups.. This study confirmed the occurrence of M540 bodies in semen and that male factor infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that the presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.

Topics: Adult; Annexin A5; Apoptosis; Biomarkers; Case-Control Studies; Cross-Sectional Studies; DNA Fragmentation; Ejaculation; Flow Cytometry; Fluorescent Dyes; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Prospective Studies; Pyrimidinones; Spermatozoa

Recent preclinical and clinical investigations indicate that phototherapy and photochemotherapy may have applications that go far beyond their "traditional" roles in the treatment of skin disorders, selected solid tumors, and neonatal hyperbilirubinemia. Bone marrow transplantation is one area that may benefit substantially from these new developments. This review focuses on new applications of phototherapy and photochemotherapy that pertain to the inactivation of tumor cells in autologous bone marrow grafts, the prevention and treatment of acute and chronic graft-versus-host disease, the prevention of transfusion-induced allosensitization and graft rejection, and the inactivation of pathogenic viruses and parasites in bone marrow grafts and blood products.

Topics: Animals; Antiviral Agents; Blood; Bone Marrow Purging; Bone Marrow Transplantation; Graft Rejection; Graft vs Host Disease; Humans; Immunization; Infection Control; Mice; Neoplasms; Neoplastic Stem Cells; Photochemotherapy; Photosensitizing Agents; Phototherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transfusion Reaction

Topics: Adolescent; Adult; Animals; Bleomycin; Bone Marrow; Bone Marrow Transplantation; Cell Separation; Child; Child, Preschool; Clinical Trials as Topic; Cyclophosphamide; Drug Evaluation; Etoposide; Graft vs Host Disease; Hematopoietic Stem Cells; Humans; Leukemia; Leukemia, Lymphoid; Lymphoma; Lysophosphatidylcholines; Methylprednisolone; Neoplasms, Experimental; Phospholipid Ethers; Pyrimidinones; Transplantation, Autologous; Transplantation, Isogeneic

Topics: Biosensing Techniques; Fluorescent Dyes; Glutamine; Humans; Limit of Detection; Pyrimidinones

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Topics: Arabidopsis; Arabidopsis Proteins; Fluorescence; Lipocalins; Meristem; Mutation; Organogenesis, Plant; Plant Roots; Protein Binding; Pyrimidinones; Retinaldehyde; Signal Transduction

Nanobubble (NB), which simultaneously enhances ultrasound (US) images and access therapeutic platforms, is required for future cancer treatment.

Topics: Animals; Cell Death; Cell Line, Tumor; Diagnostic Imaging; Gold; Humans; Hyperthermia, Induced; Lung Neoplasms; Mice; Models, Animal; Nanoparticles; Photosensitizing Agents; Phototherapy; Pyrimidinones; Theranostic Nanomedicine

The urge to discover selective fluorescent binders to G-quadruplexes (G4s) for rapid diagnosis must be linked to understand the effect that those have on the DNA photophysics. Herein, we report on the electronic excited states of a bound merocyanine dye to c-Myc G4 using extensive multiscale quantum mechanics/molecular mechanics calculations. We find that the absorption spectra of c-Myc G4, both without and with the intercalated dye, are mainly composed of exciton states and mixed local/charge-transfer states. The presence of merocyanine hardly affects the energy range of the guanine absorption or the number of guanines excited. However, it triggers a substantial amount (16%) of detrimental pure charge-transfer states involving oxidized guanines. We identify the rigidity introduced by the probe in G4, reducing the overlap among guanines, as the one responsible for the changes in the exciton and charge-transfer states, ultimately leading to a redshift of the absorption maximum.

Topics: Density Functional Theory; G-Quadruplexes; Molecular Dynamics Simulation; Molecular Structure; Photochemical Processes; Proto-Oncogene Proteins c-myc; Pyrimidinones

A lysosome-targeting dual-functional fluorescent probe was rationally designed and developed for imaging intracellular lysosomal viscosity and beta-amyloid. More importantly, the real-time tracking of the dynamic movement of lysosomes, as vesicle structures, has been achieved using Lyso-MC.

Topics: Amyloid beta-Peptides; Animals; Cell Line; Fluorescent Dyes; Humans; Lysosomes; Microscopy, Confocal; PC12 Cells; Pyrimidinones; Rats; Viscosity

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calmodulin; Cell Membrane; Flow Cytometry; Lipids; Male; Pyrimidinones; Sperm Capacitation; Spermatozoa; Swine

Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan's emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms.

Topics: Animals; Cell Membrane; CHO Cells; Cricetulus; Diphenylhexatriene; Escherichia coli; Fluorescent Dyes; HEK293 Cells; Humans; Lipopolysaccharides; Mechanotransduction, Cellular; Membrane Lipids; Microscopy, Confocal; Microscopy, Fluorescence; Pyrimidinones; Recombinant Proteins; Salmonella enterica; Transfection; TRPA1 Cation Channel; Unilamellar Liposomes

The shape and intensity of fluorescence emission spectra of Merocyanine 540 embedded in dipalmitoylphosphatidylcholine bilayers differ depending on the thermal history of the sample. This apparent hysteresis in fluorescence emission was most prominent in the temperature range of 20 to 35°C. Analysis of kinetic and temperature cycling experiments suggested that Merocyanine 540 slowly (half time of about 30min) assumes a metastable configuration as temperature is raised above the phospholipid pre-transition point. When the sample was cooled below the pre-transition temperature, the metastable state slowly depopulated (half time of about 15min). The rate of merocyanine exchange among these states was influenced more by membrane lipid mobility than by lipid order since cholesterol increased the rate of transition to the metastable state by a factor of 11.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Kinetics; Lipid Bilayers; Membrane Lipids; Phospholipids; Pyrimidinones; Thermodynamics; Transition Temperature

Sucralose, an artificial sweetener, displays very different behavior towards membranes than its synthetic precursor sucrose. The impact of both sugars on model dipalmitoylphosphatidylcholine model membranes was investigated using absorbance and flourescence spectroscopy and the membrane probe merocyanine 540. This probe molecule is highly sensitive to changes in membrane packing, microviscosity and polarity. This work focuses on the impact of sugars on the outer leaflet of unilamellar dipalmitoyl phosphatidylcholine model membranes. The choice of lipid permits access to the gel phase at room temperature and incorporation of the dye after liposome formation allows us to examine the direct impact of the sugar on the outer leaflet while maximizing the response of the dye to changes in the bilayer. The results demonstrate that sucrose has no impact on the packing efficiency of lipids in unilamellar DPPC vesicles in the gel phase. Conversely sucralose decreases the packing efficiency of lipids in the gel phase and results in decreased microviscosity and increased membrane fluidity, which may be as a result of water disruption at the membrane water interface.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Liposomes; Membranes, Artificial; Pyrimidinones; Sucrose

Spectral-fluorescent properties of a series of merocyanine dyes comprising the barbituric acid residue as the electron-accepting terminal group are investigated in comparison with those of their N,N-methylated analogues in media of various polarity. It is revealed that in polar aprotic solvents the electronic absorption spectra of the studied compounds are influenced dramatically by the formation of hydrogen bonds between the NH-groups of barbituric residue and solvent molecules. An effect of such nucleophilic solvation on the electronic structure of the studied dyes is analysed using both the spectral data obtained and the DFT/B3LYP quantum chemical simulation. It is found also, that solvation has comparatively weak influence on the shape and position of the fluorescence bands of the studied merocyanines while the fluorescence quantum yield changes substantially in solvents of various polarity.

Topics: Barbiturates; Fluorescent Dyes; Hydrogen Bonding; Molecular Structure; Pyrimidinones; Quantum Theory; Solvents; Spectrometry, Fluorescence

The side effects of antitumor drugs and low treatment efficacy are two major challenges of current chemotherapy. To address these issues, we developed a new kind of smart nanocarriers that combine pH-responsive chemotherapy and near-infrared (NIR) light triggered photodynamic therapy. These nanocarriers were based on upconversion nanoparticle (UCN)-loaded folate-conjugated polymeric lipid vesicles (UFPLVs). Merocyanine 540 (MC540), as a photosensitizer, was loaded in the UFPLVs; doxorubicin hydrochloride (DOX), as an antitumor drug, was conjugated to the surfaces of UFPLVs by pH-sensitive hydrazone bonds. The newly developed MC540&DOX-UFPLVs had a nanosized structure with targeting ligand modification, so they had the potential to accumulate into tumor sites via a combination of passive and active targeting effects. An in vitro singlet oxygen test showed that the nanocarriers can generate cytotoxic singlet oxygen successfully under the irradiation of NIR light. In vitro DOX release profiles demonstrated that the nanocarriers can achieve a pH-triggered drug release. It has been demonstrated by a cellular uptake study that the nanocarriers can efficiently deliver drugs and photosensitizers into tumor cells. In vitro and in vivo combination treatments evidenced the high antitumor effects of MC540&DOX-UFPLVs under NIR light irradiation. These results suggest that the MC540&DOX-UFPLVs may be promising nanocarriers for tumor combination therapy applications.

Topics: Antineoplastic Agents; Cell Line, Tumor; Combined Modality Therapy; Drug Carriers; Humans; Hydrogen-Ion Concentration; Infrared Rays; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Polymers; Pyrimidinones

Two identical or different merocyanine dyes were tethered by a rigid diphenylacetylene spacer unit that enables the folding of the two dyes into co-facially π-stacked structures in solvents of low polarity. Whilst the solvent-dependent absorption spectra of homodimers of identical dyes are easily interpreted as H-aggregates by exciton theory, the spectra of the heterodimers constitute a novel and more interesting case.

Topics: Dimerization; Pyrimidinones; Spectrophotometry, Ultraviolet

The aim of this study was to preserve albumin nanoparticle structure/function during the lyophilisation process. Bovine serum albumin nanoparticles were obtained by γ-irradiation. Nanoparticles were lyophilised in buffer, miliQ water or in trehalose/miliQ solution. The size and charge of the nanoparticles were tested after lyophilisation by light scattering and Z potential. The most relevant results in size of BSA nanoparticle were those lyophilised in PBS between 20 and 350nm, assembled in different aggregates, and negative Z potential obtained was 37±8mV in all, and those nanoparticles lyophilised with trehalose had a size range of 70±2nm and a negative Z potential of 20±5mV. Structure determination of surface aminoacids SH groups in the BSA NP lyophilised in PBS showed an increase in the free SH groups. Different aggregates had different amount of SH groups exposure from 55 to 938 (from smaller to bigger aggregates), whereas BSA NP lyophilised with trehalose showed no significant difference if compared with BSA NP. The binding properties of the BSA nanoparticle with a theragnostic probe (merocyanine 540) were studied after lyophilisation. Results showed more affinity between the BSA NP lyophilised with trehalose than that observed with non lyophilised BSA NP. As a result, the lyophilisation condition in trehalose 100μM solution is the best one to preserve the BSA NP structure/function and the one with the enhance binding affinity of the BSA NP.

Topics: Drug Carriers; Fluorescent Dyes; Freeze Drying; Nanoparticles; Particle Size; Pyrimidinones; Serum Albumin, Bovine; Trehalose

Merocyanine dyes, owing to their unique photochemical properties, are widely used to fabricate probes for the detection of biologically active small molecules and bioimaging. In this paper, merocyanine-based probes were proved of undergoing unwanted hydrolysis. To explore the strategies toward avoiding the hydrolysis, the detailed hydrolysis mechanism was first investigated, which was also confirmed by density functional theory (DFT) calculation. Then a series of merocyanine dyes were rationally designed. Influences of molecular structures of the probes, the analytical media such as pH and components of the solution on the hydrolysis were systematically studied. The experimental results suggest that merocyanine based probes with low electron density are more likely to suffer the hydrolysis, which could be exacerbated by the well-accepted strategy for constructing type-II probes. It is worth noting that chemical surroundings could also exert distinctive influence on the hydrolysis. The hydrolysis could be obviously aggravated when fetal calf serum or DMSO was deployed. Our findings will definitely provide an effective and reliable approach for guiding the rational design of highly robust merocyanine-based probes and the optimization of the analytical media, which is helpful in terms of avoiding the hydrolysis of the probes and hydrolysis caused analytical errors.

Topics: Animals; Benzopyrans; Biosensing Techniques; Cattle; Coloring Agents; Dimethyl Sulfoxide; Hydrolysis; Indoles; Models, Molecular; Pyrimidinones; Quantum Theory; Serum

We have developed a visual marker for the investigation of hydrogen bonding (HB) effects. The decoloration rate of a photochromic dye that incorporates a latent intra-molecular HB feature can be linked to the HB character of the media. Kinetic and thermodynamic parameters of this simple decoloration approach for HB sensing are investigated both experimentally and by high level theoretical studies. This principle has been applied for the detection of changes in the HB character of stationary and fluidic systems. A major finding is the observation of a shear-related perturbation of the balance between intra- and inter-molecular HB within a dynamic thin film.

Topics: Fluorescent Dyes; Hydrogen Bonding; Molecular Structure; Pyrimidinones

Ultrafast fluorescence resonance energy transfer (FRET) from a merocyanine dye to a Rhodamine 6G (R6G) molecule in micelles formed by the surfactants SDS and DTAB and also in a catanionic vesicle formed by SDS and DTAB has been studied by picosecond time resolved emission spectroscopy. Here the dye acts as a donor molecule and R6G acts as the acceptor molecule. Multiple timescales of FRET have been detected, namely, an ultrafast component of 100-500 ps and relatively long component (1800-3300 ps). The different time scales are attributed to different donor-acceptor distances.

Topics: Dynamic Light Scattering; Fluorescence Resonance Energy Transfer; Microscopy, Atomic Force; Pyrimidinones; Rhodamines; Time Factors

The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (-BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., -BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.

Topics: Animals; Dogs; Insemination, Artificial; Male; Pyrimidinones; Semen Analysis; Sperm Capacitation; Spermatozoa; Staining and Labeling

Merocyanine 540-mediated photodynamic therapy (MC540-PDT) has been used in clinical trials for the purging of autologous hematopoietic stem cell grafts. When the same combinations of dye and light were applied to human peripheral blood lymphocytes, a broad range of T- and B-cell functions were impaired, prompting speculations about a potential role of MC540-PDT in the prophylaxis of graft-versus-host disease (GVHD). We here report on the effects of MC540-PDT on in vitro functions of murine lymphocytes as well as a preliminary evaluation of MC540-PDT for the prevention of GVHD in murine models of allogeneic bone marrow transplantation. Mixed lymphocyte reactions, proliferative responses to lectins, interleukin-2 and lipopolysaccharide, T-cell-mediated lysis, and NK activity were all inhibited by moderate doses of MC540-PDT. Whether MC540-PDT reduced the incidence and/or the severity of GVHD in murine models of allogeneic hematopoietic stem cell transplantation depended on the composition of the mismatched grafts and the intensity of the preparative regimen. MC540-PDT was only beneficial (i.e. reduced the incidence and/or severity of GVHD) when the spleen cell content of grafts was low and/or the radiation dose of the preparative regimen was not myeloablative, and, therefore, may have encouraged mixed chimerism.

Topics: Animals; B-Lymphocytes; Body Weight; Bone Marrow Cells; Bone Marrow Transplantation; Cell Proliferation; Cell Survival; Female; Graft vs Host Disease; Killer Cells, Natural; Light; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; T-Lymphocytes; Transplantation, Homologous

This report presents evidence that the following Solanum steroids: solasodine, diosgenin and solanine interact with human erythrocytes and molecular models of their membranes as follows: a) X-ray diffraction studies showed that the compounds at low molar ratios (0.1-10.0mol%) induced increasing structural perturbation to dimyristoylphosphatidylcholine bilayers and to a considerable lower extent to those of dimyristoylphosphatidylethanolamine; b) differential scanning calorimetry data showed that the compounds were able to alter the cooperativity of dimyristoylphosphatidylcholine, dimyristoylphosphatidylethanolamine and dimyristoylphosphatidylserine phase transitions in a concentration-dependent manner; c) in the presence of steroids, the fluorescence of Merocyanine 540 incorporated to the membranes decreased suggesting a fluidization of the lipid system; d) scanning electron microscopy observations showed that all steroids altered the normal shape of human erythrocytes inducing mainly echinocytosis, characterized by the formation of blebs in their surfaces, an indication that their molecules are located into the outer monolayer of the erythrocyte membrane.

Topics: Calorimetry, Differential Scanning; Dimyristoylphosphatidylcholine; Diosgenin; Erythrocyte Membrane; Fluorescent Dyes; Humans; Lipid Bilayers; Microscopy, Electron, Scanning; Phase Transition; Phosphatidylethanolamines; Phosphatidylserines; Pyrimidinones; Scattering, Small Angle; Solanaceous Alkaloids; Solanine; X-Ray Diffraction

The work introduces the quenching of silica coated Tb(III) complexes by merocyanine 540 (MC540) and copper ions as a tool to reveal the adsorption of phosphatidylcholine (PC) and phosphatidylserine (PS) at various PS:PC ratio onto silica nanoparticles doped with Tb(III) complex. The binding of MC540 with PC-based bilayers and copper ions with PS-based ones are the basis of their use as organic and inorganic probes to sense PS:PC ratio in silica supported mixed bilayers. The enrichment of mixed bilayers by PS results in the displacement of MC540 anions, while it enhances the binding with copper ions. The displacement or binding of probe ions results in the diverse luminescence response of Tb(III)-centred luminescence. The reestablishment of the steady state and time resolved luminescence is observed, when MC540 anions are applied as probes. The use of copper ions as probes results in the opposite quenching effect. The developed route enables to discriminate the formation of the phospholipids bilayers onto silica surface from those in the bulk of solution under various concentration conditions.

Topics: Absorption; Colloids; Copper; Energy Transfer; Ions; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylserines; Pyrimidinones; Silicon Dioxide; Solutions; Spectrometry, Fluorescence; Terbium

In clinic, the application of photodynamic therapy (PDT) in deep tissue is severely constrained by the limited penetration depth of visible light needed for activating the photosensitizer (PS). In this Article, a merocyanine 540 (MC540) and upconverting nanoparticle (UCN) coloaded functional polymeric liposome nanocarrier, (MC540 + UCN)/FPL, was designed and constructed successfully for solving this problem in PDT. Compared with the conventional approaches using UCNs absorbing PSs directly, the combination of UCN and polymeric liposome has unique advantages. The UCN core as a transducer can convert deep-penetrating near-infrared light to visible light for activating MC540. The functional polymeric liposome shell decorated with folate as a nanoshield can keep the UCN and MC540 stable, protect them from being attacked, and help them get into cells. The results show that (MC540 + UCN)/FPL is an individual nanosphere with an average size of 26 nm. MC540 can be activated to produce singlet oxygen successfully by upconverting fluorescence emitted from UCNs. After (MC540 + UCN)/FPL was modified with folate, the cell uptake efficiency increased obviously. More interestingly, in the PDT effect test, the (MC540 + UCN)/FPL nanocarrier further improved the inhibition effect on tumor cells by anchoring targeting folate and transactivating transduction peptide. Our data suggest that the (MC540 + UCN)/FPL nanocarrier may be a useful nanoplatform for future PDT treatment in deep-cancer therapy based on upconversion mechanism.

Topics: Cell Line, Tumor; Cells; Humans; Infrared Rays; Liposomes; Nanoparticles; Neoplasms; Photochemotherapy; Photosensitizing Agents; Polymers; Pyrimidinones

The application of photodynamic therapy in deep tissue is constrained by some pending problems, such as the limited penetration depth of excitation light and lacking of targeting ability. In this paper, a new kind of lipid coated upconverting nanoparticles consisiting of upconerting nanocrystal core and targeted lipid polymer shell was first reported for NIR triggered photodynamic therapy and cell imaging simultaneously. The lipid coated upconverting nanoparticles offers advantages to overcome the problem mentioned above. The UCN core works as a transducer to convert deeply penetrating near-infrared light to visible lights for activating photosensitizer and cell fluorescence imaging simultaneously. The amphiphilic lipid polymer RGD peptide conjugated poly (maleic anhydride-alt-1-octadecene) grafted dioleoyl l-α-phosphatidylethanolamine (RGD-PMAO-DOPE) acts as a shield. It can protect the system from catching by RES and target the whole system to the lesions. The experiment results show that the lipid coated upconverting nanoparticle is individual nanosphere with an average size of 20 nm. The drug loading can reach 9%. After NIR exposed, the MC540 was activated to produce singlet oxygen (ROS) successfully by the upconverting fluorescence emitted from UCN. Importantly, compared with nanoparticle without RGD decoration, the lipid coated upconverting nanoparticle can co-deliver the MC540 and UCNs into the same cell with higher efficiency. Besides, the MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles showed significant inhibitory effect on tumor cells after NIR shining. Our data suggests that MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle may be a useful nanoplatform for future PDT treatment in deep-cancer therapy.

Topics: Biological Transport; Cell Survival; Diagnostic Imaging; Drug Carriers; Humans; Infrared Rays; Maleic Anhydrides; MCF-7 Cells; Nanoparticles; Oligopeptides; Particle Size; Phosphatidylethanolamines; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Reactive Oxygen Species; Robotics; Spectroscopy, Fourier Transform Infrared

The colon cancer tissues from DMH treated rats exhibited higher membrane potential, fluidity and changed lipid order as examined by Merocyanine 540 and 1,6-diphenyl-1,3,5-hexatriene, respectively. A transition from gel to liquid crystalline state was observed by Laurdan fluorescence and also reduced fluorescence quenching of NBD-PE as contributed in the decreased membrane lipid phase separation. With piroxicam, a traditional NSAID and c-phycocyanin, a biliprotein from Spirulina platensis, these effects were normalized. An augmented intracellular Ca(+2) had contributed to the drug mediated apoptosis which is supported by an elevated calpain-9 expression. Histopathologically, a large pool of secreted acid/neutral mucopolysaccrides as well as the presence of blood vessels and dysplastic crypts signifies invasive mucinous adenocarcinoma while both the drugs reduced these neoplastic alterations. Wnt/β-catenin pathway was also found to be up-regulated which served as a crucial indicator for cancer cell growth. A concomitant down regulation of PPARγ was noted in DMH treatment which is associated with tumor progression. The expression of PPARα and δ, the other two isoforms of PPAR family was also modulated. We conclude that piroxicam and c-phycocyanin exert their anti-neoplastic effects via regulating membrane properties, raising calpain-9 and PPARγ expression while suppressing Wnt/β-catenin signaling in experimental colon carcinogenesis.

Topics: 2-Naphthylamine; Animals; Apoptosis; Calcium; Calpain; Carcinogenesis; Colonic Neoplasms; Fluorescence Polarization; Fura-2; Intracellular Space; Laurates; Ligands; Male; Membrane Fluidity; Membrane Potentials; Phase Transition; Phosphatidylethanolamines; Phycocyanin; Piroxicam; PPAR gamma; Pyrimidinones; Rats, Sprague-Dawley; Up-Regulation; Wnt Signaling Pathway

Hyper-Rayleigh scattering experiments and quantum chemical calculations are combined to investigate the second-order nonlinear optical responses of a series of three-arm merocyanine derivatives. They exhibit an octupolar hyperpolarizability response with lower amplitude than crystal violet due to a lower extent of the photoinduced charge transfer and reduced bond length alternation. Strong effects on the second-order optical response measured close to the two-photon absorption level are clearly evidenced; for example, the effective measured polarization ratio deviates below the ideal octupolar value of 3/2 even at very low excitation power. These effects are attributed to two-photon absorption resonance, which we believe modifies dynamically the population of the ground state versus that of the excited state.

Topics: Models, Molecular; Photons; Pyrimidinones; Quantum Theory

In this paper, magnetic and fluorescent bifunctional YbPO4:Er,Dy microspheres were synthesized via a simple solvothermal method. The prepared microspheres exposed to 980 nm near-infrared (NIR) laser light emitted bright upconversion fluorescence (450-570 nm) after calcination at high temperatures (>800°C). Results of magnetic resonance studies demonstrated that the YbPO4:Er,Dy microspheres are more suitable to be used as a transverse relaxation time (negative) contrast magnetic resonance imaging agent. The microspheres successfully entered the human hepatocellular carcinoma cells and presented low toxicity. A well-selected photodynamic therapy (PDT) drug, merocyanine 540 (MC540) with an ultraviolet-visible spectroscopy absorption maximum of 540 nm, was loaded onto the microspheres to obtain YbPO4:Er,Dy-MC540. Since the upconversion fluorescence emitting from the microspheres could be absorbed by MC540 with a small absorption/emission disparity, YbPO4:Er,Dy-MC540 could kill the hepatocellular carcinoma cells via PDT mechanism effectively. In other words, being upconverting particles, the prepared microspheres acted as light transducers in the NIR light-triggered PDT process. A chemotherapy drug, doxorubicin, was further loaded onto YbPO4:Er,Dy-MC540 to achieve enhanced antitumor effect based on synergistic therapeutic efficacy of PDT and chemotherapy. It is expected that the prepared YbPO4:Er,Dy microspheres have applications in tumor theranostics including magnetic-fluorescent bimodal imaging and NIR light-triggered PDT.

Topics: Biocompatible Materials; Cell Survival; Drug Synergism; Hep G2 Cells; Humans; Lanthanoid Series Elements; Light; Luminescent Agents; Magnetics; Microspheres; Photochemotherapy; Pyrimidinones

To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis.. ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and β5) and β-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvβ5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy.. Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and β5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type.. After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.

Topics: Blotting, Western; Cell Survival; Cells, Cultured; Flow Cytometry; Humans; Integrin alphaV; Integrin beta Chains; Oxidative Stress; Phagocytosis; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Receptor Protein-Tyrosine Kinases; Retinal Photoreceptor Cell Outer Segment

Spectroscopic and crystallographic studies reveal that Merocyanine 540 (MC 540), a well-known therapeutically important anionic cyanine dye, interacts with hen egg white lysozyme in ground state. The formation of the complex is validated by two isosbestic points in absorption spectra of lysozyme with varied concentration of MC 540 and appearance of an isodichroic point in induced CD spectra of MC 540 with lysozyme. The blue shift of fluorescence maximum of lysozyme in presence of MC 540 shows hydrophobic effect on Trp due to complex formation probably through cooperative binding. Above 1:3M stoichiometric ratio (lysozyme:MC 540) an additional fluorescence hump arises because of structural changes in protein, where MC 540 acts as self-denaturant, inducing non-linearity in Stern-Volmer plot. The van't Hoff isotherms with negative changes in enthalpy at lower concentration and positive changes in entropy for entire concentration range of MC 540 depict the binding forces as hydrogen bonding/van der Waal's and ionic/hydrophobic respectively. Finally X-ray crystallographic structure of the complex shows that MC 540 adopts two conformations, cis and trans, while it binds to lysozyme. Benzoxole moiety of MC 540 interacts with Trp123 through π-stacking and SO3(2-) group is stabilized by ionic interaction/H-bonding with Arg125 of lysozyme.

Topics: Animals; Circular Dichroism; Crystallography, X-Ray; Muramidase; Protein Binding; Pyrimidinones; Spectrometry, Fluorescence; Thermodynamics

Nanoscale transport of merocyanine 540 within/near the plasmon field of gold nanoparticles was recognized as an effective inducer of single-excitation dual-emission ratiometric properties. With a high concentration of the signal transducer (ammonium), a 700% increase in fluorescence was observed at the new red-shifted emission maximum, compared to a nanoparticle free sensor membrane. A previously nonrecognized isosbestic point is demonstrated at 581.4 ± 0.1 nm. The mechanism can be utilized for enhanced and simplified ratiometric optical chemical sensors and potentially for thin film engineering to make solar cells more effective and stable by a broader and more regulated absorption.

Topics: Fluorescent Dyes; Materials Testing; Nanoparticles; Pyrimidinones; Spectrometry, Fluorescence; Surface Plasmon Resonance

Investigation of protein activation in living cells is fundamental to understanding how proteins are influenced by the full complement of upstream regulators they experience. Here, we describe the generation of a biosensor based on the DARPin binding scaffold suited for intracellular applications. Combining library selection and knowledge-based design, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK with a solvatochromatic merocyanine dye, whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. The DARPin-based biosensor will serve as a useful tool for studying biological functions of ERK in vitro and in vivo.

Topics: Animals; Biosensing Techniques; Butadienes; Enzyme Activation; HEK293 Cells; Humans; Mice; Mitogen-Activated Protein Kinase 1; Muscle Proteins; Mutagenesis, Site-Directed; NIH 3T3 Cells; Nitriles; Nuclear Proteins; Phosphorylation; Protein Binding; Pyrimidinones; Substrate Specificity

Three new molecular building blocks 1 a-c for supramolecular polymerization are described that feature two dipolar merocyanine dyes tethered by p-xylylene spacers. Concentration- and temperature-dependent UV/Vis spectroscopy in chloroform combined with dynamic light scattering, capillary viscosimetry and atomic force microscopy investigations were applied to elucidate the mechanistic features of the self-assembly of these strongly dipolar dyes. Our detailed studies reveal that the self-assembly is very pronounced for bis(merocyanines) 1 a,b bearing linear alkyl chains, but completely absent for bis(merocyanine) 1 c bearing sterically more bulky ethylhexyl substituents. Both temperature- and concentration-dependent UV/Vis data provide unambiguous evidence for a cooperative self-assembly process for bis(merocyanines) 1 a,b, which was analyzed in detail by the Meijer-Schenning-Van-der-Schoot model (applicable to temperature-dependent data) and by the Goldstein-Stryer model (applicable to concentration-dependent data). By combining both methods all parameters of interest to understand the self-assembly process could be derived, including in particular the nucleus size (8-10 monomeric units), the cooperativity factor (ca. 0.006), and the nucleation and elongation constants of about 10(3) and 10(6)  M(-1) in chloroform at room temperature, respectively.

Topics: Macromolecular Substances; Models, Chemical; Molecular Structure; Polymerization; Pyrimidinones; Xylenes

The effect of 1:1 complex formation on the photochromic behavior of the merocyanine isomer of a nitro-substituted spirobenzopyran dye was studied in aqueous solution using 4-sulfonatocalixarene (SCXn) cavitands possessing four or eight phenol units. The binding constants were independent of the size of the macrocycle, and about 7-fold more stable associates were produced at pH 2.3 than in slightly alkaline solution. The complexation with SCXn diminished the acidity of protonated merocyanine (trans-MCH(+)) and precluded its photoinitiated transition to spirobenzopyran form, but did not affect the reactions in basic media. Upon exposure to light, the complexed trans-MCH(+) was converted to cis isomer. The association with 4-sulfonatocalix[4]arene slowed down the thermal back reaction in the dark to a larger extent than the confinement to 4-sulfonatocalix[8]arene. Both the activation energy and the Arrhenius A factor were significantly larger when the smaller, more rigid macrocycle served as a host.

Topics: Calixarenes; Hydrogen-Ion Concentration; Isomerism; Kinetics; Light; Protons; Pyrimidinones; Spiro Compounds; Sulfonic Acids; Water

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.

Topics: Animals; Biosensing Techniques; cdc42 GTP-Binding Protein; Cell Survival; Enzyme Activation; Fluorescent Dyes; Mice; Models, Molecular; NIH 3T3 Cells; Photobleaching; Protein Binding; Pyrimidinones

Photophysical studies on binding interactions of a negatively charged anti-tumor photosensitizer, Merocyanine 540 (MC 540), with serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA), have been performed using absorption and steady-state as well as time-resolved fluorescence techniques. Formation of ground state complex has been confirmed from the detailed studies of absorption spectra of MC 540 in presence of SAs producing isosbestic points. Binding between the proteins and MC 540, which perturbs the existing equilibrium between the fluorescent monomer and its non-fluorescent dimer, induces a remarkable enhancement in fluorescence anisotropy and intensity of MC 540 along with a red shift of its maximum. The binding stoichiometry of MC 540 and SAs are more than 1.0 which depicts that two types of complexes, i.e., 1:1 and 2:1 are formed with addition of varied concentration of protein. Both the steady-state and time-resolved fluorescence results show that in 2:1 complex one of the MC 540 molecules is exposed towards aqueous environment with a greater extent when bound with HSA compared to BSA due to the structural flexibility of that protein. Thermodynamic analyses using van't Hoff plot indicate that the binding between MC 540 and individual SA is an entropy-driven phenomenon. The probable hydrophobic binding site has been located by denaturation of proteins, micropolarity measurement and Förster resonance energy transfer and that is further supported by molecular docking studies. Changes in circular dichroism spectra of BSA in presence of MC 540 depict secondary structural changes of the protein. The induced-CD shows that BSA due to its rigid structure generates chirality in MC 540 much more efficiently compared to HSA.

Topics: Animals; Binding Sites; Cattle; Circular Dichroism; Computer Simulation; Fluorescence Resonance Energy Transfer; Humans; Hydrophobic and Hydrophilic Interactions; Photosensitizing Agents; Protein Binding; Protein Denaturation; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrimidinones; Serum Albumin; Serum Albumin, Bovine; Thermodynamics; Urea

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Topics: Adult; Bacteroides Infections; Benzimidazoles; Carbocyanines; Cell Membrane; Cell Survival; Escherichia coli Infections; Humans; Infertility, Male; Male; Mitochondria; Pyrimidinones; Spermatozoa; Staphylococcal Infections; Staphylococcus haemolyticus

A set of new octupolar merocyanine chromophores was designed and synthesized. These compounds were prepared from the reaction of 1,3,5-triformyl-2,4,6-trihydroxybenzene with heterocyclic nucleophiles. Octupolar dyes were formed exclusively in their open-dye form. The one- and two-photon-absorption spectra of the dyes consist of two bands: The long-wavelength band in the two-photon absorption spectrum (a few hundreds GM above 1000 nm) matches well with the intense, long-wavelength-absorption band that is located in the visible region in the linear spectrum. Interestingly, an additional, much-more-intense TPA band in the NIR region is observed at higher energy, which corresponds to a weakly allowed one-photon electronic transition. Changing the peripheral heterocyclic moieties allows tuning of the optical properties to approach the cyanine limit (i.e., polymethine state), thus resulting in a red-shift of the low-energy one-photon-absorption band as well as to the rise of an intense two-photon-absorption band in the NIR region. To the best of our knowledge, this is the first synthesis and TPA characterization of octupolar merocyanine chromophores with typical low-bond-length alternation.

Topics: Absorption; Fluorescent Dyes; Heterocyclic Compounds; Photochemistry; Photons; Pyrimidinones; Spectrometry, Fluorescence; Spectroscopy, Near-Infrared

The photophysical properties of 4-[2-(6-hydroxy-2-naphthalenyl)-ethenyl]-1-methyl-pyridinium (HNEP(+)) and its deprotonated form (NEP), a benzofused derivative of Brooker's merocyanine (BM), were investigated through a combined spectroscopic and computational approach. Despite their structural similarities and similar pK(a) values, HNEP(+)/NEP and BMH(+)/BM differ in the extent of charge delocalization in the ground and excited states. NEP exhibits the spectral characteristics of a charge transfer species in solvents in which BM exists in a charge-delocalized quinoid; however, quantum chemical calculations show that the CT absorption of NEP is not necessarily a consequence of the zwitterionic character. HNEP(+) displays larger Stokes shifts than BMH(+), and NEP demonstrates enhanced solvatochromism relative to BM as a consequence of benzofusion.

Topics: Coloring Agents; Hydrogen-Ion Concentration; Models, Molecular; Molecular Conformation; Protons; Pyridines; Pyrimidinones; Quantum Theory; Solvents; Spectrum Analysis

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Algorithms; Binding, Competitive; Cell Membrane; Chlorobenzenes; Cholesterol; Dimyristoylphosphatidylcholine; Gels; Kinetics; Lipid Bilayers; Microarray Analysis; Microscopy, Fluorescence; Molecular Structure; Phase Transition; Phosphatidylcholines; Pyrimidinones; Reproducibility of Results; Spectrometry, Fluorescence

Sub-diffraction optical imaging with nanometer resolution of lipid phase-separated regions is reported. Merocyanine 540, a probe whose fluorescence is sensitive to the lipid phase, is combined with super-resolution imaging to distinguish the liquid- and gel-phase nanoscale domains of lipid bilayers supported on glass. The monomer-dimer equilibrium of MC540 in membranes is deemed responsible for the population difference of single-molecule fluorescence bursts in the different phase regions. The extension of this method to other binary or ternary lipid models or natural systems provides a promising new super-resolution strategy.

Topics: Fluorescent Dyes; Lipid Bilayers; Particle Size; Pyrimidinones; Spectrometry, Fluorescence

According to "fluid-mosaic model," plasma membrane is a bilayer constituted by phospholipids which regulates the various cellular activities governed by many proteins and enzymes. Any chemical, biochemical, or physical factor has to interact with the bilayer in order to regulate the cellular metabolism where various physicochemical properties of membrane, i.e., polarization, fluidity, electrostatic potential, and phase state may get affected. In this study, we have observed the in vivo effects of a pro-carcinogen 1,2-dimethylhydrazine dihydrochloride (DMH) and the two non steroidal anti-inflammatory drugs (NSAIDs); sulindac and celecoxib on various properties of the plasma membrane of colonocytes, i.e., electric potential, fluidity, anisotropy, microviscosity, lateral diffusion, and phase state in the experimentally induced colorectal cancer. A number of fluorescence probes were utilized like membrane fluidity and anisotropy by 1,6-diphenyl-1,3,5-hexatriene, membrane microviscosity by Pyrene, membrane electric potential by merocyanine 540, lateral diffusion by N-NBD-PE, and phase state by Laurdan. It is observed that membrane phospholipids are less densely packed and therefore, the membrane is more fluid in case of carcinogenesis produced by DMH than control. But NSAIDs are effective in reverting back the membrane toward normal state when co-administered with DMH. The membrane becomes less fluid, composed of low electric potential phospholipids whose lateral diffusion is being prohibited and the membrane stays mostly in relative gel phase. It may be stated that sulindac and celecoxib, the two NSAIDs may exert their anti-neoplastic role in colorectal cancer via modifying the physicochemical properties of the membranes.

Topics: 1,2-Dimethylhydrazine; 2-Naphthylamine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Celecoxib; Cell Membrane; Chemical Phenomena; Colorectal Neoplasms; Cyclooxygenase 2; Laurates; Male; Membrane Fluidity; Microscopy, Fluorescence; Phosphatidylethanolamines; Pyrazoles; Pyrimidinones; Rats; Rats, Sprague-Dawley; Spectrometry, Fluorescence; Sulfonamides; Sulindac; Viscosity

2,6-Diphenyl-4-(2,4,6-triphenylpyridinium-1-yl)phenolate (1a) and 4-[(1-methyl-4(1H)-pyridinylidene)-ethylidene]-2,5-cyclohexadien-1-one (2a) were protonated in organic solvents (dichloromethane, acetonitrile, and DMSO) to form 1b and 2b, respectively. The appearance of the solvatochromic bands of 1a and 2a was studied UV-vis spectrophotometrically by deprotonation of 1b and 2b in solution in the presence of the following amines: aniline (AN), N-methylaniline (NMAN), N,N-dimethylaniline (NDAN), n-butylamine (BA), diethylamine (DEA), and triethylamine (TEA). Titrations of 1b and 2b with the amines were carried out and the binding constants were determined from the titration curves in each solvent, using a mathematical model adapted from the literature which considers the simultaneous participation of two dye: amine stoichiometries, 1:1 and 1:2. The data obtained showed the following base order for the two compounds in DMSO: BA>DEA>TEA, while aromatic amines did not cause any effect. In dichloromethane, the following base order for 1b was verified: TEA>DEA>BA≫NDAN, while for 2b the order was: TEA>DEA>BA, suggesting that 1b is more acidic than 2b. The data in acetonitrile indicated for 1b and 2b the following order for the amines: DEA>TEA>BA. The diversity of the experimental data were explained based on a model that considers the level of interaction of the protonated dyes with the amines to be dependent on three aspects: (a) the basicity of the amine, which varies according to their molecular structure and the solvent in which it is dissolved, (b) the molecular structure of the dye, and (c) the solvent used to study the system.

Topics: Amines; Aniline Compounds; Binding Sites; Butylamines; Drug Interactions; Hydrogen-Ion Concentration; Models, Biological; Molecular Conformation; Protons; Pyrimidinones; Solutions; Solvents; Spectrophotometry; Stereoisomerism; Titrimetry

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G0/G1 phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.

Topics: Analysis of Variance; Animals; Antioxidants; Apoptosis; Caspase 3; Cell Cycle; Cell Proliferation; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytochromes c; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Female; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurons; Photosensitizing Agents; Pregnancy; Proto-Oncogene Proteins c-bcl-2; Pyrimidinones; Rats; Rats, Sprague-Dawley; Serine; Signal Transduction; Thioctic Acid; Time Factors; Zinc

The synthesis, properties and applications of a novel boronate-functioned styryl dye, BSD, as a colorimetric sensor for hydrogen peroxide is presented. The dye displayed remarkable color change from colorless (lambda(max)=391 nm) to deep red (lambda(max)=522 nm) in the presence of H(2)O(2) and the behavior could be rationalized by the chemoselective H(2)O(2)-mediated transformation of arylboronate to phenolate, resulting in the release of the merocyanine dye which featured with strong intramolecular charge transfer (ICT) absorption band. The absorption increment of merocyanine at lambda(max)=522 nm (epsilon=87000 L mol(-1) cm(-1)) is linear with the concentration of H(2)O(2) in the range of 1.0 x 10(-7)-2.5 x 10(-5) mol L(-1) with the detection limit of 6.8 x 10(-8) mol L(-1) under optimum conditions. There is almost no interference by other species that commonly exist due to the specific deprotection of H(2)O(2) towards arylboronate group on BSD. The chromogenic sensor has been applied to the detection of trace amounts of hydrogen peroxide in rain water.

Topics: Boronic Acids; Colorimetry; Hydrogen Peroxide; Hydrogen-Ion Concentration; Pyrimidinones; Radiation-Sensitizing Agents; Spectrophotometry, Ultraviolet; Styrenes

The adsorption potential of the blast furnace slag of a ferrosilicon firm in Aswan Governorate, Egypt, to decolorize aqueous solutions of 3-methyl-1-phenylpyrazol-5-one 4[2] merocyanine dye (1) was investigated at room temperature. The influence of the solution pH, the quantity of adsorbent, the initial concentration of 1, and the applied contact time were studied with the batch technique. The maximum percentage of removal of 1 was observed at pH 4. The adsorption data were better fitted by the Freundlich than by the Langmuir adsorption isotherm model, confirming the formation of monolayers of 1 on the adsorbent surface. Kinetic rate constants and the transient behavior at different initial concentrations of 1 were determined with both the Lagergren pseudo-first-order and the Ho and McKay pseudo-second-order kinetic models. The calculated kinetic parameters revealed that the adsorption of 1 on blast furnace slag followed a second-order chemisorption process.

Topics: Adsorption; Egypt; Hydrogen-Ion Concentration; Industrial Waste; Kinetics; Pyrimidinones; Silicon Compounds; Spectrophotometry, Ultraviolet; Temperature; Water Pollutants, Chemical

Identically configured bulk heterojunction organic solar cells based on merocyanine dye donor and fullerene acceptor compounds (see figure) are manufactured either from solution or by vacuum deposition, to enable a direct comparison. Whereas the former approach is more suitable for screening purposes, the latter approach affords higher short-circuit current density and power conversion efficiency.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Fluorescent Dyes; Fullerenes; Polymers; Polystyrenes; Pyrimidinones; Quantum Theory; Solar Energy

The photobehavior of merocyanine 540 (MC) was studied in homogeneous media (ethanol, buffer and glycerol), and in microheterogenous systems (Triton X-100 micelles and in the presence of human serum albumin) using stationary and time-resolved techniques. Merocyanine 540 in aqueous solution mostly forms aggregates, which in the presence of Triton X-100 or HSA are disaggregated. The extent of binding to HSA and its characteristics were estimated from dye absorption and fluorescence changes following protein addition; the Trp-214 fluorescence quenching was also employed to assess the extent of dye association, and physical separation was employed to evaluate the dye's apparent association constant. These results showed that dye adsorption on HSA takes place at both main protein-binding sites (I and II). This adsorption leads to dye monomerization, changing its photobehavior remarkably, with a noticeable increase in fluorescence and triplet lifetimes. These slower decays can be ascribed to a reduction of the dye photoisomerization rate. In addition, the adsorption of the dye partially protects it from the oxygen present in solution, thus reducing the apparent dye triplet-quenching rate constant. However, singlet oxygen and MC triplet quantum yields remain very low in all the systems tested. Finally, we found that the photoconsumption of merocyanine bound to HSA takes place predominantly by a type I mechanism, being more than seven times more efficient than that taking place in ethanol.

Topics: Adsorption; Humans; Isomerism; Octoxynol; Photochemical Processes; Photolysis; Photosensitizing Agents; Protein Binding; Pyrimidinones; Quantum Theory; Serum Albumin; Spectrometry, Fluorescence; Tryptophan

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type-WT) occur at 4-7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.

Topics: Arabidopsis; Arabidopsis Proteins; Circular Dichroism; Galactolipids; Galactosyltransferases; Light-Harvesting Protein Complexes; Microscopy, Fluorescence; Plants, Genetically Modified; Pyrimidinones; Temperature; Thylakoids

The packaging of heterochromatin during spermatogenesis has been correlated with the expression of residual apoptotic bodies (which stain with merocyanine A) that will impact on sperm function in the fertilization process; as well as the joint expression of the transmembrane translocation phosphatidyl serine and oligonucleosomes.. To evaluate the expression of bodies stained with merocyanine in the functional processes of sperm and their level of agreement with apoptotic Annexin V and TUNEL biomarkers.. We performed a prospective, cross, including 11,000 cells belonging to semen samples from infertile men, were evaluated according to WHO criteria (1999), bounded by the lines of Tygerberg. The biomolecular transformation processing of the membrane and the expression of oligonucleosomes in the terminal cascade of apoptosis were quantified by cytometry flow, using an argon lasser as a reading source of 480 nm, discriminating the degree of cellularity, both negative and positive for each indicators.. Because of the study design was found low quantification in semen parameters, motility, morphology and sperm concentration. The average expression of cells [DNA-PI(+) / dUTP-FITC(+)] (quantification of TUNEL) and [Annexin-V(+) / PI(-)] was 36.5 +/- 17.4% and 31.2 +/- 17.4%, respectively. By comparing the expression of TUNEL without the effect of M540 bodies (36.3 +/- 1.7% vs. 36 +/- 1.7%) a significant difference was not determined.. This study shows that there is a remnant of the primary processes of spermiation, which can take an important role in apoptotic and functional processes of the sperm. However, its expression does not affect measurement of biomarkers of apoptosis seminal, whose determination changed the diagnosis and functional perception of reproductive parameters in the sperm.

Topics: Annexin A5; Apoptosis; Artifacts; Awards and Prizes; Cross-Sectional Studies; DNA Fragmentation; False Positive Reactions; Fertilization; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fluorometry; Gynecology; Heterochromatin; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Membrane Lipids; Mexico; Nucleosomes; Obstetrics; Phosphatidylserines; Prospective Studies; Pyrimidinones; Sperm Motility; Spermatozoa; Staining and Labeling

The molar transition energy (E(T)) polarity values for the solvatochromic probes 2,6-diphenyl-4-(2,4,6-triphenylpyridinium)phenolate (1), 4[(1-methyl-4-(1H)-pyridinylidene)-ethylidene]-2,5-cyclohexadien-1-one (2), and 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (3) were collected in binary mixtures comprising chloroform and a hydrogen-bond accepting (HBA) solvent [dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), acetone or acetonitrile], aiming to investigate the ability of the chlorinated component to act as hydrogen-bond donating (HBD) solvent. Plots of E(T) as a function of X(2), the mole fraction of chloroform, were obtained and the data were analysed to investigate the preferential solvation (PS) of each probe in terms of both solute-solvent and solvent-solvent interactions. For dyes 1 and 2 a strong synergistic behavior was observed for all mixtures studied, indicating that the dyes are preferentially solvated by complexes formed through hydrogen bonding between chloroform and the HBA component in the mixtures. A study of 1 in deuterated chloroform with an HBA component (DMF and DMA) demonstrated that while almost no differences occur with the DMF mixtures, the presence of deuterated chloroform in its mixtures with DMA increases the synergistic effect, suggesting that it interacts more strongly with DMA, making its mixtures more polar. These data were successfully fitted to a model based on solvent-exchange equilibria. The features of the mixtures with dye 3 revealed a very different profile in comparison with the other two dyes, which suggests that in mixtures containing chloroform, the microenvironment of the dye seems to be important in determining the contribution of the structure resonances responsible for the stability of the dye.

Topics: Chloroform; Complex Mixtures; Drug Combinations; Drug Synergism; Hydrogen Bonding; Models, Biological; Models, Chemical; Pyrimidinones; Solvents

Photoinduced calcium release from the crown ether-linked merocyanine DCM-crown is reexamined by femtosecond transient absorption spectroscopy with sub-100 fs time resolution. Photodisruption of the bond linking the cation to the nitrogen atom shared by the crown and the chromophore is found to take place in 130 fs. Confirming our previous reports, the photoinduced intraligand charge transfer is observed in the picosecond regime but kinetics involving three-components (1 ps, 8 ps and 77 ps), together with a 56 ps stimulated-emission time-resolved red shift, are found in the present study. Both delayed intraligand charge transfer and cation release are assumed to occur in this time range due to repulsion effects between the positively charged nitrogen of the crown ether moiety and the cation. In the subnanosecond regime, a 670 ps time-resolved red shift of the stimulated-emission spectrum of the charge-transfer state, similar to the shift previously observed with Sr(2+), demonstrates the motion of the cation away from the crown to the bulk. A thorough examination of the present data allows us to conclude that calcium ion is photoejected to the bulk in a multistep process.

Topics: Calcium; Crown Ethers; Ion Transport; Kinetics; Photochemistry; Pyrimidinones; Time Factors

Brooker's merocyanine (BM), which changes its emission and absorption maxima upon protonation, was introduced into oligodeoxyribonucleotide (ODN) via d-threoninol by postsynthetic modification on a CPG (controlled-pore glass) support. The pK(a) of BM in the modified ODN increased from 9.5 to 10.1 upon hybridization. As a result, absorption maxima shifted from 492 to 432 nm at pH 10.0 by the presence of its complementary strand. This spectral shift was sufficiently large so that DNA hybridization could easily be discriminated even by the naked eye; the color of the solution changed from orange to yellow upon hybridization. In addition, the fluorescence emission was strongly quenched upon hybridization, demonstrating that this probe can also detect the target DNA by the fluorescence change. Ratiometric detection of hybridization was also possible by simultaneous excitation of both protonated and deprotonated BMs. Furthermore, we could also modulate its pK(a) by the antiparallel stacking of two BM molecules in the duplex; the pK(a) of BM decreased from 10.1 to 9.7 by the stacking of two BMs in an antiparallel manner. Thus, control of the microenvironment around the BM molecule allowed modulation of its pK(a), which is applicable to the sequence-specific recognition of target DNA.

Topics: Absorption; Amino Alcohols; Base Sequence; Butylene Glycols; DNA; DNA, Single-Stranded; Fluorescence; Fluorescent Dyes; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Protons; Pyrimidinones

The solvatochromic behavior of N-(2,5-di-tert-butylphenyl)-9-pyrrolidinoperylene-3,4-dicarboximide () was investigated by measuring the excitation and emission spectra over a wide range of temperature in 2-methyltetrahydrofuran (MTHF). The temperature induced spectral changes can be compared with the changes caused by changing solvent polarity using different solvents at room temperature. In both cases a strong positive solvatochromism is observed both in absorption/excitation and in emission. The difference between excitation and emission energies decreases with increasing solvent polarity. The behavior of can be rationalized in terms of a change in electronic structure with solvent polarity. Although has the typical molecular structure of a push-pull substituted aromatic system, in which the solvatochromic shift in emission is normally larger than that in absorption, in its solvent-induced electronic structure change it resembles a merocyanine.

Topics: Absorption; Fluorescent Dyes; Perylene; Piperidines; Pyrimidinones; Solvents; Spectrophotometry, Ultraviolet; Temperature

During apoptosis, physical changes in the plasma membrane prepare the cell for clearance by phagocytes and hydrolysis by secretory phospholipase A(2) (sPLA(2)). The relationships among these changes have not been adequately established, especially for hormone-stimulated apoptosis. This study addresses these issues for glucocorticoid-induced apoptosis in S49 lymphoma cells. Flow cytometry, microscopy, and fluorescence spectroscopy were used to assess merocyanine 540 emission, laurdan generalized polarization, phosphatidylserine exposure, caspase activation, and membrane permeability to propidium iodide in the absence and presence of sPLA(2). The earliest event observed was activation of cellular caspases. Results with membrane probes suggest that interlipid spacing also increases early during apoptosis and precedes transbilayer migration of phosphatidylserine, DNA fragmentation, and a general increase in lipid order associated with blebbing and dissolution of the cells. The activity of sPLA(2) appeared to be linked more to lipid spacing than to loss of membrane asymmetry. The early nature of some of these events and their ability to promote activity of a proinflammatory enzyme suggests the possibility of an inflammatory response during T-lymphocyte apoptosis.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Membrane; Cell Survival; Enzymes; Flow Cytometry; Fluorescent Dyes; Glucocorticoids; Hydrolysis; Lipid Metabolism; Lymphoma; Microscopy; Phosphatidylserines; Phospholipases A2; Pyrimidinones; Spectrometry, Fluorescence; Time Factors; Water

GroEL(SP/MC), prepared by genetic and chemical modifications of group I chaperonin protein GroEL, site-specifically possesses approximately 28 photochromic (spiropyran [SP] and merocyanine [MC]) units in the entrance parts of its cavity. Addition of divalent metal ions such as Mg(2+) to a tris-HCl buffer solution of GroEL(SP/MC) results in one-dimensional (1D) assembly of GroEL(SP/MC), affording cylindrical hollow fibers with a very large aspect ratio; the longest fiber was approximately 2.5 microm long, corresponding to a 170-mer of GroEL(SP/MC) (MW approximately 1.4 x 10(8)). When such long fibers are mixed with EDTA, they are cut into short-chain oligomers and eventually into monomeric GroEL(SP/MC). Similar to GroEL, GroEL(SP/MC) possesses a large binding affinity toward denatured proteins. When GroEL(SP/MC) undergoes 1D assembly after incubation with a denatured protein, guest-containing cylindrical fibers result.

Topics: Benzopyrans; Cations, Divalent; Chaperonin 60; Indoles; Magnesium; Nitro Compounds; Protein Denaturation; Protein Engineering; Pyrimidinones

An earlier proposed strategy to orient two merocyanine dyes in the desirable head-to-tail parallel fashion through multiple hydrogen bonds has been examined by molecular dynamics simulations and quantum chemical calculations. Two different merocyanine dyes dissolved in chloroform solution under various conditions are simulated by molecular dynamics. It is found that two dipolar units can be well connected through various numbers of hydrogen bonds. Although the probability to form the desirable head-to-tail dimer is unfortunately small even under strong poling electric field and low temperature, the formation of unwanted antiparallel structure has been effectively reduced. Typical hydrogen bonded dimers obtained from molecular dynamics simulations have been studied by hybrid density functional calculations. It is found that only the most probable complex can lead to the optical absorption spectrum that is in close agreement with the corresponding experiments. Calculated results for dipole moments of ground and charge transfer states, as well as first hyperpolarizabilities, of three typical complexes have also been provided.

Topics: Absorption; Hydrogen Bonding; Models, Molecular; Molecular Conformation; Optical Phenomena; Photons; Pyrimidinones; Quantum Theory; Thermodynamics

A new "dual-mode" chromogenic and fluorescent turn-on probe (2) for the selective sensing of biological thiols is reported. In MeOH-H(2)O cosolvent at physiological pH 7.40 (MeOH-H(2)O = 3:7), biological thiols cleave the 2,4-dinitrobenzenesulfonyl group to release the chromo- and fluorophore merocyanine (3).

Topics: Colorimetry; Fluorescent Dyes; Hydrogen-Ion Concentration; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds

Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (alpha-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.

Topics: Apoptosis; Benzimidazoles; Down-Regulation; Humans; Ketocholesterols; Microscopy, Electron, Transmission; Oxazines; Phosphatidylinositol 3-Kinases; Phospholipids; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidinones; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; U937 Cells; Vitamin E

Density Functional Theory (DFT) calculations have been performed on the TTC-->TTT isomerization reaction of the open forms of the 1',3'-dihydro-8-bromo-6-nitro-1',3',3'-trimethylspiro[2H-1-benzopyran-2,2'-(2H)indole (8-Br-6-nitro-BIPS) system. The calculations were carried out in vacuo and in methylene chloride solution at different temperatures. Results are compared with the available experimental values of free energy difference and activation energy in solution.

Topics: Benzopyrans; Fluorescent Dyes; Indoles; Methylene Chloride; Models, Molecular; Models, Theoretical; Nitro Compounds; Pyrimidinones; Solutions; Stereoisomerism; Thermodynamics; Vacuum

Conflicting results are reported by recent studies comparing flow cytometry (FCM) and fluorescence microscopy (FM) for detecting sperm DNA fragmentation by TUNEL assay. Each of the two technologies has specific peculiarities and limitations, but whereas the limitations of FM observation are well known, the biases due to the inability of FCM to recognize morphologically analyzed cells are less explored. In particular, so far, FCM analysis of sperm DNA fragmentation have included in the analyses M540 bodies, round semen structures exhibiting FSC/SSC properties similar to sperm. Semen M540 bodies, altogether with the occurrence of two sperm populations with different nuclear staining, concur to an underestimation of values of sperm DNA fragmentation by FCM. However, even considering such bias, the observed discrepancies between the performance of FM and FCM are not fully explained. We discuss here the possible variables that may affect the results of each of the two technologies and the necessary efforts to be taken to address this issue.

Topics: Cell Nucleus; DNA; DNA Fragmentation; Flow Cytometry; Humans; In Situ Nick-End Labeling; Intranuclear Inclusion Bodies; Male; Microscopy, Fluorescence; Pyrimidinones; Sensitivity and Specificity; Spermatozoa

Photodynamic treatment (PDT) has been proposed as a new approach for inactivation of biofilms associated with medical devices that are resistant to chemical additives or biocides. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against Staphylococcus epidermidis biofilms. Effect of the combined photodynamic action of MC 540 and 532 nm laser was investigated on the viability and structure of biofilms of two Staphylococcus epidermidis strains, RP62A and 1457. Significant inactivation of cells was observed when biofilms were exposed to MC 540 and laser simultaneously. The effect was found to be light dose-dependent but S. epidermidis 1457 biofilm proved to be slightly more susceptible than S. epidermidis RP62A biofilm. Furthermore, significant killing of both types of cells was attained even when a fixed light dose was delivered to the biofilms. Confocal laser scanning microscope (CLSM) analysis indicated damage to bacterial cell membranes in photodynamically treated biofilms, while disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).

Topics: Anti-Bacterial Agents; Biofilms; Cell Membrane; Dose-Response Relationship, Radiation; Lasers; Microscopy, Confocal; Microscopy, Electron, Scanning; Photosensitizing Agents; Pyrimidinones; Staphylococcus epidermidis

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.

Topics: Fluorescent Dyes; Membrane Lipids; Molecular Structure; Pyrimidinones; Spectrometry, Fluorescence; Temperature; Thylakoids; Time Factors

Time-dependent Hartree-Fock and Møller-Plesset second-order calculations have been used to unravel the relationships between structure and first hyperpolarizability in spiropyran/merocyanine couples and therefore to design efficient second-order nonlinear optical switching compounds. Large first hyperpolarizabilities for the merocyanine form as well as large contrasts of first hyperpolarizability have been obtained when, on the same species, (i) substituents at R(1) and R(2) positions on the phenolate ring of the merocyanine form are strong acceptor and donor substituents, respectively, (ii) the ethylenic bridge is substituted by donor groups, (iii) the other aromatic part of the system is benzimidazolo rather than indolino or benzothiazolo, and (iv) strong donor substituents are placed on the benzimidazolo moiety.

Topics: Benzimidazoles; Benzopyrans; Ethylenes; Indoles; Kinetics; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Structure; Nitro Compounds; Nonlinear Dynamics; Pyrimidinones; Quantum Theory; Structure-Activity Relationship

Counterions affect on the substructures formation in the case of the merocyanine dye, 1-methyl-4-[2-(4-hydroxyphenyl)ethenyl)]piridinium] hydrogensquarate both in gas and condense phase. Spectroscopically and structural elucidation of these aggregates have been performed, using solid-state conventional and linear-polarized IR-spectroscopy of oriented colloids as a nematic liquid crystal suspension, UV-vis spectroscopy, HPLC tandem ESI mass spectrometry, 1H and 13C NMR, TGV and DSC. Quantum chemical DFT calculations have been carried out as well. Experimental and theoretical data are compared with analogous ones of corresponding iodide salt of dye studied.

Topics: Electrons; Models, Molecular; Molecular Structure; Pyrimidinones; Solvents; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry

S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.

Topics: Animals; Biosensing Techniques; Calcium; Cell Movement; Cell-Free System; Circular Dichroism; Cysteine; Egtazic Acid; Fibroblasts; Fluorescence Polarization; Humans; Iodoacetamide; Lysophospholipids; Mice; Models, Molecular; Molecular Structure; Nonmuscle Myosin Type IIA; Phenothiazines; Protein Binding; Protein Conformation; Pyrimidinones; Recombinant Proteins; S100 Calcium-Binding Protein A4; S100 Proteins; Spectrometry, Fluorescence; Trifluoperazine

Photoisomerization of two cyanine derivatives, 3,3(')-diethyloxadicarbocyanine iodide (DODCI) and merocyanine 540 (MC 540), has been investigated in an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate and aqueous glycerol (93 wt % glycerol +7 wt % water) by measuring fluorescence lifetimes and quantum yields. The aim of this work is to understand how the rates of photoisomerization of DODCI and MC 540 are influenced by specific solute-solvent interactions besides the viscosity of the medium. For DODCI, it has been observed that the nonradiative rate constants, which represent the rates of photoisomerization, are almost identical in the ionic liquid and aqueous glycerol at given temperature, indicating that viscosity is the sole parameter that governs the rate of photoisomerization. In contrast, the photoisomerization rate constants of MC 540 have been found to be a factor of 2 higher in aqueous glycerol compared to the ionic liquid. The observed behavior is due to the zwitterionic character of MC 540, a consequence of which, the twisted state gets stabilized by the solute-solvent hydrogen bonding interactions in aqueous glycerol, thus lowering the barrier for isomerization.

Topics: Carbocyanines; Fluorescence; Glycerol; Imidazoles; Ionic Liquids; Isomerism; Light; Molecular Structure; Photochemistry; Pyrimidinones; Quantum Theory; Sensitivity and Specificity; Temperature; Water

Structural and spectroscopic elucidation of merocyanine dye, 2,5-[1-metyl-4-[2-(4-hydroxyphenyl)ethenyl)]piridinium]-hexane tetraphenylborate, is performed in gas and condense phase by means of solution and solid-state conventional and linear-polarized IR-spectroscopy of oriented colloids in nematic liquid crystal suspension, UV-vis and fluorescence methods, HPLC MS/MS tandem and ESI mass spectrometry, (1)H, (13)C and (1)H-(1)H COSY NMR, TGV and DSC methods. Quantum chemical DFT calculations are performed for structural optimization and spectroscopic properties prediction.

Topics: Chromatography, High Pressure Liquid; Dimerization; Magnetic Resonance Spectroscopy; Molecular Structure; Pyrimidinones; Quantum Theory; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Tandem Mass Spectrometry; Thermodynamics

Topics: Aptamers, Nucleotide; Benzopyrans; Indoles; Light; Molecular Structure; Nitro Compounds; Photochemistry; Pyrimidinones; RNA; Substrate Specificity; Surface Plasmon Resonance

To devise and evaluate a protocol for monitoring lipid packing and membrane permeability in live cells in whole peripheral blood, and to assess these properties in blood from controls and patients.. Samples were stained simultaneously with merocyanine 540 and Sytox Green, diluted, and analyzed by three-color flow cytometry.. Membrane changes characteristic of apoptosis/necrosis were detected in cells in culture and in blood that had been "aged" in vitro, with sensitivity and specificity, which was comparable to that obtained using fluoresceinated Annexin-V and ethidium bromide or propidium iodide. Merocyanine 540 also reported increases in membrane lipid disorder when cells in whole blood were activated by the Ca(2+) ionophore, A23187. Very few (<2%) leukocytes or platelets in the blood of healthy subjects (n = 14) had disordered and/or permeable membranes, However, if blood was stored with heparin the microparticle to platelet ratio increased and membrane lipids of microparticles and platelets became disordered within hours. In the blood of patients with idiopathic thrombocytopenia (n = 4), the microparticle to platelet ratio (1.5 +/- 1.1 vs 0.14 +/- 0.06; p = 0.000), and the percentages of microparticles (67.3% +/- 34.9% vs 20.4% +/- 12.6%; p = 0.000) and platelets (47.0% +/- 18.8% vs 1.9 +/- 2.0%; p = 0.000) with disordered membrane lipids were all markedly increased by comparison with the controls.. This stain combination enabled important membrane characteristics to be assessed simply and quickly in unfixed, whole blood; and revealed significant differences in patient blood.

Topics: Adult; Aged; Apoptosis; Blood Platelets; Cell Line; Cell Membrane Permeability; Female; Flow Cytometry; Fluorescent Dyes; Humans; Ionophores; Leukocytes; Male; Membrane Lipids; Middle Aged; Organic Chemicals; Pyrimidinones; Reference Values; Staining and Labeling; Thrombocytopenia

The structure of sarcoplasmic reticulum membranes was studied in the presence of modeled transmembrane Ca2+ gradient corresponding to the status of Ca2+ depot at different stages of the muscle contraction-relaxation cycle in health and disease. Various sites of the membrane were characterized using spectral analysis of tryptophan, pyrene, and merocyanine-540 fluorescence without evaluating specific changes in the molecules of membrane components (Ca2+ -ATPase, ryanodine receptor, and lipids). The transmembrane Ca2+ gradient modulates the protein-lipid interactions and structural characteristics of the membrane. The proposed model can be used for studies of the effects of pharmacologically active substances and endogenous regulators.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Fluorescence; Intracellular Membranes; Membrane Lipids; Models, Theoretical; Protein Binding; Pyrenes; Pyrimidinones; Rabbits; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Spectrometry, Fluorescence; Tryptophan

The fluorescence spectra of Merocyanine 540 (MC 540), an anionic dye have been studied in aqueous solution of different nonionic surfactants. The results show the enhancement and red shift of fluorescence bands, indicating electron transfer from the surfactants to the excited dye. This is also supported by the photovoltage generation by the dye-surfactant systems in a photoelectrochemical cell. Possible mechanisms of the excited state interaction and photovoltage generation have been suggested. From the thermodynamic, spectrophotometric and photogalvanic results, it can be concluded that the electron donating abilities of the nonionic surfactants towards MC 540 are in the order: Tween 80 approximately Tween 60>Tween 40>Tween 20>Triton X-100. The spectral studies (both absorption and fluorescence) of Merocyanine 540 have been carried out in solvents of varying polarities as well as in an aqueous micellar dispersions of nonionic surfactants. The Stokes shifts of the fluorescence from the absorption have been found to increase with increasing polarity of the solvents. An attempt has been made to ascertain the polarity of the microenvironment of Merocyanine 540 in the nonionic surfactant media from the photophysical characteristics of the dye in different solvents of known polarities.

Topics: Electrochemistry; Kinetics; Micelles; Photochemistry; Pyrimidinones; Solutions; Solvents; Spectrometry, Fluorescence; Surface-Active Agents; Temperature; Thermodynamics

To directly compare distinct assays proposed to monitor human sperm quality and possibly preselect sperm populations for assisted reproductive technology (ART).. Analysis of human sperm sample quality using several methodologies.. Academic and clinical institutions.. Samples from consenting patients undergoing routine semen analysis or ART.. Human sperm samples were analyzed in terms of World Health Organization parameters and processed for annexin V, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA (TUNEL), and the sperm-ubiquitin tag immunoassay (SUTI). Samples were analyzed both by flow cytometry and fluorescence microscopy.. Correlations among apoptotic markers (outer leaflet phosphatidylserine exposure, membrane integrity, and DNA fragmentation), external ubiquitination, and semen parameters in human spermatozoa.. Nonviable sperm, TUNEL-positive cells, and ubiquitin fluorescence intensity means inversely correlate with semen parameters. Apoptotic markers do not correlate with sperm surface ubiquitination. Normozoospermic samples have a higher number of viable cells and lower DNA fragmentation compared with samples with abnormal parameters. Nonviable sperm are more prevalent in samples with low counts and poor morphology but not low motility. Not all sperm with morphologic abnormalities present surface ubiquitination.. Sperm quality is inversely correlated with lack of viability, DNA fragmentation, and ubiquitin fluorescence intensity means. However, none of the apoptotic markers correlate with ubiquitin labeling. Elimination of defective sperm cells prior to ART using surface markers (annexin V, ubiquitin) seems unwarranted at this stage.

Topics: Annexin A5; Apoptosis; Cell Survival; DNA Fragmentation; Flow Cytometry; Humans; In Situ Nick-End Labeling; Male; Microscopy, Fluorescence; Phosphatidylserines; Pyrimidinones; Sperm Motility; Spermatozoa; Ubiquitin

(1)H and (13)C NMR spectra of two series of malononitrile-based merocyanines, which possess positive and negative solvatochromism have been in detail investigated in low polar chloroform and polar dimethyl sulfoxide (DMSO). Careful attribution of signals in spectra has been made with the help of two-dimensional NMR experiments (COSY, NOESY, HMBC, and HMQC). Hence, the dependence of merocyanines electronic structure on their chemical structure and solvent nature has been studied by this powerful method. It has been shown that there exists a good correlation between the calculated charges on carbon atoms of a polymethine chain and their chemical shifts in (13)C NMR spectra. The influence of solvent polarity on bond orders for dyes with positive and negative solvatochromism is also observed. The comparison of (13)C NMR spectra of merocyanines and corresponding parent ionic dyes allows to determine their sign of solvatochromism irrespectively of electronic spectra, and also to find the key atoms of chromophore whose signals in (13)C NMR spectra are most informative.

Topics: Absorption; Carbon; Chemistry; Electrochemistry; Electronics; Electrons; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Models, Chemical; Pyrimidinones; Solvents; Spectrophotometry; Spectrophotometry, Ultraviolet

The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells. Non-apoptotic PS externalization is induced by several activation stimuli, including engagement of immunoreceptors. Immune cells can also be activated by aggregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs). However, it is unknown whether cell triggering through these proteins, lacking transmembrane and cytoplasmic domains, also leads to PS externalization. Here we show that engagement of GPI-APs in rodent mast cells induces a rapid and reversible externalization of PS by a non-apoptotic mechanism. PS externalization triggered by GPI-AP-specific monoclonal antibodies was dependent on the activity of H(+)-ATP synthase and several other enzymes involved in mast cell signaling but was independent of cell degranulation, free cytoplasmic calcium up-regulation, and a decrease in lipid packing as determined by merocyanine 540 binding. Surprisingly, disruption of actin cytoskeleton by latrunculin B or plasma membrane integrity by methyl-beta-cyclodextrin had opposite effects on PS externalization triggered through GPI-AP or the high affinity IgE receptor. We further show that PS externalization mediated by GPI-APs was also observed in some other cells, and its extent varied with antibodies used. Interestingly, effects of different antibodies on PS externalization were additive, indicating that independent stimuli converge onto a signaling pathways leading to PS externalization. Our findings identify the cell surface PS exposure induced through GPI-AP as a distinct mechanism of cell signaling. Such a mechanism could contribute to "inside-out" signaling in response to pathogens and other external activators and/or to initiation of other functions associated with PS externalization.

Topics: Animals; Apoptosis; beta-Cyclodextrins; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic; Cell Degranulation; Cell Line, Tumor; Fluorescent Dyes; Glycosylphosphatidylinositols; Humans; Male; Mast Cells; Mice; Mitochondrial Proton-Translocating ATPases; NIH 3T3 Cells; Phosphatidylserines; Pyrimidinones; Rats; Rats, Wistar; Receptors, IgE; Signal Transduction; Thiazolidines

Topics: Benzopyrans; Indoles; Light; Methacrylates; Micelles; Nitro Compounds; Photochemistry; Polyethylene Glycols; Polymers; Pyrimidinones; Ultraviolet Rays

The thermal and photochemical processes associated with the acid-induced conversions of 6-nitroBIPS, SP-1, to form the protonated merocyanine (MC-OH+) were investigated via UV/vis spectrophotometric studies in acetone. It was found that the mechanism of trifluoroacetic acid (TFA)-induced ring-opening of the SP and the rate of MC-OH+ formation follows a general acid catalysis mechanism. In accord with this mechanism, the thermal growth of the acid-induced ring-opened form (MC-OH+) was retarded as the concentration of TFA in the medium was increased. The N-protonated SP, i.e., SP-NH+, is formed in a competing side-equilibrium process as an unreactive "sink", with the nitrogen lone-pair no longer available to drive the ring-opening process and resulting in the inverse rate dependence as a linear 1/kobs vs [HA] plot. Addition of a tertiary amine to MC-OH+ regenerated MC which underwent thermal ring closure to the SP, thus restoring its function as a molecular switch. NMR titration of SP samples showed a downfield shift of the N-substituent peak upon increasing the TFA concentration. However, a saturation behavior could not be observed with SP-1 up to 1 M acid, unlike the model compound, N,N-dimethylaniline (N,N-DMA), which indicates a base strength order of N,N-DMA > SP-1. Further, we have demonstrated that in solvent acetone, on acidification, the normal photo- and thermochromic behavior is reversed; now MC-OH+ is photochemically transformed into SP-H+, which undergoes thermal ring-opening to MC-OH+.

Topics: Acids; Benzopyrans; Free Radicals; Hydroxylation; Indoles; Magnetic Resonance Spectroscopy; Molecular Structure; Nitro Compounds; Pyrimidinones

During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis was elevated at all enzyme concentrations, the total amount of reaction product was increased fourfold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by availability of substrate rather than adsorption of enzyme. Fluorescence experiments identified three membrane alterations during apoptosis that might affect substrate access to the sPLA(2) active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting an increase in lipid spacing. Second, laurdan detected increased solvation of the lower headgroup region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it is proposed that the membranes of apoptotic cells become susceptible to sPLA(2) through a reduction in lipid-neighbor interactions that facilitates migration of phospholipids into the enzyme active site.

Topics: Animals; Apoptosis; Binding Sites; Biophysics; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Flow Cytometry; Group II Phospholipases A2; Hydrolysis; Ionophores; Kinetics; Mice; Models, Chemical; Phospholipases A; Phospholipases A2; Pyrimidinones

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl-L-alpha phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.

Topics: Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Erythrocytes; Humans; Liposomes; Membrane Fluidity; Membrane Microdomains; Propofol; Pyrimidinones; Spectrum Analysis; Spin Labels

Merocyanine dyes have proven valuable for live cell fluorescence imaging applications, but many structures have been limited by rapid photobleaching. We show that photostability is substantially enhanced for merocyanines having a cyano group at a specific position in the central polymethine chain. Evidence is presented that this is due to reduction in reactivity of the dyes with singlet oxygen. These results point toward cyano-substitution as a general strategy for improving dye performance in imaging applications.

Topics: Fluorescence; Photochemistry; Pyrimidinones

Our group has recently identified, in human semen, round bodies of different size and density which were termed M540 bodies due to their staining with the fluorochrome merocyanine 540. Here, we investigate the hypothesis that such structures represent apoptotic bodies. To this aim, by both fluorescence-activated cell sorting (FACS) and fluorescence microscopy, we examined the occurrence of apoptotic markers such as caspase activity, Fas, p53 and Bcl-x in M540 bodies. In addition, we evaluated their ultrastructure by transmission electron microscopy. We found that M540 bodies express all the investigated markers, strongly supporting our hypothesis. We also found that M540 bodies contain fragmented DNA, another evidence of their apoptotic derivation. We investigated also the presence of M540 bodies in the different categories of patients. With respect to normozoospermic subjects, a higher content of M540 bodies was found in oligoasthenoteratozoospermic and asthenoteratozoospermic, but not in asthenozoospermic and teratozoospermic men. Interestingly, these subjects are those whose semen shows the highest levels of apoptotic signs. The variable occurrence of apoptotic bodies in semen may thus be considered a sign of abortive apoptosis in male reproductive organs. Of interest, since M540 bodies exhibit a similar size and density to sperm, they represent a confounding factor in FACS studies on ejaculated sperm.

Topics: Apoptosis; bcl-X Protein; Biomarkers; Caspases; DNA Fragmentation; Fas Ligand Protein; Flow Cytometry; Fluorescent Antibody Technique; Humans; In Situ Nick-End Labeling; Inclusion Bodies; Male; Microscopy, Electron, Transmission; Pyrimidinones; Semen; Spermatozoa; Tumor Suppressor Protein p53

Glycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0-30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.

Topics: Acrosome Reaction; Animals; Cattle; Chlortetracycline; Dermatan Sulfate; Egg Proteins; Flow Cytometry; Fluorescent Dyes; Heparin; Male; Membrane Glycoproteins; Pyrimidinones; Receptors, Cell Surface; Sperm Capacitation; Spermatozoa; Zona Pellucida Glycoproteins

Topics: Gels; Macromolecular Substances; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Molecular Structure; Particle Size; Pyrimidinones; Spectrophotometry, Ultraviolet

A wave packet approach to the calculation and interpretation of circular dichroism (CD) spectra is applied to the spectroscopy of aggregates of a merocyanine dye. A combined analysis of absorption and CD spectra allows for the extraction of geometric information and excited state electronic coupling. It is shown that in the case of dimer aggregates of a chiral merocyanine dye, it is possible to infer the dynamics of an exciton transfer directly from the CD spectrum. This relation is established via the Fourier relation to a time-dependent correlation function reflecting the quantum dynamics in the dye aggregate.

Topics: Circular Dichroism; Dimerization; Molecular Structure; Pyrimidinones; Quantum Theory; Spectrophotometry, Ultraviolet; Time Factors

The objective of this study was to investigate the response of Na(+)/K(+)-ATPase of human erythrocytes to green laser irradiation. Effects of green laser light of fluences 9.5-63.3 J.cm(-2) and merocyanine 540-mediated laser light treatment were studied. Isolated erythrocyte membranes (protein concentration of 1 mg/ml) were irradiated by Nd:YAG laser (532 nm, 30 mW) and then incubated in a medium with 2 mM ATP for 30 min. Activity of ATPase was determined colorimetrically by measuring the colored reaction product of liberated inorganic phosphate and malachite green at 640 nm. Contribution of Na(+)/K(+)-ATPase to overall phosphate production was determined using ouabain. A positive effect of green laser light on Na(+)/K(+)-ATPase activity was observed. The dependence of enzymatically liberated inorganic phosphate on light fluence showed a linear correlation (R(2)=0.96, P=0.0005) for all fluences applied (9.5-63.3 J.cm(-2)). On the other hand, MC 540-mediated phototreatment caused a suppression of enzyme activity.

Topics: Color; Erythrocytes; Fluorescent Dyes; Humans; Lasers; Low-Level Light Therapy; Pyrimidinones; Sodium-Potassium-Exchanging ATPase

In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p<0.05), and membrane integrity (p<0.01). Following the SU selection, motility, membrane integrity (p<0.001), and membrane instability increased (p<0.01), as did stability (p<0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p<0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p<0.01), average path velocity (VAP) (p<0.001), and percentage of spermatozoa with unstable plasma lemma (p<0.05), had a significant relationship with non-return rate (NRR). The results indicate that a triple combination of the fluorophores M540/Yo-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in a homogenous sire population.

Topics: Aging; Animals; Benzimidazoles; Benzoxazoles; Cattle; Cell Membrane; Cryopreservation; Fertility; Flow Cytometry; Fluorescent Dyes; Hot Temperature; Insemination, Artificial; Male; Pyrimidinones; Quinolinium Compounds; Semen Preservation; Sperm Head; Sperm Motility; Spermatozoa

This chapter details quantitative imaging of the Mero-CBD biosensor, which reports activation of endogenous Cdc42 in living cells. The procedures described are appropriate for imaging any biosensor that uses two different fluorophores on a single molecule, including FRET biosensors. Of particular interest is an algorithm to correct for fluorophore photobleaching, useful when quantitating activity changes over time. Specific topics include procedures and caveats in production of the Mero-CBD sensor, image acquisition, motion artifacts, shading correction, background subtraction, registration, and ratio imaging.

Topics: Biosensing Techniques; cdc42 GTP-Binding Protein; Cell Movement; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Green Fluorescent Proteins; Image Interpretation, Computer-Assisted; Peptide Fragments; Pyrimidinones; Software; Wiskott-Aldrich Syndrome Protein

Monoterpenes (MTs) are highly hydrophobic substances present in essential oils. They cover a wide spectrum of biological effects with a membrane interaction as a common point. Here we studied the surface activity of camphor, cineole, thymol, menthol and geraniol, and their ability to reach and incorporate into model membranes affecting some features of their dynamic organization. All the MTs studied self-aggregated in water with critical micellar concentrations (CMC) between 3 and 8 microM. Their octanol-water and membrane-water partition coefficients were correlated with one another. They all penetrated in monomolecular layers of dipalmitoyl-phosphatildylcholine at the air-water interface, even at surface pressures (pi) above the equilibrium lateral pressure of bilayers; thymol exhibited the highest (61.3 mN/m) and camphor the lowest (37 mN/m) pi(cut-off) value. They affected the self-aggregation of Triton X-100, increasing its CMC from 0.16 mM in the absence of MTs up to 0.68 mM (e.g. for geraniol), and the topology of sPC vesicles, increasing its surface curvature, suggesting their location at the polar head group region of the membrane. The latter was supported by their ability to increase differentially the polarity of the membrane environment sensed by two electrochromic dyes. Dipole moment values (between 1.224 and 2.523 D) and solvation areas (between 80 and 97 A(2)) were calculated from their energy-minimized structures. The relative contribution of each experimental, theoretical and structural property to determine MTs' effects on membrane dynamics were evaluated by a principal component analysis.

Topics: Air; Bromthymol Blue; Diffusion; Membranes, Artificial; Micelles; Molecular Structure; Octanols; Octoxynol; Particle Size; Permeability; Phosphatidylcholines; Principal Component Analysis; Pyrimidinones; Surface Properties; Terpenes; Water

Whether a pulsed laser is superior to a continuous wave (CW) light source in photodynamic therapy (PDT) of cancer is still unclear and contradictory in the literature. Although photosaturation of a sensitizer and oxygen depletion in tumor have been considered to be involved during pulsed laser irradiation, there is a lack of experimental data. In the present work several parameters such as the amount of merocyanine 540 (MC540) in cells, the oxygen concentration in cells, and the amount of photos reaching cells during pulsed laser irradiation, were studied to compare the MC540-mediated PDT effects of a pulsed laser and a CW light source on murine myeloid WEH-3B (JCS) cells in vitro. The results showed that the pulsed laser was less effective at cell inactivation than the CW light under the same irradiation dose. However, when the energy of the pulsed laser was reduced from 0.25 to 0.06 mJ/cm2 while keeping the total irradiation dose unchanged, the photoinactivation of cells was increased significantly. Based on the measurements and calculations for the present experimental conditions, each cell has about 108 MC540 molecules bound (5 microg/ml MC540 for 1 hr) and receives about 109 photos from 0.25 mJ/cm2 of the pulsed laser. The results indicate that the photosaturation of MC540 occurs in the present conditions due to the fact that the photons received by one cell in one laser pulse were much more than the numbers of MC540 molecules bound to one cell. Thus, the photosaturation of the photosensitizer is one of the reasons to explain the different efficiency in cell inactivation between the pulsed laser and CW light.

Topics: Animals; Cell Line, Tumor; Leukemia; Light; Mice; Photochemotherapy; Photosensitizing Agents; Pyrimidinones

Polymer nanoparticles of 40-400 nm diameter with spiropyran-merocyanine dyes incorporated into their hydrophobic cavities have been prepared; in contrast to their virtually nonfluorescent character in most environments, the merocyanine forms of the encapsulated dyes are highly fluorescent. Spiro-mero photoisomerization is reversible, allowing the fluorescence to be switched "on" and "off" by alternating UV and visible light. Immobilizing the dye inside hydrophobic pockets of nanoparticles also improves its photostability, rendering it more resistant than the same dyes in solution to fatigue effects arising from photochemical switching. The photophysical characteristics of the encapsulated fluorophores differ dramatically from those of the same species in solution, making nanoparticle-protected hydrophobic fluorophores attractive materials for potential applications such as optical data storage and switching and biological fluorescent labeling. To evaluate the potential for biological tagging, these optically addressable nanoparticles have been delivered into living cells and imaged with a liquid nitrogen-cooled CCD.

Topics: Acrylamides; Benzopyrans; Fluorescence; Humans; Hydrophobic and Hydrophilic Interactions; Indoles; Luminescence; Microscopy, Electron, Transmission; Models, Molecular; Nanostructures; Nitro Compounds; Photochemistry; Polystyrenes; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Compatible solutes are accumulated by diverse organisms in response to environmental stresses such as drought, salt, or cold. Glycinebetaine (Bet) is such a solute that is accumulated by many plants and microorganisms to high concentrations under stress conditions. It is an osmoprotectant in bacteria and stabilizes both soluble and peripherally membrane-bound proteins in vitro. Here, the effects of Bet on the stability of model lipid membranes are compared to the effects of two other compatible solutes, sucrose and trehalose. Both in the presence of 1M NaCl and during freezing to -20 degrees C, Bet is highly destabilizing to liposomes containing nonbilayer lipids, while the disaccharides are either protective or, in some cases, much less destabilizing. The destabilizing effect of Bet is more pronounced in membranes containing the nonbilayer galactolipid monogalactosyldiacylglycerol from plant chloroplasts than in membranes containing the nonbilayer phospholipid phosphatidylethanolamine. The most dramatic differences between the sugars and Bet were observed in liposomes made from a combination of lipids resembling plant chloroplast thylakoid membranes. Measurements with the dye merocyanine 540 indicate that the water-membrane interface was affected in opposite directions by the presence of high concentrations of sucrose or Bet. The dynamics of the lipids, however, were not differentially affected by the solutes, making direct solute-lipid interactions an unlikely explanation for the different effects on stability. The data offer an explanation, why Bet at high concentrations achieved during exogenous feeding of leaf tissues can be detrimental to cellular stability and survival under stress, while bacterial membranes that contain phosphatidylethanolamine instead of monogalactosyldiacylglycerol, or cyanobacteria that contain highly saturated monogalactosyldiacylglycerol are less susceptible.

Topics: Betaine; Fluorescence Polarization; Freezing; Galactolipids; Liposomes; Phosphatidylethanolamines; Pyrimidinones; Sucrose; Thylakoids; Trehalose

We isolated and characterized cell lines resistant to aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) derived from a murine adenocarcinoma and studied cross resistance with other injuries. The most resistant clones were numbers 4 and 8, which exhibited 6.7- and 4.2-fold increase in resistance respectively. Several characteristics were altered in these clones. A 2-fold increase in cell volume, higher cell spreading, and a more fibroblastic, dendritic pattern, were the morphology features that led us to think they could have different adhesive, invasive or metastatic phenotypes. The amount of porphyrins synthesized per cell in the resistant clones was similar to the parental line but, when it was expressed per mg protein, there was a 2-fold decrease, with a higher proportion of hydrophilic porphyrins. These cells were not cross-resistant to photosensitization with Benzoporphyrin derivative and Merocyanine 540, but exhibited a slight resistance to exogenous protoporphyrin IX treatment. Both clones displayed higher protein content and increased number of mitochondria, together with a higher oxygen consumption. The distinctive features found in the resistant lines led as to think how to exploit the changes induced by PDT treatment to target surviving cells. Those hypoxic cells can be also a preferential target of bioreductive drugs and hypoxia-directed gene therapy, and would be sensitive to treatment with other photosensitizers.

Topics: Aminolevulinic Acid; Animals; Cell Line, Tumor; Light; Mice; Oxygen Consumption; Photochemotherapy; Photosensitizing Agents; Porphyrins; Protoporphyrins; Pyrimidinones; Tetrazolium Salts; Thiazoles

The efficiency of photodynamic effect (PDE) for Photofrin II (PfII), Verteporfin, and Merocyanine 540 (MC540) was compared against neoplastic cells. Triplet state lifetimes and singlet molecular oxygen quantum yields were correlated with biological effect. PfII triplet lifetime was two times longer than that of Verteporfin, however, its singlet molecular oxygen quantum yield was two times lower in comparison with Verteporfin. High singlet molecular oxygen quantum yield of Verteporfin resulted in high biological efficacy. To achieve 50% mortality of cells four times lower light dose and five times lower concentration of Verteporfin were applied in comparison with PfII. The same level of cell damage was reached using 10 times higher light dose and two times higher concentration of MC540 in comparison with PfII. Our results confirm that singlet molecular oxygen based mechanism, prevalent for Verteporfin and PfII, was highly effective against melanoma cells. Verteporfin can be used at small doses with high cellular damage efficiency.

Topics: Animals; Cell Line, Tumor; Cell Survival; Dihematoporphyrin Ether; Dose-Response Relationship, Radiation; Kinetics; Melanoma; Mice; Oxygen; Photochemotherapy; Photosensitizing Agents; Porphyrins; Pyrimidinones; Signal Transduction; Verteporfin

The properties of liquid-ordered, solid-ordered, and liquid-disordered phases were investigated by steady-state fluorescence spectroscopy in liposomes composed of mixtures of dipalmitoylphosphatidylcholine and cholesterol (0-40 mol %) as a function of temperature (24-51 degrees C). The fluorescent probes used (bis-pyrene, nystatin, prodan, and merocyanine) were chosen because they differ in the location they occupy in the membrane and in the types of properties they sense. Comparison of phase diagrams with contour plots of the fluorescence data suggested that bis-pyrene is sensitive primarily to lipid order. In contrast, nystatin fluorescence intensity responded to changes in lipid fluidity. The shape of the prodan emission spectrum detected both liquid-solid and order-disorder transitions in the phase diagram. Merocyanine's behavior was more complex. First, it was more sensitive than any of the other probes to the membrane pretransition that occurs in the absence of cholesterol. Second, regardless of whether emission intensity, anisotropy, or spectral shape was observed, the probe appeared to distinguish two types of liquid-ordered phases, one with tightly packed lipids and one in which the apparent spacing among lipids was increased. The prodan data supported these results by displaying modest versions of these two observations. Together, the results identify eight regions within the phase diagram of distinguishable combinations of these physical properties. As an example of how this combined analysis can be applied to biological membranes, human erythrocytes were treated similarly. Temperature variation at constant cholesterol content revealed three of the eight combinations identified in our analysis of liposomes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Anisotropy; Biophysics; Cholesterol; Fluorescent Dyes; Lipids; Microscopy, Fluorescence; Nystatin; Phospholipids; Pyrenes; Pyrimidinones; Spectrometry, Fluorescence; Temperature

The free volume properties of phospholipid bilayers have been determined using a new assay that applies the photochromic and solvatochromic properties of merocyanines. The orientation and embedding depth of the merocyanines in the bilayer are controlled using substitution on the merocyanine indole moiety. The free volume changes at the aqueous interface (region 1), the phospholipid headgroup (region 2), and the aliphatic interior (region 3) of the bilayer are compared by analyzing the rate constants for the merocyanine ring-closing reaction. Free volume variations during the P(beta)(')(gel) <--> L(alpha)(liquid) phase transition are observed in region 1, in accordance with large structural rearrangements between the gel and the liquid phases in this region. The largest free volume is found in region 3, and the smallest is found in region 2. This distribution of free volume in the bilayer agrees with computational studies of these systems. Comparison of the free volume in region 2 of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids shows that this method is sensitive to small structural differences between lipids. In region 2, the free volume is found to be approximately 2 times larger in DPPC bilayers, which could be related to different merocyanine interactions with the two phosphatidylcholines. Free volume properties determined on picosecond and second time scales are compared based on an analysis of merocyanine formation and decoloration reactions.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Dimyristoylphosphatidylcholine; Fluorescent Dyes; Kinetics; Lipid Bilayers; Models, Biological; Models, Chemical; Phospholipids; Pyrimidinones; Thermodynamics

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.

Topics: Adsorption; Agkistrodon; Animals; Apoptosis; Barium; Binding Sites; Biophysical Phenomena; Biophysics; Calcium; Cell Membrane; Crotalid Venoms; Diffusion; Dose-Response Relationship, Drug; Erythrocyte Membrane; Erythrocytes; Group II Phospholipases A2; Humans; Hydrolysis; Ionomycin; Ionophores; Kinetics; Lipid Bilayers; Lipids; Membrane Lipids; Microscopy, Fluorescence, Multiphoton; Models, Chemical; Phosphatidylcholines; Phospholipases A; Phospholipases A2; Phospholipids; Protein Binding; Pyrimidinones; Temperature

The lipophilic dye merocyanine 540 (MC540) localizes primarily in the plasma membrane (PM) of tumor cells, where it can sensitize lethal photoperoxidative damage of potential therapeutic importance. We postulated (i) that chain peroxidation triggered by iron-catalyzed turnover of nascent hydroperoxides (LOOHs) generated by singlet oxygen ((1)O(2)) attack on PM lipids contributes significantly to overall cytolethality, and (ii) that nitric oxide (NO), a known scavenger of organic free radicals, would suppress this and, thus, act cytoprotectively. In accordance, irradiation of MC540-sensitized L1210 cells produced 5alpha-OOH, a definitive (1)O(2) adduct of PM cholesterol, which decayed during subsequent dark incubation with appearance of other signature peroxides, viz. free-radical-derived 7alpha/beta-OOH. Whereas chemical donor (SPNO or SNAP)-derived NO had little or no effect on post-irradiation 5alpha-OOH disappearance, it dose-dependently inhibited 7alpha/beta-OOH accumulation, consistent with interception of chain-carrying radicals arising from one-electron reduction of primary LOOHs. Using [(14)C]cholesterol as an L1210 PM probe, we detected additional after-light products of chain peroxidation, including diols (7alpha-OH, 7beta-OH) and 5,6-epoxides, the yields of which were enhanced by iron supplementation, but strongly suppressed by NO. Correspondingly, photoinitiated cell killing was significantly inhibited by NO introduced either immediately before or after light exposure. These findings indicate that prooxidant LOOH turnover plays an important role in photokilling and that NO, by intercepting propagating radicals, can significantly enhance cellular resistance.

Topics: Animals; Cell Line, Tumor; Cell Survival; Iron; Leukemia; Lipid Peroxidation; Membrane Lipids; Mice; Nitric Oxide; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Pyrimidinones

[reaction: see text] We describe the design, synthesis, and characterization of a family of thiol-reactive optical switches for labeling proteins and other biomolecules. Site-selective introduction of photochromic probes within biomolecules is being used as part of a new approach for optical control of biomolecular interactions and activities within cells. The thiol-reactive photochromic probes described in this report include a spironaphthoxazine and five spirobenzopyrans. The location of the thiol-reactive group on the spirobenzopyran is different for each probe; this feature can be used to control the geometry of the optical switch within a bioconjugate. The photochromes undergo rapid and reversible, optically driven transitions between a colorless spiro (SP) state and a brightly colored merocyanine (MC) state. The MC absorption of a spironaphthoxazine conjugate is red shifted by more than 100 nm compared to the equivalent spirobenzopyran, which may be exploited for the independent control of the MC to SP transition for up to two different spironaphthoxazine and spirobenzopyran conjugates within the same sample.

Topics: Binding Sites; Molecular Conformation; Molecular Probes; Photochemistry; Pyrimidinones; Spiro Compounds

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter (S) and correlation time (tau(c)) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl-l-alpha phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 A were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 degrees C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl-l-alpha phosphatidylcholine liposomes above the phase-transition temperature (23.9 degrees C) did not change microviscosity. At 10 degrees C, an increase of propofol concentration from 0 to 1.0 x 10(-2) M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10(-2) M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 A) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10(-2) M was identified as the saturation limit of propofol in dimyristoyl-l-alpha phosphatidylcholine liposomes.

Topics: Biophysical Phenomena; Biophysics; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Liposomes; Membrane Fluidity; Nitrogen Oxides; Propofol; Pyrimidinones; Spin Labels; Surface Properties; Temperature; Time Factors; Viscosity

In this contribution we report studies of the nature of solvation and resonance energy transfer processes in a reverse micelle (RM) upon encapsulation of a digestive enzyme, alpha-chymotrypsin (CHT). We have used one donor, Coumarin 500 (C500), and three acceptors Rhodamine 123 (R123, cationic), ethidium bromide (EtBr, cationic), and Merocyanine 540 (MC540, anionic). By selectively exciting the donor at the surface of the RM with a proper excitation wavelength we have examined solvation dynamics in the microenvironment. The solvation correlation function in the RM without CHT exhibits single-exponential decay with time constant approximately 660 ps, which is similar to that of the CHT-included RM. However, in the case of CHT-included RM (w(0)=10), the time-resolved anisotropy and spectral linewidth analysis of the surface-bound donor reveal the existence of an annular aqueous channel of thickness approximately 2.5 A between the enzyme surface and the inner surface of the RM. The aqueous channel is a potential host for the water-soluble substrate and also is involved in maintaining the proper functionality of RM encapsulated CHT. The studies use both steady-state and time-resolved fluorescence resonance energy transfer (FRET) techniques to measure donor-acceptor distances in the RM and also emphasize the danger of using steady-state fluorescence quenching as a method in careful estimation of the distances. The local geometrical restriction on the donor and acceptor molecules was estimated from time-resolved polarization (anisotropy) measurements. The time-resolved anisotropy of the donor and acceptor molecules also revealed significant randomization of the relative orientation of transition dipoles of the donor and acceptor, justifying the use of 2/3 as the value of the orientation factor kappa2. These studies attempt to elucidate the excellence of the RM as a nanohost of biological macromolecules.

Topics: Adsorption; Chymotrypsin; Coumarins; Dioctyl Sulfosuccinic Acid; Enzyme Activation; Ethidium; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Micelles; Molecular Structure; Nanostructures; Particle Size; Pyrimidinones; Rhodamine 123; Solubility; Structure-Activity Relationship; Surface Properties; Time Factors

Topics: Circular Dichroism; Kinetics; Microscopy, Atomic Force; Molecular Structure; Nanostructures; Pyrimidinones; Spectrophotometry, Ultraviolet; Stereoisomerism; Thermodynamics

In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.

Topics: Animals; beta-Cyclodextrins; Cell Membrane; Cells, Cultured; Cholera Toxin; Cholesterol; Dogs; Fertilization in Vitro; Flow Cytometry; Fluorescence; Male; Phospholipids; Pyrimidinones; Sperm Capacitation; Sus scrofa

Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V.. Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively.. Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.

Topics: Acrosome; Acrosome Reaction; Amino Acid Chloromethyl Ketones; Apoptosis; Biomarkers; Calcimycin; Calcium; Caspase Inhibitors; Cell Membrane; DNA Fragmentation; Enzyme Inhibitors; Flow Cytometry; Fluorescein-5-isothiocyanate; Humans; In Situ Nick-End Labeling; Ionophores; Male; Membrane Cofactor Protein; Microscopy, Fluorescence; Phosphatidylserines; Propidium; Pyrimidinones; Sperm Capacitation; Spermatozoa

The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more "fluid" plasma membranes than the sensitive cells. Because of this observed difference in membrane "fluidity," we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance.

Topics: Carcinoma; Cell Division; Cell Line, Tumor; Cell Membrane; Cisplatin; Clone Cells; Cyclic N-Oxides; Drug Resistance, Neoplasm; Fatty Acids; HeLa Cells; Humans; Isoxazoles; Membrane Fluidity; Membrane Lipids; Membrane Potentials; Membrane Proteins; Neoplasms; Potassium Channels; Pyrimidinones

The ET polarity values of 4-[(1-methyl-4(1H)-pyridinylidene)-ethylidene]-2,5-cyclohexadien-1-one (Brooker's merocyanine) were collected in mixed-solvent systems comprising a formamide [N,N-dimethylformamide (DMF), N-methylformamide (NMF) or formamide (FA)] and a hydroxylic (water, methanol, ethanol, propan-2-ol or butan-1-ol) solvent. Binary mixtures involving DMF and the other formamides (NMF and FA) as well as NMF and FA were also studied. These data were employed in the investigation of the preferential solvation (PS) of the probe. Each solvent system was analyzed in terms of both solute-solvent and solvent-solvent interactions. These latter interactions were responsible for the synergism observed in many binary mixtures. This synergistic behaviour was observed for DMF-propan-2-ol, DMF-butan-1-ol, FA-methanol, FA-ethanol and for the mixtures of the alcohols with NMF. All data were successfully fitted to a model based on solvent-exchange equilibria, which allowed the separation of the different contributions of the solvent species in the solvation shell of the dye. The results suggest that both hydrogen bonding and solvophobic interactions contribute to the formation of the solvent complexes responsible for the observed synergistic effects in the PS of the dye.

Topics: 1-Butanol; 1-Propanol; Amides; Dimethylformamide; Formamides; Hydrogen Bonding; Hydrolysis; Methanol; Models, Chemical; Photosensitizing Agents; Pyrimidinones; Solvents; Spectrophotometry; Temperature; Water

The antioxidant action of carotenoids is believed to involve quenching of singlet oxygen and scavenging of reactive oxygen radicals. However, the exact mechanism by which carotenoids protect cells against oxidative damage, particularly in the presence of other antioxidants, remains to be elucidated. This study was carried out to examine the ability of exogenous zeaxanthin alone and in combination with vitamin E or C, to protect cultured human retinal pigment epithelium cells against oxidative stress. The survival of ARPE-19 cells, subjected to merocyanine 540-mediated photodynamic action, was determined by the MTT test and the content of lipid hydroperoxides in photosensitized cells was analyzed by HPLC with electrochemical detection. We found that zeaxanthin-supplemented cells, in the presence of either alpha-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. Cells with added antioxidants exhibited increased viability and accumulated less lipid hydroperoxides than cells without the antioxidant supplementation. Such a synergistic action of zeaxanthin and vitamin E or C indicates the importance of the antioxidant interaction in efficient protection of cell membranes against oxidative damage induced by photosensitized reactions.

Topics: alpha-Tocopherol; Antioxidants; Ascorbic Acid; beta Carotene; Biological Assay; Cell Line; Cell Survival; Humans; Iron; Lipid Peroxidation; Oxidative Stress; Photochemistry; Photosensitizing Agents; Pigment Epithelium of Eye; Pyrimidinones; Singlet Oxygen; Tetrazolium Salts; Thiazoles; Xanthophylls; Zeaxanthins

The 13C NMR of two solvatochromic dyes derived from a barbituric acid acceptor and dimethylaminophenyl donor fragments, compound 1 and the related merocyanine 2, were recorded in various solvents. The observed chemical-shift variations were used to interpret their structural differences and solvatochromic behavior in solution.

Topics: Barbiturates; Coloring Agents; Electrochemistry; Magnetic Resonance Spectroscopy; Molecular Structure; Pyrimidinones; Solutions

Two highly dipolar merocyanine dyes were tethered by a rigid tris(n-dodecyloxy)xylylene unit that preorganizes the dyes for a supramolecular polymerization process through intermolecular aggregation of the dyes. UV/vis spectroscopy revealed a solvent dependent equilibrium between monomeric dyes and two different types of dye aggregates that are characterized by hypsochromically shifted D- and H-type absorption bands. Taking into account the ditopic nature of the supramolecular building blocks, the occurrence of the D-band indicates the formation of an oligomeric/polymeric supramolecular chain whereas the observation of the H-band suggests a higher order assembly. For the H-aggregated dyes, intrinsic viscosities exceed 0.65 L g(-1) in methylcyclohexane, values typically found for macromolecular solutions. At higher concentration, further association of these aggregates takes place by entanglement of the alkyl groups leading to a substantial increase in viscosity and gelation. Rheology studies show linear viscoelastic behavior which was attributed to the formation of an entangled dynamic network. AFM and cryo-TEM studies of the gel reveal long and stiff rod-type assemblies. X-ray diffraction studies for a solid film show columnar mesomorphism. Based on these results, a structural model is proposed in which six helically preorganized strands of the supramolecular polymer intertwine to form a rod with a diameter of about 5 nm. Within these rods all dyes are tightly aggregated in a tubular fashion giving rise to delocalized excitonic states, and the pi-conjugated tube is jacketed by the tridodecyloxy groups.

Topics: Coloring Agents; Gels; Hydrogen Bonding; Kinetics; Macromolecular Substances; Models, Chemical; Molecular Structure; Pyrimidinones; Spectrophotometry, Ultraviolet; Viscosity

In the present work, we studied the ability of thymol to affect the organization of model membranes and the activity of an intrinsic membrane protein, the GABA(A) receptor (GABA(A)-R). In this last aspect, we tried to elucidate if the action mechanism of this terpene at the molecular level, involves its binding to the receptor protein, changes in the organization of the receptor molecular environment, or both. The self-aggregation of thymol in water with a critical micellar concentration approximately = 4 microM and its ability to penetrate in monomolecular layers of soybean phosphatidylcholine (sPC) at the air-water interface, even at surface pressures above the equilibrium, lateral pressure of natural bilayers were demonstrated. Thymol affected the self-aggregation of Triton X-100 and the topology of sPC vesicles. It also increased the polarity of the membrane environment sensed by the electrochromic dye merocyanine. A dipolar moment of 1.341 Debye was calculated from its energy-minimized structure. Its effect on the binding of [3H]-flunitrazepam ([3H]-FNZ) to chick brain synaptosomal membranes changed qualitatively from a tendency to the inhibition to a clear activatory regime, up on changing the phase state of the terpene (from a monomeric to a self-aggregated state). Above its CMC, thymol increased the affinity of the binding of [3H]-FNZ (K(d-control)= 2.9, K(d-thymol)= 1.7 nM) without changing the receptor density (B(max-control)= 910, B(max-thymol)= 895 fmol/mg protein). The activatory effect of thymol on the binding of [ [3H]-FNZ was observed even in the presence of the allosteric activator gamma-aminobutyric acid (GABA) at a concentration of maximal activity, and was blocked by the GABA antagonist bicuculline. Changes in the dipolar arrangement and in the molecular packing of GABA(A)-R environment are discussed as possible mediators of the action mechanism of thymol.

Topics: Air; Animals; Bicuculline; Cell Membrane; Chickens; Flunitrazepam; GABA Antagonists; Glycine max; Kinetics; Lipid Bilayers; Micelles; Octoxynol; Phosphatidylcholines; Protein Binding; Pyrimidinones; Spectrophotometry; Surface-Active Agents; Thymol; Time Factors; Water

Signaling proteins are tightly regulated spatially and temporally to perform multiple functions. For Cdc42 and other guanosine triphosphatases, the subcellular location of activation is a critical determinant of cell behavior. However, current approaches are limited in their ability to examine the dynamics of Cdc42 activity in living cells. We report the development of a biosensor capable of visualizing the changing activation of endogenous, unlabeled Cdc42 in living cells. With the use of a dye that reports protein interactions, the biosensor revealed localized activation in the trans-Golgi apparatus, microtubule-dependent Cdc42 activation at the cell periphery, and activation kinetics precisely coordinated with cell extension and retraction.

Topics: Actins; Algorithms; Animals; Biosensing Techniques; cdc42 GTP-Binding Protein; Cell Adhesion; Cell Line; Cell Membrane; Cell Polarity; Cell Surface Extensions; Endothelial Cells; Fibroblasts; Fluorescence; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Luminescent Proteins; Mice; Microtubules; Neutrophil Activation; Neutrophils; Proteins; Pseudopodia; Pyrimidinones; rho GTP-Binding Proteins; Sensitivity and Specificity; trans-Golgi Network; Wiskott-Aldrich Syndrome Protein

Previous studies of our group demonstrated that flunitrazepam is a lipophilic drug capable of interacting with membranes through a partition equilibrium phenomenon. Its localization at the phospholipid polar head region could explain the decrease in the size of dipalmitoylphosphatidylcholine (dpPC) vesicles, through a mechanism that involves the increment in the relative volume of this region with a subsequent increase in the vesicle's surface curvature. In the present work, we investigated if flunitrazepam can affect the L(alpha)-H(II) phase transition of phosphatidylethanolamine through a similar mechanism. This study was approached by using merocyanine 540, a dye sensitive to the molecular packing of membrane lipids. A detailed analysis of merocyanine absorption and fluorescence emission and excitation spectra was performed. The results indicated that the fluorescence emitted came mainly from the monomeric form of merocyanine and that it resulted a good indicator of this phase transition, as was previously described. Flunitrazepam did not affect significantly the onset of the phase transition but showed a tendency to diminish the dye fluorescence emission intensity, which could involve a lower partition of merocyanine in the vesicles. Moreover, the results suggest that this drug produced a delay in the completeness of the phase transition and a decrement in the cooperativity of this phenomenon.

Topics: Dimerization; Flunitrazepam; Fluorescent Dyes; Indicators and Reagents; Membrane Lipids; Phase Transition; Phosphatidylethanolamines; Pyrimidinones; Surface Properties; Temperature

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Topics: Animals; Antioxidants; Benzoxazoles; Chromans; Cryopreservation; Ejaculation; Fluorescent Dyes; In Vitro Techniques; Male; Membrane Fluidity; Membrane Lipids; Pyrimidinones; Quinolinium Compounds; Semen Preservation; Spermatozoa; Swine

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 mug of MC 540 per ml and a light dose of 600 J/cm2 or to 20 mug of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.

Topics: Anti-Bacterial Agents; Biofilms; Light; Microbial Sensitivity Tests; Plankton; Pyrimidinones; Radiation-Sensitizing Agents; Staphylococcus aureus

We report the preparation of aqueous liposome dispersions of J-aggregates formed by the amphiphilic merocyanine dye (MD). A series of liposome-forming lipids were dispersed together with MD J-aggregates at different molar ratios of MD to lipid. The MD J-aggregate dispersions prepared with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at the MD to DMPC ratio of 0.16 exhibit good dispersibility; that is, they can be readily redispersed without any flocculation even after their precipitation. By use of different counterions for the MD molecules, two types of J-aggregate dispersions, one that exhibits an absorption band (J-band) at 635 nm (type I) and the other at 600 nm (type II), were obtained. As an example of the use of MD J-aggregates liposome dispersions, the thermochromic transformation of MD J-aggregates was demonstrated. When the dispersions are heated, J-aggregates of type I transformed into type II at a certain temperature (T(disp)). The parameters that control the speed of the transformation and the value of T(disp) were determined.

Topics: Cations, Divalent; Dimyristoylphosphatidylcholine; Lipids; Liposomes; Models, Molecular; Molecular Structure; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry; Surface Properties; Water

We have identified a strategy to communicate a chemical signal between two independent molecular components. One of them is a photoactive merocyanine that switches to a spiropyran, releasing a proton, when stimulated with visible light. The other is a 4,4'-pyridylpyridinium monocation that captures the released proton, producing an electroactive 4,4'-bipyridinium dication. Under the irradiation conditions employed, the photoinduced transformation requires ca. 15 min to reach a photostationary state. In the dark, the ensemble of communicating molecules reequilibrates to the original state in ca. 5 days. These processes can be monitored following the photoinduced enhancement and thermal decay, respectively, of the current for the monolectronic reduction of the 4,4'-bipyridinium dication. The pronounced difference in time scale for the current enhancement and decay steps can be exploited to implement a memory element with a bit retention time of 11 h. A bit of information can be written optically in the chemical system and it can be read electrically and nondestructively. The memory can be reset, extending its permanence in the dark beyond the bit retention time. A binary logic analysis of the signal transduction operated by the communicating molecules reveals the characteristic behavior of sequential logic operators, which are the basic components of digital memories.

Topics: Benzopyrans; Biomimetic Materials; Indoles; Nitro Compounds; Photochemistry; Protons; Pyridinium Compounds; Pyrimidinones

Paediatric solid tumours exhibit steep dose-response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms' tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Dose-Response Relationship, Drug; Hematopoietic Stem Cell Transplantation; Humans; Mice; Neuroblastoma; Osteosarcoma; Phospholipid Ethers; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Rhabdomyosarcoma; Tumor Cells, Cultured; Wilms Tumor

Covalent attachment of solvent-sensitive fluorescent dyes to proteins is a powerful tool for studying protein conformational changes, ligand binding, or posttranslational modifications. We report here new merocyanine dyes that make possible the quantitation of such protein activities in individual living cells. The quantum yield of the new dyes is sharply dependent on solvent polarity or viscosity, enabling them to report changes in their protein environment. This is combined with other stringent requirements needed in a live cell imaging dye, including appropriate photophysical properties (excitation >590 nm, high fluorescence quantum yield, high extinction coefficient), good photostability, minimal aggregation in water, and excellent water solubility. The dyes were derivatized with iodoacetamide and succinimidyl ester side chains for site-selective covalent attachment to proteins. A novel biosensor of Cdc42 activation made with one of the new dyes showed a 3-fold increase in fluorescence intensity in response to GTP-binding by Cdc42. The dyes reported here should be useful in the preparation of live cell biosensors for a diverse range of protein activities.

Topics: Biosensing Techniques; cdc42 GTP-Binding Protein; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Photochemistry; Protein Conformation; Pyrimidinones; Solubility; Spectrometry, Fluorescence; Water

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.

Topics: Adenocarcinoma; Blood Proteins; Cell Membrane Structures; Colonic Neoplasms; Culture Media; Humans; Hydrogen-Ion Concentration; Ions; Lipid Bilayers; Liposomes; Photosensitizing Agents; Pyrimidinones; Spectrometry, Fluorescence; Tumor Cells, Cultured

Fructans are a group of fructose-based oligo- and polysaccharides. They are proposed to be involved in membrane protection of plants during dehydration. In accordance with this hypothesis, they show an interaction with hydrated lipid model systems. However, the structural requirements for this interaction are not known both with respect to the fructans as to the lipids. To get insight into this matter, the interaction of several inulins and levan with lipids was investigated using a monomolecular lipid system or the MC 540 probe in a bilayer system. MD was used to get conformational information concerning the polysaccharides. It was found that levan-type fructan interacted comparably with model membranes composed of glyco- or phospholipids but showed a preference for lipids with a small headgroup. Furthermore, it was found that there was an inulin chain-length-dependent interaction with lipids. The results also suggested that inulin-type fructan had a more profound interaction with the membrane than levan-type fructan. MD simulations indicated that the favorable conformation for levan is a helix, whereas inulin tends to form random coil structures. This suggests that flexibility is an important determinant for the fructan-lipid interaction.

Topics: Computer Simulation; Crystallography; Fructans; Glycolipids; Lipid Bilayers; Liposomes; Macromolecular Substances; Membrane Fluidity; Models, Molecular; Molecular Conformation; Phospholipids; Pyrimidinones; Reproducibility of Results; Sensitivity and Specificity

The relationship between tumor cell differentiation and photosensitizer accumulation used in PDT is poorly defined. In the present work, specific cell differentiation of colon carcinoma CT26 cells induced by sodium butyrate was manifested by morphological changes, proliferation and protein expression and was correlated with the accumulation of endogenous and exogenous photosensitizes. Reduced accumulation of the endogenous protoporphyrin IX and the exogenous hypericin and MC540 was detected in differentiated cells. In contrast, a differentiation-dependent increase was measured with TPPS4, TMPyP, the pheophorbides (C5, C6, C12), HypS4 and helianthrone. In conclusion, PpIX, Hypericin and MC540 show specific binding and accumulation in poorly differentiated tumors, giving these tumors tissue-specific advantage in photo-diagnostic PDT applications.

Topics: Animals; Anthracenes; Butyrates; Carcinoma; Cell Cycle; Cell Differentiation; Colonic Neoplasms; Mice; Perylene; Photosensitizing Agents; Protoporphyrins; Pyrimidinones; Tumor Cells, Cultured

A pulsed (17 nanoseconds) Nd:YAG laser (1064 nm) was used to inject impermeable dyes (propidium iodide andiodide and merocyanine 540) and a plasmid (pEGFP-N1) encoding green fluorescent protein (GFP) into human breast adenocarcinoma cells (MCF-7). The cell membrane integrity and viability were fully preserved in this laser-assisted transfer.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Membrane Permeability; Cell Survival; Drug Delivery Systems; Green Fluorescent Proteins; Humans; Lasers; Luminescent Proteins; Microinjections; Plasmids; Propidium; Pyrimidinones; Transfection; Transformation, Genetic; Tumor Cells, Cultured

Membranotropic amphiphilic chromophore merocyanine 540 sensitized photodynamic inhibition of drug-resistant and sensitive variants of type I herpes simplex virus in cultured Vero cell. Optimal conditions of photodamage to virus particles and infected cells were determined (merocyanine 540 concentration 1 microM, illumination dose 32.5-65.0 kJ/m(2), exposure at early stages of infection). Infected cells actively bind the photosensitizer, which explains their selective photodamage.

Topics: Animals; Antiviral Agents; Chlorocebus aethiops; Drug Resistance, Viral; Herpes Simplex; Herpesvirus 1, Human; Humans; Photosensitizing Agents; Pyrimidinones; Vero Cells; Virus Replication

The purpose of this study was to determine in a preclinical purging model, how effective crystal violet-mediated photodynamic therapy (CV-PDT) is against solid tumor and drug-resistant mutant tumor cells, and if certain limitations of CV-PDT can be overcome by using crystal violet (CV) in combination with the membrane-active photosensitizer, Merocyanine 540 (MC540). When used under conditions that preserved an adequate fraction of normal human granulocyte/macrophage progenitors (CFU-GM), CV-PDT failed to achieve meaningful reductions of DU145 prostate, H69 small cell lung cancer, and MDA-MB-435S breast cancer cells. Melphalan-resistant L1210/L-PAM1, adriamycin-resistant P388/ADR, and adriamycin-resistant HL-60/ADR leukemia cells were markedly less sensitive to CV-PDT than their wild-type counterparts, whereas cisplatin-resistant H69/CDDP cells were more sensitive than wild-type H69 cells. Sequential exposure to MC540- and CV-PDT under conditions that preserved an adequate fraction (73% and 29%, respectively) of normal CD34-positive hematopoietic stem cells and granulocyte/macrophage progenitors was highly effective against H69 (99.997% reduction) and H69/CDDP (99.999% reduction) cells, but ineffective against HL-60/ADR, MDA-MB-435S, and DU145 cells. CV thus shows only limited promise as a single-modality purging agent. However, in certain situations, clinically meaningful tumor cell depletions can be obtained by using CV in combination with a second photosensitizer such as MC540.

Topics: Animals; Bone Marrow Purging; Caspases; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Gentian Violet; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Mice; Molecular Structure; Necrosis; Neoplasms; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Receptors, Peptide

The ability of several natural terpenes to affect benzodiazepine (BZD)-micelle interaction through the membrane dipolar organization was investigated. The acid-base equilibrium of chlorodiazepoxide (CDX) and the spectroscopic behavior of the electrochromic dye merocyanine were tested in the presence and in the absence of Triton X-100 micelles (used to mimic a membrane environment) containing or not cineole, menthol, geraniol or camphor. CDX's apparent pK increased in the environment of terpene-containing micelles compared with pure Triton X-100 micelles. Decrements in electric potentials (between -111 and -128 mV with respect to pure detergent) were calculated from Boltzmann equation. This result suggested, that in the presence of terpenes, the tendency of CDXH(+) to remain in the membrane phase increased. The dielectric constant (D) of the microenvironment sensed by merocyanine within Triton X-100 micelles, determined from lambda(max,2) of merocyanine monomer, was D=9 and increased in the presence of all the terpenes assayed (D congruent with 11). The decrease in merocyanine partitioning (A(peak1)/A(peak2) increased) also reflected an increment in the negative dipole potential. The present results suggest that terpenes contributed to the whole dipolar arrangement of the micelle with a dipole moment vector which had an intense component oriented parallel to the intrinsic dipole of the Triton X-100 molecules in the micelles. This led to a more negative environment of the interface region where CDX was located, and increased the net polarity of the deepest micelle regions sensed by merocyanine.

Topics: Acid-Base Equilibrium; Chlordiazepoxide; Micelles; Monoterpenes; Octoxynol; Pyrimidinones; Static Electricity

The study revealed that beta-adrenoceptor blockade with propranolol (0.40 mg/100 g/day, s.c.) in adult male DA rats: (i) increased the thymocyte proliferation and apoptosis, (ii) caused disturbances in kinetics of T cell differentiation leading to distinguishable changes in relative proportion of thymocytes at distinct maturational steps and to an expansion of the most mature single positive (CD4+, CD8+) thymocyte pool, (iii) affected the relative proportion of neither CD4+ nor CD8+ peripheral blood lymphocytes (PBL), and (iv) augmented the relative number of CD8+CD25+ cells. Thus, the results suggest the role of beta-adrenoceptors in fine-tuning of T cell maturation, and, possibly, distribution and activation of distinct PBL subsets.

Topics: Adrenergic beta-Antagonists; Animals; Apoptosis; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Count; Cell Differentiation; Cells, Cultured; Flow Cytometry; Lymphocyte Activation; Male; Organ Size; Phenotype; Propidium; Propranolol; Pyrimidinones; Rats; Rats, Inbred Strains; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin-2; T-Lymphocyte Subsets; T-Lymphocytes; Thymus Gland

The condensation of the CH acidic heterocycles 4-alkyl-2,6-dioxo-1,2,5,6-tetrahydropyridine-3-carbonitrile (5a and b) and barbituric acid (15) with electron-rich thiophene aldehydes and benzaldehyde derivatives affords the respective monomethine dyes 10-13 and 17-19. The formylation of 5a,b and 15 with N,N'-diphenylformamidine or dibutylformamide in acetic anhydride and further reaction with 4-picolinium salts 9a,b provide the dimethine dyes 14 and 20a,b. Triple hydrogen bonding of the imide groups of merocyanine dyes 10-14 has been investigated by NMR titration experiments with melamine 21. Despite rather pronounced variations of the charge-transfer properties within the given series of dyes, minor changes of their binding constants have been observed. These results could be rationalized by semiempirical calculations that reveal small changes in the charge density at the oxygen functionalities involved in hydrogen bonding upon variation of the electron-donating carbocyclic or heterocyclic groups at the terminal double bond. Although the binding constants for triple hydrogen bonding between imides and melamines are rather weak in chloroform, they proved to be strong enough to facilitate dissolution of some of these dyes in aliphatic solvents by coordination to amphiphilic melamines and dipolar aggregation. UV-vis spectral changes observed in methylcyclohexane vs chloroform suggest the formation of colloidal assemblies through noncovalent polymerization.

Topics: Hydrogen Bonding; Magnetic Resonance Spectroscopy; Pyrimidinones; Triazines

The special physical state of the sphingolipid-enriched membranes with characteristic lipid composition, presently one of the most controversial foci in cell biology, provides the essential environment for the proteins inside to be involved in the related physiological processes. The role of gangliosides, an important component of the membranes, deserves attention. The present investigation using several biophysical techniques indicates that ganglioside GM(1) induces the phase separation in the sphingomyelin membrane with 5 mol% cholesterol and regulates the membrane structure. The results of differential scanning calorimetry show that a higher T(m), GM(1)-rich phase emerges behind the lower T(m), sphingomyelin-rich phase with the incorporation of GM(1) into the sphingomyelin/cholesterol bilayers; and the GM(1)-rich phase dominates the membrane when the proportion of GM(1) reaches about 20 mol%. Fluorescence quenching further shows that the separation of the two domains is independent of temperature, occurring both in the gel phase and in the liquid phase. Laser Raman spectroscopy and fluorescence polarization suggest that the order of hydrocarbon chains increases and membrane fluidity decreases with increase in GM(1) content. Use of the fluorescence probe merocyanine-540 and electron microscopy reveals that the insertion of GM(1) leads to an increase in the spatial density of the lipid headgroups and a decrease in the curvature of the sphingomyelin/cholesterol bilayers. In sums, both the hydrophilic sugar heads and the hydrophobic hydrocarbon chains of GM(1) contribute to the regulation of membrane architecture. We suggest that the convex curvature of ganglioside-enriched membrane could be involved in forming and maintaining the characteristic flask-shaped invagination of caveolae.

Topics: Calorimetry, Differential Scanning; Ceramides; Cholesterol; G(M1) Ganglioside; Gangliosides; Lipid Bilayers; Lipids; Liposomes; Membrane Microdomains; Microscopy, Electron; Microscopy, Fluorescence; Pyrimidinones; Spectrum Analysis, Raman; Sphingomyelins; Temperature

Topics: 1,2-Dipalmitoylphosphatidylcholine; Fluorescent Dyes; Indicators and Reagents; Liposomes; Pyrimidinones; Spectrometry, Fluorescence; Surface Properties; Thermodynamics

Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V).. Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced.. Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents.. (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.

Topics: Adult; Annexin A5; Biomarkers; Blood Coagulation Factors; Blood Platelets; Cell Membrane; Collagen; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Flow Cytometry; Fluorescent Dyes; Humans; Liposomes; Phosphatidylserines; Platelet Activation; Proteins; Pyrimidinones; Receptors, Thrombin; Solubility; Vanadates

High-dose chemotherapy combined with autologous stem cell support has improved response rates in high-risk and metastatic breast cancer, but has failed to improve long-term survival. Breast cancer has a tendency to metastasize to the bone marrow, and live tumor cells are known to circulate in the peripheral blood of breast cancer patients. Sensitive immunohistochemical, culture-based, and reverse transcriptase polymerase chain reaction (RT-PCR)-based methods have shown that about 50% of histologically normal stem cell grafts from breast cancer patients are contaminated with occult tumor cells, which may cause or contribute to tumor recurrences. Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Unfortunately, most solid tumor cells (including breast cancer cells) are only moderately sensitive or refractory to MC540-PDT. We report here that if MC540-PDT is followed by a 1-h incubation with the alkyl-lysophospholipid, Edelfosine (ET-18-OCH(3)), the depletion of murine and human breast cancer cells is greatly enhanced whereas the recovery of normal hematopoietic stem and progenitor cells is only minimally degraded. When used under conditions that reduce CD34-positive human bone marrow cells only 5.1-fold, and murine and human granulocyte/macrophage progenitors 6.8- and 3-fold, respectively, combination purging with MC540-PDT and Edelfosine depletes murine (Mm5MT) and human (MDA-MB-435S) breast cancer cells >17,000- and >125,000-fold, respectively. These data suggest that combination purging with MC540-PDT and Edelfosine may offer a simple, safe and effective method for the ex vivo purging of autologous stem cell grafts from breast cancer patients.

Topics: Animals; Antigens, CD; Antigens, CD34; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cryopreservation; Female; Hematopoietic Stem Cells; Humans; Mice; Phospholipid Ethers; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Tumor Stem Cell Assay

Several factors, including the exposure of the negatively charged PL and transmembrane potential (TMP), may affect the binding of merocyanine 540 dye (MC540) to membrane lipids. Our aim was to quantify the significance of each of these two determinants in MC540 interactions with phosphatidylserine:phosphatidylcholine (PS/PC) vesicles. The effects of the altered PS content (PS/PC molar ratio: 5:95, 10:90 and 20:80) and TMP on MC540 binding were monitored using flow cytometry. Rapid [K(+)] flux across the vesicle membrane lipid bilayer was generated using valinomycin. We showed that the increased PS content leads to attenuated MC540 binding, while having no influence on the dynamic parameters of PS/PC vesicle membranes (electron spin resonance (ESR) spectrometry). Higher [K(+)](out) makes PS/PC liposomes bind more MC540, which implies that TMP-which becomes more positive inside the vesicles-favours the interactions of MC540 with the PL bilayer. Overall, the variability attributed to MC540-PL interactions is explained only to a minor extent by the generated TMP (7%) and largely by the variations in PS content (by up to 60%). In conclusion, the content of negatively charged PL is more important than TMP in determining the interactions of MC540 with PS/PC membranes.

Topics: Electron Spin Resonance Spectroscopy; Flow Cytometry; Lipid Bilayers; Liposomes; Membrane Potentials; Phosphatidylserines; Potassium; Pyrimidinones

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.

Topics: Cell Membrane; Cell Survival; Female; HeLa Cells; Humans; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Uterine Cervical Neoplasms

In this work, phospholipid liposomes were used to investigate the influence of lipid negative charge on the interaction of merocyanine 540 (MC540) with model membranes. Liposomes were prepared from a mixture of neutral dimyristoyl lecithin (DMPC) and negatively charged dimyristoyl phosphatidic acid (DMPA). A strong dependence between the presence of charges on the membrane and dye association was found. The affinity of the dye to liposomes was decreased with an increasing content of DMPA in liposomes. Changes in absorption spectra of MC540 suggest that the decrease in affinity of MC540 to charged membranes is accompanied by a hypsochromic solvatochromic shift and changes in monomer/dimer equilibrium of MC540 incorporated in the membrane.

Topics: Dimyristoylphosphatidylcholine; Glycerophospholipids; Liposomes; Membranes, Artificial; Pyrimidinones

Since apoptosis plays many roles in development, immune function, and disease, there is an ongoing need to identify inexpensive and reliable fluorochromes for the quantitation of apoptosis. Merocyanine 540 (MC540) binds to the outer membrane of cells and readily fluoresces in the highly disordered membranes of apoptotic cells making them readily detectable by flow cytometry. Protocols for the effective labeling and gating of MC540br apoptotic cells are provided. For example, MC540br cells from dexamethasone (Dex) treated thymocytes were found to be equivalent in proportion to apoptotic cells noted in the propidium iodide (PI) stained and annexin-V stained populations. Sorting of the MC540br cells followed by counterstaining with PI demonstrated that these cells resided in the low DNA fluorescent or sub-G1 region and were small in size based on light scatter. Dexamethasone, etoposide, irradiation, and a calcium ionophore were used to induce cell death with equivalent numbers of apoptotic cells obtained with MC540 and PI. Moreover, apoptotic human bone marrow (BM) B cells, neutrophils, Jurkat T cells, and testicular cells could readily be identified with MC540. The latter is particularly noteworthy since some of the standard methods for identifying cell death have not worked well with human cells. The versatility of this dye is such that it was also possible to phenotypically label cells stained with MC540 to analyze apoptosis in heterogenous populations of cells. Finally, the rate of detection of apoptotic cells after treatment of thymocytes with dexamethasone at 2, 4, 6, and 8 h with MC540 was shown to be equivalent to PI and annexin-V. Taken together, the data demonstrate that when proper precautions are taken, MC540 is a reliable, versatile, and inexpensive fluorochrome that can be used to identify apoptotic cells of human or murine origin even in heterogenous populations that require multicolor labeling.

Topics: Animals; Annexin A5; Apoptosis; Bone Marrow Cells; Cell Line; DNA; Female; Flow Cytometry; Fluorescent Dyes; Humans; Jurkat Cells; Mice; Mice, Inbred A; Neutrophils; Propidium; Pyrimidinones; Staining and Labeling; T-Lymphocytes

A method for the analysis of aliphatic carboxylic acids (ACAs) in nonaqueous capillary electrophoresis (NACE) in conjunction with indirect laser-induced fluorescence (ILIF) using merocyanine 540 (MC 540) is described. Performing the analysis in organic solvent is advantageous when using MC 540, because of its greater quantum yield in aprotic solvent. To achieve a high dynamic reserve (DR) and optimize resolution, we have tested a number of aqueous mixtures containing alcohols and acetonitrile (ACN). The optimum buffer for the analysis of C2-C18 ACAs, in terms of sensitivity, resolution, and speed, is an aqueous mixture of 40% ACN, 30% ethanol, and 1 mM Tris at apparent pH 7.4 (adjusted with ascorbic acid). Under this condition, the DR is greater than 1000, thereby the limits of detection for acids are in the range of sub-microM to microM. Linear plots show that the dynamic ranges for the analysis of ACAs are at least two decades in concentration, with regression coefficients all greater than 0.98. The relative standard deviations of the migration times and peak heights for all ACAs are less than 2.0%. Furthermore, this simple and cost-effective method has been applied to the analysis of marine lipid concentrate, with the concentrations of 1.67+/-0.03 and 4.50+/-0.05 mM (n = 5) for C14 and C16 acids, respectively, in a tablet of marine lipid concentrate sample.

Topics: Carboxylic Acids; Decanoic Acids; Electrophoresis, Capillary; Fatty Acids, Omega-3; Fluorescence; Fluorescent Dyes; Hydrogen-Ion Concentration; Ions; Molecular Structure; Pyrimidinones; Solvents

The effects of several biologically important inorganic salts, including NaCl, NaI, NaBr, KCl, MgCl2, MgSO4 and CaCl2 on the electronic absorption and fluorescence spectra of Merocyanine 540 (MC-540) have been investigated in aqueous media at 25 degrees C. Depending on both the MC-540 concentration and the nature of salt, a new absorption band appears at about 515 nm, above the critical salt concentration (CSC), corresponding to salt-induced MC-540 aggregation. Several types of MC-540 fluorescence quenching by the salts are observed, according to their cationic charge and the nature of anion: in the case of monovalent ions (Na+, K+), a non-linear Stern-Volmer behaviour is observed, indicating variable contributions of dynamic and static quenching mechanisms, whereas for divalent alkaline-earth (Mg2+, Ca2+) ions, linear Stern-Volmer relationships are obtained. Using these results, an analytical quenchofluorimetric approach is proposed for the determination of magnesium ions.

Topics: Anions; Calcium; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Magnesium; Magnesium Sulfate; Models, Chemical; Potassium; Pyrimidinones; Salts; Sodium; Sodium Chloride; Spectrometry, Fluorescence; Temperature

The capacitating agent bicarbonate/CO(2) has been shown to induce profound changes in the architecture and dynamics within the sperm's plasma membrane lipid bilayer via a cAMP-dependent protein phosphorylation signaling pathway. Here we have investigated the effect of bicarbonate on surface exposure of endogenous aminophospholipids in boar spermatozoa, detecting phosphatidylserine (PS) with fluorescein-conjugated annexin V and phosphatidylethanolamine (PE) with fluorescein-conjugated streptavidin/biotinylated Ro-09-0198. Flow cytometric analyses revealed that incubation with 15 mM bicarbonate induced 30%-70% of live acrosome-intact cells to expose PE very rapidly; this exposure was closely related to a decrease in lipid packing order as detected by enhanced binding of merocyanine 540. PS exposure was detectable in the same proportion of cells, though its expression was slower. Confocal microscopy revealed that exposure of aminophospholipids in intact cells was restricted to the anterior acrosomal region of the head plasma membrane. Aminophospholipid exposure, merocyanine stainability, and a subsequent migration of cholesterol to the apical region of the head plasma membrane, were all under the control of the cAMP-dependent protein phosphorylation pathway. The close coupling of decreased lipid packing order with exposure of PE led us to conclude that bicarbonate was inducing phospholipid scrambling (i.e., collapse of asymmetric transverse distribution), and that the scrambling was a prerequisite for cholesterol relocation. There was no evidence whatever that the bicarbonate-induced scrambling was an apoptotic process. It was not accompanied by major loss of viability or by DNA degeneration or by loss of mitochondrial function, and it could not be blocked by the broad-specificity caspase inhibitors zVAD-fmk and BocD-fmk. In the absence of bicarbonate, scrambling could not be induced by the apoptotic agents UV, staurosporine, or cycloheximide. Bicarbonate-induced phospholipid scrambling thus appears to be an important and early physiological event in the capacitation process.

Topics: Acridine Orange; Animals; Annexin A5; Anti-Bacterial Agents; Apoptosis; Bicarbonates; Caspase Inhibitors; Cell Membrane; Cholesterol; Coloring Agents; Cyclic AMP; DNA Fragmentation; Enzyme Inhibitors; Filipin; In Situ Nick-End Labeling; In Vitro Techniques; Male; Membrane Potentials; Microscopy, Confocal; Peptides; Peptides, Cyclic; Phospholipids; Pyrimidinones; Sperm Capacitation; Sperm Head; Spermatozoa; Swine

Topics: Fluorescent Dyes; Lipid Bilayers; Liposomes; Membrane Fluidity; Membrane Potentials; Phosphatidylcholines; Phosphatidylserines; Pyrimidinones

Topics: Drug Carriers; Liposomes; Phospholipids; Photosensitizing Agents; Pyrimidinones

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.

Topics: Apoptosis; Caspase Inhibitors; Caspases; Cyclosporine; Cytochrome c Group; Enzyme Activation; Flow Cytometry; Free Radical Scavengers; HL-60 Cells; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Ion Channels; Light; Membrane Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; NADPH Oxidases; Protein Transport; Pyrimidinones; Reactive Oxygen Species; Superoxides

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147-153, 2001)

Topics: Alkaline Phosphatase; Animals; Butyrates; Cell Differentiation; Cell Division; Colonic Neoplasms; Flow Cytometry; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Mice; Pyrimidinones; Spectrometry, Fluorescence; Subcellular Fractions; Tumor Cells, Cultured

Lung cancer has long been considered a disease that might benefit from the dose escalation of radio/chemotherapy afforded by a stem cell transplant. However, the clinical experience with high-dose chemotherapy and autologous bone marrow transplantation in lung cancer has been disappointing, with most trials showing little or no improvement in long-term survival. Unfortunately, lung cancer has a tendency to metastasize to the bone marrow, and lung cancer cells are known to circulate in the peripheral blood. Therefore, there is concern that autologous stem cell grafts from lung cancer patients may reinoculate recipients with live tumor cells. Photochemical purging of stem cell grafts with Merocyanine 540 (MC540) is highly effective against a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Most solid tumor cells (including lung cancer cells), however, are only moderately sensitive or refractory to MC540-mediated photodynamic therapy (PDT). We report here that postirradiation hyperthermia (< or = 42 degrees C, 3 h) potentiates the MC540-mediated photoinactivation of both wild-type (H69) and cisplatin-resistant mutant (H69/CDDP) small cell lung cancer cells by several orders of magnitude, while only minimally enhancing the depletion of normal human granulocyte/macrophage progenitor cells. Our data suggest that postirradiation hyperthermia provides a simple and effective means of extending the utility of MC540-PDT to the purging of stem cell grafts contaminated with lung cancer and possibly other solid tumor cells.

Topics: Bone Marrow Purging; Carcinoma, Small Cell; Hematopoietic Stem Cell Transplantation; Humans; Hyperthermia, Induced; Lung Neoplasms; Photosensitizing Agents; Pyrimidinones; Transplantation, Autologous; Tumor Cells, Cultured

The present study deals with photomodification of the electrical properties of the plasma membrane of an epithelial cell line (opossum kidney (OK) cells). The effect of photofrin II (previously investigated) is compared with that of 5 other membrane-active sensitizers: sulfonated Zn-phthalocyanine, merocyanine 540, rose bengal, methylene blue and protoporphyrin IX (an endogenous sensitizer induced by addition of its biosynthetic precursor 5-aminolaevulinic acid). The study was performed in order to investigate whether photomodification of the ion transport properties of the plasma membrane by membrane-active sensitizers is a general and early event in cellular photosensitization. The changes in the electrical properties were monitored by application of the whole-cell and the inside-out configuration of the patch-clamp technique. Illumination in the presence of the compounds (apart from merocyanine 540) gave rise to similar changes of the electrical properties of the membrane: depolarization of the membrane potential, inactivation of a large-conductance, Ca2+-dependent K+-channel (maxi-KCa), and a strong increase of the leak conductance of the membrane. This similarity indicates the general character of the functional photomodifications by membrane-active sensitizers previously reported for photofrin II.

Topics: Aminolevulinic Acid; Animals; Cell Line; Cell Membrane; Dihematoporphyrin Ether; Electric Conductivity; Indoles; Ion Transport; Isoindoles; Kidney; Light; Membrane Potentials; Methylene Blue; Opossums; Organometallic Compounds; Patch-Clamp Techniques; Photochemistry; Photosensitizing Agents; Protoporphyrins; Pyrimidinones; Rose Bengal; Zinc Compounds

The preparation and the solvatochromic behavior of two dyes, obtained by condensation of N,N'-dimethylbarbituric acid with dimethylaminobenzaldehyde and with 4,4'-bis(N,N-dimethylamino)benzophenone (Michler's ketone) are described. The latter dye is rather sensitive to the polarity of the medium, and in particular, to the hydrogen-bond-donor ability of protic solvents. The solvatochromism of both compounds is discussed in terms of the pi* and E(T)(30) solvent polarity scales and their differences in behavior interpreted with the aid of semiempirical calculations.

Topics: Barbiturates; Benzophenones; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Molecular Structure; Pyrimidines; Pyrimidinones; Solvents; Spectrophotometry, Ultraviolet

Alzheimer's disease pathology has demonstrated amyloid plaque formation associated with plasma membranes and the presence of intracellular amyloid-beta (A beta) accumulation in specific vesicular compartments. This suggests that lipid composition in different compartments may play a role in A beta aggregation. To test this hypothesis, we have isolated cellular membranes from human brain to evaluate A beta 40/42-lipid interactions. Plasma, endosomal, lysosomal, and Golgi membranes were isolated using sucrose gradients. Electron microscopy demonstrated that A beta fibrillogenesis is accelerated in the presence of plasma and endosomal and lysosomal membranes with plasma membranes inducing an enhanced surface organization. Alternatively, interaction of A beta with Golgi membranes fails to progress to fibril formation, suggesting that A beta-Golgi head group interaction stabilizes A beta. Fluorescence spectroscopy using the environment-sensitive probes 1,6-diphenyl-1,3,5-hexatriene, laurdan, N-epsilon-dansyl-L-lysine, and merocyanine 540 demonstrated variations in the inherent lipid properties at the level of the fatty acyl chains, glycerol backbone, and head groups, respectively. Addition of A beta 40/42 to the plasma and endosomal and lysosomal membranes decreases the fluidity not only of the fatty acyl chains but also the head group space, consistent with A beta insertion into the bilayer. In contrast, the Golgi bilayer fluidity is increased by A beta 40/42 binding which appears to result from lipid head group interactions and the production of interfacial packing defects.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Anisotropy; Brain; Cell Membrane; Dimerization; Endosomes; Fluorescent Dyes; Golgi Apparatus; Humans; Lipids; Lysosomes; Male; Microscopy, Electron; Models, Chemical; Peptides; Phospholipids; Protein Binding; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca(2+)-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO(2) on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca(2+) ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus approximately 3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10,000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca(2+) ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.

Topics: Acrosome Reaction; Animals; Bicarbonates; Calcimycin; Chlortetracycline; Coloring Agents; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Horses; Ionophores; Male; Microscopy, Fluorescence; Peanut Agglutinin; Pyrimidinones; Sperm Capacitation; Sperm Motility; Spermatozoa

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes.

Topics: Animals; Cell Membrane; Fluorescence Polarization; Lipid Metabolism; Liposomes; Osteoblasts; Pyrimidinones; Rats; Spectrometry, Fluorescence; Tumor Cells, Cultured; Vanadium Compounds

By means of recording a simple serie of merocyanine 540 spectra, we present a method to calculate the value proportional to co-operative unit size of membranes (n). Our calculations, applied to different liposomal samples processed in the presence or absence of sugars, in high or low ionic strength showed two main results. First, that any temperature cycling in high ionic strength of rigid DPPC bilayers will modify the membrane cooperativity. Second, the presence of polysaccharide Neu-5-ac in solution will always produce a strong drop in co-operativity of a rigid membrane of DPPC, whenever the negative charge is fully exposed. This last result indicates a differential ability of charged Neu-5-ac to disrupt a rigid membrane structure, even in the absence of a covalent linkage and--remarkably-in fully hydrated media.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Entropy; Fluorescent Dyes; Lipid Bilayers; Neuraminic Acids; Osmolar Concentration; Pyrimidinones; Spectrophotometry; Spectrum Analysis, Raman; Thermodynamics; Trehalose

Multichromophoric hydrogen-bonded assemblies 1(3) small middle dot(BAR)(6) are studied that bear a remarkably close resemblance to commelinin, a naturally occurring assembly responsible for an intense blue color of flowers. The incorporated chromophores exhibit a hypsochromic shift in the UV/visible (Vis) absorption maximum (Delta lambda(max) = 14 nm) compared with the free chromophores. In addition, the chiroptical properties of incorporated chromophores can be rationally controlled by changing the supramolecular chirality of the assembly. These properties have been used to study the stability of this type of assembly with UV and CD spectroscopy at concentrations far below the NMR sensitivity threshold (10(-4) M). The determined C(50%) values of 2-3 microM in benzene show the extremely high stability of these hydrogen-bonded assemblies.

Topics: Circular Dichroism; Crystallography, X-Ray; Hydrogen Bonding; Macromolecular Substances; Magnetic Resonance Spectroscopy; Models, Molecular; Pigments, Biological; Plants; Pyrimidinones; Spectrophotometry, Ultraviolet

Apoptotic leukocytes undergo cellular changes that are used as markers for "early" versus "late" stages of apoptosis. To ascertain if the order for acquisition of these changes is unique to specific hematopoietic cell types, we compared four leukocyte cell types and the following five apoptotic characteristics: MC540 incorporation, annexin V-FITC binding, propidium iodide (PI) labeling of hypodiploid nuclei, DNA fragmentation by a colorimetric assay, and cell membrane permeability to PI. The order for acquisition of these apoptotic characteristics was significantly different for each of the leukocyte cell types and for the mode of induction of apoptosis. It is interesting that the nuclear changes but not the membrane changes studied in mouse spleen cells required caspase activity. In summary, the acquisition of these apoptotic characteristics occurs through caspase-dependent and caspase-independent mechanisms, and importantly, the order for acquisition of the characteristics is specific for the cell type and for the mode of induction of apoptosis.

Topics: Animals; Annexin A5; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Membrane; Cell Membrane Permeability; Cell Nucleus; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA Fragmentation; Indicators and Reagents; Kinetics; Leukocytes; Mice; Mice, Inbred BALB C; Oligopeptides; Propidium; Pyrimidinones; Tumor Cells, Cultured

The kinetic and structural behavior of a photochromic compound, 3-(2-fluorophenyl)-3-phenyl-3H-naphtho[2,1-b]pyran (F-Py), was investigated using 1H and 19F nuclear magnetic resonance (NMR) spectroscopy. Upon irradiation, the four theoretically predicted photomerocyanines appear along with a fifth form X, whose final structure has not been elucidated. This last form and two of the photomerocyanines are thermally labile, whereas the other two do not show any signs of decay. The system has been analyzed by NMR spectroscopy. This led to the structural assignment of each photomerocyanine. The kinetics of the thermal bleaching were monitored by directly and separately measuring the concentrations of each species at regular time intervals using 19F NMR spectroscopy. We therefore propose a plausible reaction mechanism. On the basis of this mechanism, the mathematical treatment and the study of the effects of temperature led to the determination of the kinetic and thermodynamic parameters (rate coefficients, enthalpy and entropy of activation) of this photochromic system. The leading role of the labile intermediate X on the formation of trans-transoid-cis (TTC) and cis-transoid-cis (CTC) photomerocyanines is pointed out.

Topics: Carbocyanines; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Naphthalenes; Photosensitizing Agents; Pyrans; Pyrimidinones

Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.

Topics: Animals; Calcium; Cell Membrane; Cells, Cultured; Chlortetracycline; Cold Temperature; Coloring Agents; Electrophoresis, Polyacrylamide Gel; Intracellular Fluid; Male; Membrane Fluidity; Phosphorylation; Pyrimidinones; Sperm Capacitation; Spermatozoa; Swine

Topics: Adult; Erythrocyte Membrane; Humans; In Vitro Techniques; Lasers; Lipid Peroxidation; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Thiobarbituric Acid Reactive Substances

The effect of low-intensity ultrasound on HL-60 cells (human promyelocytic leukemia cells) in the presence of the photo sensitizing drug merocyanine 540 (MC 540) was evaluated morphologically, using a high-resolution scanning electron microscope and a transmission electron microscope. Exposure of HL-60 cells to ultrasound without MC 540 resulted in a decrease of finger-like processes in the cells. The cells showed many undulating ruffles on the surface. Distinct pits or holes in the membrane were not observed in these cells. The surface of HL-60 cells treated only with MC 540 was relatively smooth compared with that of control cells. HL-60 cells exposed to ultrasound in the presence of MC 540 showed apparent surface deformation. Numerous crater-like depressions of heterogeneous dimensions were observed in many cells. In addition, various-sized pores were noted in the cell membranes of more damaged cells. These results indicate that cell degeneration was induced by a rapid change in cell membrane porosity during sonication in the presence of MC540.

Topics: Apoptosis; Cell Membrane; HL-60 Cells; Humans; Microscopy, Electron, Scanning; Pyrimidinones; Ultrasonic Therapy

Bicarbonate/CO(2), a physiological effector of sperm capacitation, has been shown to induce a rapid and reversible change in the lipid architecture of the plasma membrane of live boar sperm: the change is detectable as an increase in the cells' ability to bind the fluorescent dye merocyanine, a characteristic which implied an increase in lipid packing disorder (Harrison et al. 1996. Mol Reprod Dev 45:378-391). Evidence suggested that cAMP may act as a second messenger in the system, and we have therefore investigated this cAMP-dependency in more detail. Bicarbonate stimulates cAMP levels within 1 min in a dose-dependent fashion, prior to parallel increases in merocyanine binding. Although the potent somatic cell adenylyl cyclase activator forskolin is unable to induce significant increases in cAMP or merocyanine binding, increases in merocyanine binding are inducible in a dose-dependent fashion by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate, a cAMP analogue highly specific in its ability to stimulate protein kinase A; moreover, the bicarbonate-induced membrane change is inhibited by H89, a specific protein kinase A inhibitor. Neither bisindolylmaleimide I (protein kinase C inhibitor) nor lavendustin A (protein tyrosine kinase inhibitor) are inhibitory. In the presence of low levels of the potent phosphodiesterase inhibitor papaverine, increases in merocyanine binding are enhanced by okadaic acid and (more effectively) by calyculin (both protein phosphatase inhibitors). We conclude that boar sperm plasma membrane lipid architecture is controlled via a target protein that is dynamically phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase type 1. Mol. Reprod. Dev. 55:220-228, 2000.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Bicarbonates; Cell Membrane; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Flow Cytometry; Fluorescent Dyes; Lipid Metabolism; Lipids; Male; Phosphodiesterase Inhibitors; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Pyrimidinones; Signal Transduction; Spermatozoa; Swine

Induction of mitochondrial permeability transition (MPT) and cytosolic translocation of cytochrome C are considered essential components of the apoptotic pathway. Hence, there is the realization that mitochondrial-specific drugs could have potential for use as chemotherapeutic agents to trigger apoptosis in tumor cells. Recently, we showed that photoproducts of merocyanine 540 (pMC540) induced tumor cell apoptosis. In this study, we focused on identifying mitochondrial-specific compounds from pMC540 and studied their apoptotic potential. One purified fraction, C5, induced a drop in mitochondrial transmembrane potential and cytosolic translocation of cytochrome C in HL60 human leukemia cells. Moreover, the addition of C5 to purified rat liver mitochondria induced MPT as indicated by mitochondrial matrix swelling, which was completely inhibited by cyclosporin A, an inhibitor of the inner-membrane pore. Supernatant of C5-treated mitochondria showed a dose-dependent increase in cytochrome C, which was also inhibited in the presence of cyclosporin A, strongly indicating a direct effect on the inner-membrane pore. Despite the strong mitochondrial reactivity, C5 elicited minimal cytotoxicity (less than 25%) against HL60 leukemia and M14 melanoma cells because of inefficient caspase activation. However, prior exposure to C5 significantly enhanced the apoptotic response to etoposide or the CD95 receptor. Thus, we demonstrate that MPT induction and cytochrome C release by the novel compound C5, in the absence of effective caspase activation, is insufficient for triggering efficient apoptosis in tumor cells. However, when used in combination with known apoptosis inducers, such compounds could enhance the sensitivity of tumor cells to apoptosis. (Blood. 2000;95:1773-1780)

Topics: Animals; Antineoplastic Agents; Apoptosis; Biological Transport; Caspase Inhibitors; Caspases; Cyclosporine; Cytochrome c Group; Cytosol; Enzyme Activation; Enzyme Inhibitors; Etoposide; Fluorescence; HL-60 Cells; Humans; Intracellular Membranes; Melanoma; Mitochondria; Mitochondria, Liver; Neoplasm Proteins; Oligopeptides; Permeability; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Rats; Rats, Wistar; Signal Transduction; Skin Neoplasms; Staurosporine; Tumor Cells, Cultured

Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin-lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.

Topics: Anion Exchange Protein 1, Erythrocyte; Binding Sites; Cell Membrane Permeability; Erythrocyte Membrane; Erythrocytes; Freeze Fracturing; Hemoglobins; Hemolysis; Humans; Lipid Bilayers; Membrane Proteins; Pyrimidinones; Saponins; Spectrin

Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.

Topics: Erythrocyte Membrane; Erythrocytes; Histidine; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Indoles; Organometallic Compounds; Photobiology; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Vesicular stomatitis Indiana virus

Merocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic environment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluorescent transmembrane probe remains controversial. In order to determine the exact spectra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phospholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 microM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K(1) and K(2), for the assumed stacking-model reactions, M+M <--> M(2) and M+M(2) <--> M(3), were 3.1 x 10(-4) M and 5.7 x 10(-4) M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking-model reaction, M(*)+M(*) <--> *M(2), was 1.9x10(-5)M. The fluorescent intensities of MC540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the temporal increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change.

Topics: Fluorescent Dyes; Molecular Conformation; Multivariate Analysis; Phosphatidylcholines; Phospholipids; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry; Water

In order to evaluate the combined effect of Amifostine and Merocyanine 540 during photoirradiation in neoplastic cells, bone marrow cells from children with acute leukemia (AL), age-matched controls as well as HL-60 cell line were studied. Cell suspensions were incubated with Amifostine, then with MC 540 and they were subsequently exposed to different irradiation doses by Argon Laser 514 nm. Cell survival was estimated by trypan blue supravital stain following a 24-h incubation. The leukemic cell line was studied in continuous liquid cell cultures for 4 weeks. The survival of normal bone marrow progenitors has been estimated by colony formation assay in methylcellulose cultures. Our results showed that Amifostine enhances the photokilling effect of MC 540 on leukemic cells and significantly protects bone marrow nucleated and committed progenitors (BFU-E and CFU-GM) from children with AL under chemotherapy. In conclusion, Amifostine seems to be a promising cytoprotective agent in the clinical use of purging with MC 540 mediated phototherapy.

Topics: Amifostine; Bone Marrow; Bone Marrow Purging; Cell Survival; Drug Therapy, Combination; HL-60 Cells; Humans; Photochemotherapy; Photosensitizing Agents; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidinones; Radiation-Protective Agents

A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior of C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.

Topics: 4-Chloro-7-nitrobenzofurazan; Acrosome; Animals; Bicarbonates; Calcium; Cell Membrane; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Fluorescent Dyes; Lipid Bilayers; Male; Oxadiazoles; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Phosphorylation; Pyrimidinones; Sperm Capacitation; Spermatozoa; Sphingomyelins; Swine

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. In this study, two clinical photosensitizers, Temoporfin (meta-tetra-hydroxyl-phenyl-chlorin; mTHPC) and merocyanine 540 (MC540), were selected to explore for their photocytotoxic and genotoxic effects on nasopharyngeal carcinoma cells (NPC/HK1 and CNE2). Results of tetrazolium reduction assay showed that 80% cell killing were achieved for both cell lines at 0.4 microg/ml mTHPC for 24 h incubation and then with 40 kJ/m2 light irradiation, whereas 40 microg/ml MC540 with 50 kJ/m2 light dosage was required to attain the same level of phototoxicity for NPC/HK1. On the contrary, NPC/CNE2 was quite resistant to MC540. Hence, mTHPC-mediated PDT exerted a more potent effect than MC540-mediated PDT, even though the molar extinction coefficient of the main absorption peak for MC540 is much higher than that of mTHPC. Dark cytotoxicity remained negligible for both sensitizers. Comet assay was used to evaluate the DNA strand break and potential genotoxic effect induced by mTHPC and MC540 on the NPC cells. No DNA strand break was detected in the absence of light, and under sublethal treatment (LD25) for either sensitizer-loaded cells. Confocal laser scanning microscopy showed that mTHPC and MC540 localized in the cytoplasm but not in the nucleus of the tumor cells, which provided evidence for undetectable DNA damage under dark and low photodynamic dose.

Topics: Aged; Analysis of Variance; Antineoplastic Agents; Comet Assay; DNA; DNA Damage; Humans; Male; Mesoporphyrins; Nasopharyngeal Neoplasms; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.

Topics: Animals; Annexin A5; Apoptosis; Calcium; Cells, Cultured; Dexamethasone; Fluorescent Dyes; Glucans; Macrophage Activation; Macrophages; Male; Membrane Lipids; Mice; Phagocytosis; Phosphatidylserines; Pyrimidinones; T-Lymphocytes

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.

Topics: Animals; Apoptosis; Leukemia, Myeloid, Acute; Mice; Mitochondria; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Subcellular Fractions; Tumor Cells, Cultured

The fluorescence of Merocyanine 540 (MC 540) is sensitive to the molecular packing of membrane lipids. Therefore, the fluorescence of MC 540 is expected to be sensitive to the curvature-related packing stress at the onset of the lamellar-hexagonal phase transition. We measured the fluorescence intensity of MC 540 when the temperatures of lipid bilayers approached their lamellar-hexagonal phase transitions. The fluorescence of MC 540 in the presence of egg and dioleoylphosphatidylethanolamine bilayers increased at the respective lamellar-hexagonal phase transitions of these lipids. Furthermore, increases in fluorescence intensity were also observed at temperatures just below their phase transitions. The enhanced fluorescence was not due to the specific interaction of the dye with the ethanolamine headgroup, because no such increase was observed when the probe was exposed to phosphatidylethanolamines which do not form hexagonal phase within the range of applied temperature. In addition, when the temperature of the lamellar-hexagonal phase transition was shifted, by the addition of a small amount of phosphatidylcholine, the dependence of the fluorescence intensity on temperature was modified accordingly. We postulate that the change of MC 540 fluorescence intensity at temperatures approaching the lamellar-hexagonal phase transition reflects changes in the partition of MC 540 into the fluid lipid phase. The change in partition is influenced by the curvature stress in bilayers at temperatures just below the lamellar-hexagonal phase transition.

Topics: Fluorescent Dyes; Lipid Bilayers; Membrane Lipids; Phosphatidylethanolamines; Pyrimidinones; Temperature

As a major Ca exit system in myocytes, the electrogenic Na+-Ca2+ exchange is exposed to rapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation-contraction coupling. The dynamic aspects of the exchanger response to regulatory factors have not been resolved in the past due to technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activity of Na+-Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dye Merocyanine-540 (M540). The M540 signal of Nao-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 microM Cafree. This signal is inhibited by a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler (valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-state burst (210 s-1), followed by slow steady-state phase (

Topics: Aniline Compounds; Animals; Calcium; Cattle; Diffusion; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Myocardium; Nickel; Pyrimidinones; Sarcolemma; Signal Transduction; Sodium-Calcium Exchanger; Spectrometry, Fluorescence; Xanthenes

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25 degrees C.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Anisotropy; Carbohydrate Sequence; Cholesterol; Dansyl Compounds; Diglycerides; Diphenylhexatriene; Fatty Acids; Glycolipids; Lipid Bilayers; Molecular Sequence Data; Pyrimidinones; Spectrometry, Fluorescence; Spectrum Analysis; Surface Properties; Temperature; Trisaccharides

Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle. We rationalized that MC540 uptake could be used to distinguish between quiescent and metabolically active cells and, therefore, to fractionate normal and leukemic BM cells and normal mobilized peripheral blood (MPB) cells into functionally distinct groups of progenitors. BM and MPB cells were separated into fractions ranging in fluorescence from MC540Bright to MC540Dim. Cell cycle analysis of these fractions revealed that the MC540Dim fraction of normal and CML BM CD34+ cells constituted the most quiescent fraction, and the MC540Bright fractions from these cell types contained the most actively cycling cells. However, no differences in the percentage of cells in G/G1 were observed between MC540Bright and MC540Dim fractions of MPB CD34+ cells. To investigate if these cell cycle status differences translated into distinct functional properties, the hematopoietic potential of BM CD34+MC540Bright and CD34+MC540Dim cell fractions was analyzed in vitro in long-term BM cultures and limiting dilution analysis (LDA) assays. CD34+MC540Dim cells produced more total and committed progenitor cells in long-term cultures than did the CD34+MC540Bright fraction. The CD34+MC540Dim fraction also contained a 2-fold higher number of long-term hematopoietic culture-initiating cells (LTHCIC) than the CD34+MC540Bright fraction, as defined by LDA assays. These data demonstrate that MC540 can be a useful probe for the isolation of primitive HPC from some hematopoietic tissues and may assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation of HPC.

Topics: Bone Marrow; Cell Separation; Flow Cytometry; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Pyrimidinones

If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non-receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (triangle uppsim), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial triangle uppsim are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspases; Cytochrome c Group; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Melanoma; Membrane Potentials; Mitochondria; Mitochondria, Liver; Photochemistry; Pyrimidinones; Rats; Tumor Cells, Cultured

Merocyanine 540 (MC) is an anionic dye that is used to photopurge the bone marrow of leukemia cells. Under these conditions it is localized mostly in cell membranes, which may affect its photochemical reactivity. We investigated the photochemistry of MC dissolved as a hydrophobic ion pair with a hexadecyltrioctadecylammonium cation in cyclohexane, trimethylpentane and toluene as well as in propylene carbonate, CH3CN, C2H5OH and D2O. In organic solvents, the absorption and fluorescence spectra of MC were strongly red-shifted compared with aqueous solutions. The fluorescence was also more intense despite aggregation that occurred in some solvents. Aggregation strongly affects the spectral and photochemical properties of MC, especially in aliphatic hydrocarbons in which distinctive H-type aggregates are formed. Hydrophobic MC is a moderate photosensitizer of singlet molecular oxygen (1O2). The following values for 1O2 quantum yields were calculated based on 1O2 phosphorescence relative to 1O2 generation by Rose Bengal: approximately 0.12 in trimethylpenthane, approximately 0.13 in cyclohexane, 0.045 in EtOH, 0.039 in toluene, 0.007 in CH3CN and approximately 3 x 10(-4) in D2O. The H-aggregates of MC in cyclohexane and trimethylpentane are better 1O2 producers than monomeric MC. The above 1O2 quantum yields are corrected for self-quenching because MC is an efficient 1O2 quencher (17 x 10(7) M-1 s-1 in CH3CN, 6.8 x 10(7) M-1 s-1 in D2O, 5.2 x 10(7) M-1 s-1 in EtOH, and 1.4 x 10(7) M-1 s-1 in toluene). Merocyanine undergoes photodegradation, a solvent-dependent process that proceeds faster when the dye is aggregated. The initial photodegradation rate is much slower in organic solvents than in water, but photodegradation products accumulated during longer irradiation may increase the rate in most solvents. Higher photostability and better photosensitization by MC in hydrophobic nonpolar solvents suggest that the killing of leukemia cells via a photodynamic mechanism may operate mostly in cell membranes. In contrast, any cytotoxic products from photodecomposition may be important in hydrophilic cell compartments. Our data show the spectral and photochemical properties of MC in a pure hydrophobic environment.

Topics: Humans; Oxygen; Photochemistry; Pyrimidinones; Solubility; Solvents; Spectrometry, Fluorescence; Spectrophotometry; Surface-Active Agents

We tested the hypothesis that elevated blood pressure, a known stimulus for vascular remodeling and an independent risk factor for the development of atherosclerotic disease, can modulate basal and cytokine-induced tissue factor (TF; CD 142) expression in cultured human endothelial cells (EC). Using a chromogenic enzymatic assay, we measured basal and tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC (HVCEC) cultured at atmospheric pressure and at 170 mmHg imposed pressure for up to 48 h. Basal TF activities were 22 +/- 10 U/mg protein for HAEC and 14 +/- 9 U/mg protein for HVCEC and were upregulated in both cell types >10-fold by TNF-alpha. Exposure to pressure for 5 h induced additional elevation of basal TF activity by 47 +/- 16% (P < 0.05, n = 6) for HAEC and 17 +/- 5% (P < 0.05, n = 3) for HVCEC. Pressurization also enhanced TF activity in TNF-alpha-treated cells from 240 +/- 28 to 319 +/- 32 U/mg protein in HAEC (P < 0.05, n = 4) and from 148 +/- 25 to 179 +/- 0.8 U/mg protein (P < 0.05, n = 3) in HVCEC. Cytokine stimulation caused an approximately 100-fold increase in steady-state TF mRNA levels in HAEC, whereas pressurization did not alter either TF mRNA or cell surface antigen expression, as determined by quantitative RT-PCR methodology and ELISA. Elevated pressure, however, modulated the EC plasma membrane organization and/or permeability as inferred from the increased cellular uptake of the fluorescent amphipathic dye merocyanine 540 (33 +/- 7%, P < 0.05). Our data suggest that elevated static pressure modulates the hemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.

Topics: Aorta; Atmospheric Pressure; Cell Membrane Permeability; Cells, Cultured; Cytokines; Cytological Techniques; Endothelium, Vascular; Fluorescent Dyes; Humans; Pressure; Pyrimidinones; Recombinant Proteins; RNA, Messenger; Thromboplastin; Venae Cavae

When bursal lymphocytes are placed in cell culture, they undergo an apoptotic form of cell death that can be inhibited by phorbol esters and protein synthesis inhibitors. The goal of the current study was to evaluate the time course of this process and the inhibition of this process using several different assays to detect apoptosis: (1) terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) of lymphocyte DNA strand breaks with dUTP-FITC; (2) propidium iodide (PI) staining of lymphocyte chromatin; (3) chloromethyl-x-rosamine (CMX-Ros) binding to lymphocyte mitochondria; (4) merocyanine-540 (MC-540) binding to the lymphocyte plasma membrane; (5) flow cytometric analysis of light scatter from lymphocytes; (6) analysis of genomic DNA from lymphocytes by agarose gel electrophoresis; and (7) cellular caspase-3 activity of lymphocytes. When bursal lymphocyte apoptosis was analyzed as a function of time, or inhibited by phorbol esters or cycloheximide, all of these assays corroborated the apoptotic process. However, treatment of lymphocytes with a cytotoxic level of the proteinase inhibitor, n-ethylmaleimide (NEM) resulted in a putative, necrotic form of cell death that revealed discrepancies among the various assays in the detection of apoptotic cells. Specifically, the CMX-Ros and MC-540 assays erroneously detected the necrotic cells as being apoptotic cells following NEM treatment. These findings indicate the need for additional assays and appropriate treatment controls to verify the apoptotic process when using the CMX-Ros and MC-540 assays.

Topics: Animals; Apoptosis; B-Lymphocytes; Bursa of Fabricius; Cell Survival; Cells, Cultured; Chickens; DNA Fragmentation; Ethylmaleimide; Fluorescent Dyes; In Situ Nick-End Labeling; Nucleosomes; Organic Chemicals; Propidium; Pyrimidinones; Scattering, Radiation; Time Factors

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.

Topics: 4-Chloro-7-nitrobenzofurazan; Arbutin; Cryoprotective Agents; Diglycerides; Dose-Response Relationship, Drug; Fluorescence Polarization; Freezing; Galactolipids; Glycine max; Glycolipids; Intracellular Membranes; Liposomes; Membrane Fusion; Ovum; Phosphatidylcholines; Phosphatidylethanolamines; Plastocyanin; Pyrimidinones; Spinacia oleracea; Thylakoids

As shown in earlier work (M.M. Henszen et al., Mol. Membr. Biol. 14 (1997) 195-204), exposure of erythrocytes to single brief electric field pulses (5-7 kV cm(-1)) enhances the transbilayer mobility of phospholipids and produces echinocytes which can subsequently be transformed into stomatocytes in an ATP-dependent process. These shape transformations arise from partly reversible changes of the transbilayer disposition of phospholipids, in agreement with the bilayer couple concept. Extensive membrane modification by repetitive (

Topics: Cell Size; Electroporation; Erythrocyte Deformability; Erythrocyte Membrane; Erythrocytes; Glutathione; Humans; Lipid Peroxidation; Membrane Proteins; Phospholipids; Pyrimidinones; Serum Albumin; Stilbenes; Temperature; Time Factors; Trypsin

Intracellular superoxide (O(2)*- was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O(2)*- increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O(2)*- regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.

Topics: Apoptosis; Caspase 3; Caspases; Daunorubicin; Drug Interactions; Enzyme Activation; Etoposide; fas Receptor; Humans; Oligodeoxyribonucleotides, Antisense; Pyrimidinones; RNA, Messenger; Superoxide Dismutase; Superoxides; Tumor Cells, Cultured

Ultrasound is frequently used in medicine for diagnostic purposes. Recently, there have been numerous reports on application of ultrasound energy for controlling drug release or targeting. This new concept of therapeutic ultrasound combined with drugs has induced excitement in various areas. Ultrasound energy can enhance effects of thrombolytic agents as urokinase. Ultrasound emitting catheters are currently being developed for cardiovascular diseases. Device with ultrasound transducers implanted in transdermal drug patches are also being evaluated for possible delivery of insulin through the skin. Chemical activation of drugs by ultrasound energy for treatment of cancers is another new field recently termed as "Sonodynamic Therapy". Various examples of application of ultrasound for drug delivery systems are discussed.

Topics: Antineoplastic Agents; Drug Delivery Systems; HL-60 Cells; Humans; Insulin; Pyrimidinones; Thrombolytic Therapy; Transducers; Ultrasonic Therapy

The photosensitizing dye merocyanine 540 (MC540) has been used in preclinical models and in a phase I clinical trial in the U.S.A. for the extracorporeal purging of autologous bone marrow grafts contaminated with leukemia or lymphoma. In this communication, we report MC540-mediated photodynamic therapy (PDT) was effective in purging leukemic cells expressing P-gp. When K562 and K562/ADM were exposed to MC540 (15 micrograms/ml) and white light (145.8 kJ/m2), the concentration of K562 and K 562/ADR was reduced by 1.8 and 3.0 log, respectively. Using flow cytometry and confocal laser scan microscopy, MC540 and calcein-AM were bound intracellularly and effluxed by P-gp in K562/ADM. In K562/ADM, calcein-AM efflux was inhibited by P-gp modulator, cyclosporin A (5 microM) and verapamil (15 micrograms/ml). In contrast, MC540 efflux was inhibited by cyclosporin A but not verapamil. Furthermore, MC540-mediated PDT inhibited efflux of calcein-AM and MC540, and induced the accumulation of dyes in K562/ADM. We conclude that MC540 is a substrate of P-gp and that MC540-mediated PDT is useful for purging MDR cells through inhibition of P-gp activity.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Doxorubicin; Drug Resistance, Neoplasm; Humans; K562 Cells; Leukemia; Photochemotherapy; Photosensitizing Agents; Pyrimidinones

A new neoglycolipid (AgH-1) bearing carbohydrate units that mimics the antigenic determinant of the O-blood group was synthesized and the effect of its incorporation in dipalmitoylphosphatidylcholine (DPPC): cholesterol liposomes was evaluated. The results obtained show that AgH-1 is readily incorporated into DPPC:cholesterol liposomes. The conditions leading to the optimal incorporation are the result of a compromise between incorporation efficiency and incorporation extent. The presence of AgH-1 produces liposomes of smaller size, with only small changes in the properties of the bilayer. However, the data obtained employing diphenylhexatriene and laurodan as fluorescence probes and merocyanine 540 as optical probe suggest that AgH-1 incorporation leads to a small rigidization of the liposomes at temperatures lower than ca. 42 degrees C.

Topics: Blood Group Antigens; Carbohydrate Sequence; Cholesterol; Diphenylhexatriene; Erythrocyte Membrane; Fluorescence Polarization; Fluorescent Dyes; Glycolipids; Kinetics; Lipid Bilayers; Liposomes; Molecular Sequence Data; Naphthalenes; Particle Size; Phosphatidylcholines; Pyrimidinones; Temperature

Illumination of erythrocytes in the presence of merocyanine 540 (MC540) resulted in changed binding characteristics of MC540, i.e. a red shift in the emission maximum of bound dye with an increase in the relative fluorescence quantum yield. Aluminum phthalocyanine tetrasulfonate-mediated photodynamic treatment, before addition of MC540, resulted in a comparable change in the MC540-binding characteristics with, in addition, an increase in the concentration of MC540 in the membrane. Both photodynamic treatments induce depolarization of the red cell membrane, with a dose dependency comparable to that of changed MC540 binding. Also depolarization, induced by incubation of the cells with A23187 in the presence of Ca2+ in high [K+] buffer, resulted in similar changes in the MC540 binding characteristics. These results indicate a relation between photodynamically induced membrane depolarization and changed MC540-binding characteristics. Hyperpolarization induced by incubation with A23187 in low [K+] buffer resulted in decreased binding of MC540. In accordance, the MC540-mediated photodamage to red cells decreased upon hyperpolarization of the cells. The results indicate that the binding of MC540 to erythrocytes is strongly dependent on the membrane potential and that hyperpolarization of the membrane could be a possible protection mechanism for erythrocytes against MC540-mediated photodynamic damage.

Topics: Dose-Response Relationship, Radiation; Erythrocyte Membrane; Humans; In Vitro Techniques; Light; Membrane Potentials; Photosensitizing Agents; Pyrimidinones

The molecular events involved in tumor cell death induced by novel photoproducts of merocyanine 540 (pMC540) are poorly understood. Using HL60 leukemia and M14 melanoma cell lines we investigated the role of the apoptotic pathway in pMC540-mediated cell death. Tumor cells exposed to pMC540 showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by pMC540 in both tumor cell lines as evidenced by the externalization of phosphatidylserine. A dose-dependent increase in caspase-3 protease activity suppressed by the tetrapeptide inhibitor DEVD-CHO was observed in both cell lines. Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116 to 89 kDa) associated with apoptosis in pMC540-treated cell lysates. Furthermore, caspase inhibition blocked the externalization of membrane PS, indicating that the loss of membrane phospholipid asymmetry is a downstream event of caspase activation. These findings demonstrate that tumor cell death induced by pMC540 is mediated by caspase proteases.

Topics: Apoptosis; Cell Size; Cysteine Endopeptidases; DNA, Neoplasm; Enzyme Activation; Humans; Leukemia; Melanoma; Phosphatidylserines; Pyrimidinones; Tumor Cells, Cultured

The photodynamic inactivation of herpes simplex virus type 1 (HSV-1) by two phthalocyanines (Pcs), the cationic dye HOSi-PcOSi(CH3)2(CH2)3N+(CH3)3I-(Pc5) and the amphiphilic dye aluminum dibenzodisulfophthalocyanine hydroxide (AlN2SB2POH), has been compared with that by the anionic dye, Merocyanine 540 (Mc540). Both Pc derivatives demonstrate a remarkable virucidal activity upon light activation even 3 h after the onset of HSV-1 adsorption, while Mc540 is effective for only 30 min after adsorption. Since fusion and virus penetration are promoted by membrane glycoproteins, we have studied the damage to viral proteins following photodynamic treatment (PDT) of HSV-1 and its relation to inactivation. The effect of AlN2SB2POH PDT is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Major changes are found in the protein profile of PDT-treated HSV-1. A reduced ability of specific antibodies to react with HSV-1 major envelope proteins is detected by employing the Western blot assay. In particular, we demonstrate the related changes of glycoprotein D (gD), a structural protein of the HSV envelope. Since the envelope proteins participate in viral entry into the host cell, these alterations to viral envelope proteins may impair their ability to participate in early events of viral entry, leading to reduced infectivity of HSV-1. In contrast, no significant changes in the proteins' electrophoretic mobility could be seen after PDT with Mc540 or with Pc5. When HSV-1 purified proteins are subjected to combined electrophoretic and electro osmotic forces on cellulose acetate, there is a shift in their cathode mobility, which may indicate changes in the protein mass and protein net charges following AlN2SB2POH photosensitization. There are only minor changes in the virus proteins, assayed as above, when HSV-1 is treated with Pc5.

Topics: Animals; Chlorocebus aethiops; Gene Products, env; Indoles; Isoindoles; Kinetics; Light; Photosensitizing Agents; Pyrimidinones; Simplexvirus; Vero Cells; Viral Proteins

The photodynamic effects of temoporfin (meso-tetrahydroxyphenylchlorin, mTHPC) and merocyanine 540 (MC540) in murine myeloid leukemia M1 and WEHI 3B (JCS) cells were compared. The mTHPC was found to be more potent and selective. At a lethal dosage of 90% killing (LD90), only 1.3 microM of mTHPC and 4.2 kJ/m2 of light irradiation was required, which was a 20-fold lower drug concentration and 11-fold smaller light dose than that required when using MC540. Meanwhile, three times less, or 15%, of the coincubated erythrocytes were destroyed by mTHPC than by MC540. Confocal micrographs showed that both drugs accumulated diffusely inside the cytoplasm in a very similar fashion, but mTHPC induced a more extensive apoptosis in photosensitized JCS cells. For example, at LD90, mTHPC practically killed all JCS cells via apoptosis and cleaved the DNA to extremely small 150 base-pair fragments. In contrast, among the JCS cells killed by MC540, about 88% died via apoptosis and large DNA fragments were abundant. Relative to MC540, the ability of mTHPC to trigger large-scale and thorough apoptosis in leukemia cells may help explain its potency and selectivity.

Topics: Animals; Apoptosis; Cell Survival; Erythrocytes; Leukemia, Experimental; Leukemia, Myeloid; Light; Mesoporphyrins; Mice; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

The antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye previously used to purge malignant cells from autologous bone marrow grafts, was evaluated against a panel of Gram-positive and Gram-negative bacteria and Candida albicans in the presence and absence of light. In the absence of light, MC 540 demonstrated no antibacterial activity against any of the organisms tested. When combined with increasing intervals of photoillumination, growth inhibition was observed with all Gram-positive organisms tested except Mycobacterium fortuitum. Photosensitizing growth inhibition was also observed with Moraxella catarrhalis but not with any other Gram-negative bacilli including members of the Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophila, or Burkhoderia cepacia. These results suggested that differences in cell wall structure confer resistance to the photodamaging effects of the dye. MC 540 exhibited no antimicrobial activity against C. albicans in the presence or absence of light.

Topics: Anti-Bacterial Agents; Anti-Infective Agents; Candida albicans; Colony Count, Microbial; Gram-Negative Bacteria; Gram-Positive Bacteria; Light; Microbial Sensitivity Tests; Photosensitizing Agents; Pyrimidinones; Time Factors

The changes in ultrastructure, lipid organization, chromatin decondensation, and denaturation of rhesus monkey spermatozoa during epididymal maturation were studied. This study would provide background information that would be useful to evaluate adverse effects, if any, caused by the use of contraceptive agents.. Adult sexually mature rhesus monkeys were castrated under ketamine anesthesia. The epididymis was divided into initial segment, caput, corpus, and cauda epididymides. To study changes in lipid organization of the sperm plasma membrane during epididymal transit, spermatozoa from different epididymal segments and ejaculated spermatozoa were exposed to merocyanine 540 (MC 540). The changes in chromatin denaturation and decondensation were assessed by using the nucleic acid-specific fluorochromes, acridine orange, and ethidium bromide, respectively, prior to and after exposure to dithiothreitol (DTT).. Testicular spermatozoa (approximately 40%) showed localization of MC 540 mainly in the midpiece, whereas remaining sperm did not localize MC 540. Spermatozoa from the initial segment of the epididymis showed uniform distribution of MC 540 localization in the head and midpiece. A pattern of localization of MC 540 similar to mature caudal and ejaculated sperm in which the staining was restricted to the acrosome and the midpiece first appeared in a small percentage of caput spermatozoa and was completed during transit through the corpus epididymidis. Mature spermatozoa from cauda epididymidis, vas deferens, and ejaculate did not undergo chromatin denaturation even after exposure to 10 mM DTT, unlike sperm from testis, initial segment, and caput epididymidis. Spermatozoa exposed to DTT showed chromatin decondensation; maximum decondensation was seen in testicular sperm and a decrease in the percentage of sperm, showing decondensation, occurred during epididymal transit. Ultrastructural studies showed that spermatozoa undergo structural changes during sperm maturation.. The present study shows that rhesus monkey spermatozoa undergo reorganization of the plasma membrane lipids and stabilization of disulfide linkages during epididymal transit. The results would be of use in evaluating the action of potential male contraceptive drugs on epididymal spermatozoa.

Topics: Animals; Cell Membrane; Chromatin; Cysteine; Dithiothreitol; Ejaculation; Epididymis; Fluorescent Dyes; In Vitro Techniques; Macaca mulatta; Male; Pyrimidinones; Spermatozoa

Photodynamic action of merocyanine 540 (MC540) on the plasma membrane of human glioblastoma(U-87MG) cells has been investigated. Plasma membrane was labeled with lipid specific probe 1,(4-trimethylammonium),6-diphenyl-1,3,5-hexatriene. Steady-state anisotropy, decay time and time-dependent anisotropy of TMA-DPH in U-87MG cells have been measured as a function of light dose. A decrease in the steady-state anisotropy and decay time of TMA-DPH in MC540-treated cells was observed upon light irradiation. The time-dependent anisotropy measurements showed a decrease in the limiting anisotropy (r infinity) and an increase in the rotational relaxation time (phi) of the probe upon photosensitization of cells. Analysis of these data using wobbling in cone model for probe rotation in the membrane indicated an increase in the cone angle (theta c) and a decrease in the order parameter (S). Protein specific probe N-(1-pyrene)-maleimide was used to study the effect of photosensitization on the plasma membrane proteins. An increase in the rotational relaxation time and a decrease in the ratio of excimer to monomer fluorescence intensity of PM was observed on photosensitization. Photodynamic action of MC540 also caused an inhibition of protein SH groups and Na(+)-K(+)-ATPase activity of plasma membrane. Our results demonstrate that the photodynamic action of MC540 decreases the order of the lipid bilayer and reduces the mobility of the proteins in the plasma membrane of cells.

Topics: Cell Membrane; Diphenylhexatriene; Dithionitrobenzoic Acid; Fluorescence Polarization; Fluorescent Dyes; Glioblastoma; Humans; Light; Maleimides; Photosensitizing Agents; Pyrimidinones; Sodium-Potassium-Exchanging ATPase; Sulfhydryl Reagents; Tumor Cells, Cultured

The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care.

Topics: Adolescent; Erythrocytes; Fluorescent Dyes; Humans; Hydrogen Peroxide; Lipid Peroxidation; Oxidants; Oxidative Stress; Pyrimidinones

Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates experimental leukemia, lymphoma, and neuroblastoma cells by a singlet oxygen-mediated mechanism but is relatively well tolerated by normal pluripotent hematopoietic stem cells and granulocyte/macrophage progenitors (CFU-GM). MC540 is currently undergoing phase I clinical testing for the extracorporeal purging of autologous bone marrow and peripheral blood stem cells. We report here that performing MC540-mediated PDT at 4.7 degrees C (hypothermia) instead of at ambient temperature enhanced the photoinactivation of L1210 cells and CFU-GM but left the photoinactivation of K562 cells unchanged. Hypothermia reduced dye binding in K562 but not in L1210 cells, whereas the photogeneration of lipid hydroperoxides (LOOH) was affected in neither cell line. Post-PDT incubation at 4 degrees C delayed the decay of LOOH and enhanced the photoinactivation of CFU-GM as well as L1210 and K562 cells. Taken together, these results suggest that hypothermia interfered with the repair of potentially lethal photodynamic damage. They stress the importance of temperature control during and immediately after the photochemical purging of autologous bone marrow and peripheral blood stem cells.

Topics: Animals; Bone Marrow Cells; Bone Marrow Purging; Bone Marrow Transplantation; Cell Survival; Cold Temperature; Dose-Response Relationship, Radiation; Female; Fluorescent Dyes; Hematopoietic Stem Cell Transplantation; Humans; Kinetics; Leukemia; Leukemia L1210; Lipid Peroxidation; Lipid Peroxides; Mice; Mice, Inbred DBA; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Short-term exposure to the alkyl-lysophospholipid, rac-2-methyl-1-octadecyl-glycero-(3)-phosphocholine (ET-18-OCH3) or the photosensitizing dye, Merocyanine 540 (MC540) and light kills a wide range of leukemia and some solid tumor cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells as well as certain classes of committed progenitor cells. Both ET-18-OCH3 and MC540-mediated photodynamic therapy (PDT) have been used as purging agents in preclinical models of autologous hematopoietic stem cell transplantation and are currently undergoing phase I/II clinical testing for the same purpose. We report here that ET-18-OCH3 synergistically enhances the MC540-mediated photoinactivation of leukemia cells but only minimally reduces the survival of normal granulocyte-macrophage progenitors. Therapeutic indices are most favorable when MC540-mediated PDT precedes incubation with ET-18-OCH3 and when purging is followed by cryopreservation. Taken together, these data suggest that combination purging with alkyl-lysophospholipid and MC540-mediated PDT may provide a simple, versatile, and effective means of eliminating large numbers of leukemia cells from autologous bone marrow grafts without causing excessive damage to normal hematopoietic stem cells.

Topics: Animals; Antineoplastic Agents; Blood Component Removal; Cell Death; Combined Modality Therapy; Drug Synergism; Hematopoietic Stem Cell Transplantation; Humans; Leukemia; Mice; Phospholipid Ethers; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

Although several lines of investigation demonstrate that many heavy metals are cytotoxic to host defense cells, the mechanism of killing is poorly understood. The major focus of this investigation was to determine if organic mercuric compounds kill human lymphocytes by inducing the cells to undergo apoptosis and to evaluate possible flow cytometric systems for assessing cell death. T-cells were exposed to 0.6-5 microM MeHgCl, EtHgCl, or PhHgCl for up to 24 hr and then analyzed by flow cytometry. Mercury-treated cells exhibited increased Hoechst 33258 and 33342 fluorescence while maintaining their ability to exclude the vital stain 7-AAD. Furthermore, T-cells exposed to mercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side scatter. The light scatter and fluorescent changes were consistent with changes that cells display during apoptosis. To further evaluate cell death and to distinguish between apoptosis and necrosis, merocyanine 540 staining and annexin V binding to the plasma membrane as well as DNA fragmentation were assessed. Mercury-treated cells exhibited increased merocyanine 540 fluorescence and annexin V binding along with changes in nuclear morphology consistent with the notion of apoptosis. Conventional agarose gel electrophoresis failed to demonstrate low-molecular-weight DNA bands; however, when probed by flow cytometry using both nick translation and a modified TUNEL assay, patterns consistent with nuclear fragmentation were evident. We noted that the percentage of T-cells undergoing apoptosis was dependent upon the amount of serum present in the medium; as serum concentrations were increased from 0 to 10%, cell death declined. Apoptosis (33%) was observed within 1 hr of exposure to MeHgCl; maximum cell death (67%) occurred after 24 hr exposure. Induction of apoptosis was dependent on the mercury concentration and independent of the hydrophobicity of the mercury ligand. Finally, we assessed mercury-dependent apoptosis in activated T-cells. When treated with mitogen, mercury failed to induce apoptosis in these cells. Indeed, there was no evidence of either apoptosis nor necrosis in these populations. It was concluded that the activation process prevented development of a metabolic state that was required for induction of apoptogenic genes.

Topics: Annexin A5; Apoptosis; Cell Death; Cell Membrane; Cells, Cultured; DNA; DNA Fragmentation; Electrophoresis, Agar Gel; Flow Cytometry; Fluorescent Dyes; Humans; Lymphocyte Activation; Membrane Lipids; Microscopy, Fluorescence; Organomercury Compounds; Phytohemagglutinins; Pyrimidinones; T-Lymphocytes

The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cytometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12% vs 80% of PI-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells.

Topics: Antineoplastic Agents; Antiviral Agents; Bone Marrow Cells; Cell Survival; Fluorescent Dyes; Humans; In Vitro Techniques; Leukemia; Leukocytes, Mononuclear; Monocytes; Photochemistry; Photosensitizing Agents; Pyrimidinones; Quaternary Ammonium Compounds; Radiation Tolerance

Photodynamic action of merocyanine 540, an antileukemic sensitizing dye, on murine L1210 cells results in the formation of lipid hydroperoxides and loss of cell viability. High-performance liquid chromatography with mercury cathode electrochemical detection was used for determining lipid oxidation products, including the following cholesterol-derived hydroperoxides: 5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, and unresolved 7 alpha, 7 beta-OOH. Among these species, 5 alpha-, 6 alpha-, and 6 beta-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas 7 alpha- and 7 beta-OOH (products of free radical reactions) became so after prolonged irradiation or during dark incubation after exposure to a light dose. These mechanistic changes were studied in a unique way by monitoring shifts in the peroxide ratio, i.e., 7-OOH/5 alpha-OOH, or 7-OOH/6-OOH. When cells (10(7)/ml) were exposed to a visible light fluence of 0.6 J/cm2 in the presence of 10 microM merocyanine 540, 7-OOH/5 alpha-OOH increased by approximately 100% after 2 h of dark incubation at 37 degrees C. The increase was much larger (approximately 250%) when cells were photooxidized after treatment with 1 microM ferric-8-hydroxyquinoline, a lipophilic iron donor, whereas no increase was observed when cells were pretreated with 100 microM desferrioxamine, an avid iron chelator/redox inhibitor. Correspondingly, postirradiation formation of thiobarbituric acid-reactive material was markedly enhanced by ferric-8-hydroxyquinoline and suppressed by desferrioxamine, as was the extent of cell killing. When added to cells after a light dose, chain-breaking antioxidants such as butylated hydroxytoluene and alpha-tocopherol strongly protected against cell killing and slowed the increase in 7-OOH/5 alpha-OOH ratio. It is apparent from these results that (1) the 7-OOH/5 alpha-OOH or 7-OOH/6-OOH ratio can be used as a highly sensitive index of singlet oxygen vs. free radical dominance in photodynamically stressed cells; and (2) that postirradiation chain peroxidation plays an important role in photodynamically initiated cell killing.

Topics: Animals; Butylated Hydroxytoluene; Cell Survival; Cholesterol; Chromatography, High Pressure Liquid; Deferoxamine; Free Radicals; Iron Compounds; Leukemia L1210; Light; Lipid Peroxidation; Lipid Peroxides; Mice; Photosensitizing Agents; Pyrimidinones; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Tumor Cells, Cultured

Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6-diphenyl-1,3,5-hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5-mon) and old (24-mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n-3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet-modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA-induced increases in H+ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n-3 fatty acids. The experiments reported here indicate that incorporation of long-chain polyunsaturated n-3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.

Topics: Aging; Animals; Dietary Fats, Unsaturated; Docosahexaenoic Acids; Female; Fluorescent Dyes; Liposomes; Male; Membrane Fluidity; Membrane Fusion; Membrane Lipids; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Mitochondria, Liver; Oxygen Consumption; Permeability; Phospholipids; Protons; Pyrimidinones; Spectrometry, Fluorescence

The binding of merocyanine 540 (MC540) to murine myeloid leukemia (M1) cells and normal erythrocytes was measured by fluorescence digital imaging microscopy using an intensified charge-coupled device. It was found that, on average, about three times more MC540 were bound to a unit membrane area of M1 cells than erythrocytes, a result consistent with previous studies. However, it was shown for the first time that MC540 binding varied significantly from one M1 cell to the next, and about 15% of the sensitized M1 cells were as MC540-negative as normal erythrocytes. Using the leukemic inhibitory factor as a differentiation inducer, M1 cells were induced to differentiate into mature macrophage-like cells in vitro. Such treatment lowered the average MC540 binding by about one-third but did not affect the cell-to-cell variation significantly.

Topics: Animals; Erythrocytes; Kinetics; Leukemia, Myeloid; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Phenotype; Photosensitizing Agents; Pyrimidinones; Spectrometry, Fluorescence; Tumor Cells, Cultured

The molecular basis of the differential sensitivity of normal hematopoietic stem cells and of leukemia, lymphoma, and neuroblastoma cells to merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) is not yet completely understood. While the capacity to bind dye molecules appears to be the major determinant of a cell's susceptibility of MC540-mediated PDT, we here present evidence that under certain experimental conditions a cell's capacity to repair MC540-mediated photodynamic damage is also an important factor. Two parameters, temperature and intracellular glutathione (GSH) content, were varied to investigate the role of cellular defense mechanisms in the dye-sensitized photoinactivation of normal murine granulocyte/macrophage progenitors (CFU-GM) and K562, L1210, and melphalan-resistant L1210/L-PAM1 leukemia cells. When exposed to MC540 and light at room temperature, the three leukemia cell lines bound similar amounts of dye and accumulated similar amounts of lipid hydroperoxide (LOOH) but differed markedly in their sensitivity to MC540-mediated PDT. Performing MC540-mediated PDT at 4 degrees C instead of at room temperature reduced dye binding and LOOH generation and enhanced cytotoxicity in some but not all cell lines. A brief (< or = 120 minutes) incubation at 37 degrees C immediately following MC540-mediated PDT accelerated the decay of LOOH in all leukemic cell lines and reduced cell kill by about 2 log in both CFU-GM and leukemia cells. The effect of post-PDT incubation at 37 degrees C on LOOH decay was most pronounced in K562 and least pronounced in L1210/L-PAM1 cells, whereas its effect on cell survival was less pronounced in L1210 cells than in the remaining cell types. L1210/L-PAM1 cells whose GSH content had been reduced from 8.2 to 1.6 micrograms/mg protein by incubation with buthionine sulfoximine recovered from potentially lethal photodynamic damage as rapidly as untreated L1210/L-PAM1 cells and more rapidly than wild-type L1210 cells with a GSH content of 4.5 micrograms/mg protein. Thus, with regard to capacity of L1210/L-PAM1 cells to recover from photodynamic damage, the cells' enhanced capacity to synthesize GSH appeared more decisive than intracellular GSH levels per se. Taken together, these data suggest that temperature-dependent cellular defense mechanisms are significant determinants of a cell's susceptibility to MC540-mediated PDT. The data emphasize the need for temperature control during and immediately after the photochemi

Topics: Animals; Bone Marrow; Bone Marrow Cells; Bone Marrow Purging; Bone Marrow Transplantation; DNA Damage; DNA Repair; Hematopoietic Stem Cells; Humans; Leukemia L1210; Lipid Peroxides; Mice; Photochemotherapy; Pyrimidinones; Temperature; Tumor Cells, Cultured

Many parallel processes occur during the final stages of apoptosis. It is not clear which of these processes occur in all or most models of apoptosis and which occur only in some. In addition, the temporal relationship of these events is not always well understood. Correlated flow cytometric measurements were used to address these questions. Several models of apoptosis were studied, including thymocytes treated with dexamethasone. MOLT-4 cells treated with etoposide, U937 cells treated with anti-Fas, HL-60 cells treated with camptothecin, Raji cells grown in low serum, and aged neutrophils. All models showed a decrease in LDS-751 and fluorescein diacetate (FDA) staining, an increase in staining with dihydrorhodamine 123 (dhR123) or dihydroethidium, and an acidification of the cytoplasm. In each model, these changes were highly correlated, appearing simultaneously as multiparameter measurements. Changes in membrane status detected with merocyanin 540 (MC540) and annexin V behaved differently. A population with LDS-751 and FDA changes but without annexin V or MC540 changes could be demonstrated in some models. Several models did not show any change in annexin V binding, and HL-60 did not show a change in MC540 binding during apoptosis. The loss of cell surface antigens (CD45 and CD16) from aged neutrophils occurred in the entire LDS-751 and FDA dim population, even though other membrane changes (including the appearance of annexin V binding sites) were only apparent in a subset of these cells. These results suggest a model for the ordering of some of the terminal processes in apoptosis, with annexin V and MC540 changes trailing other events in apoptosis. These results confirm the need for caution in using a single-parameter measurement as an indicator of apoptosis for any new model being studied.

Topics: Animals; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Camptothecin; Cell Separation; Dexamethasone; Ethidium; Etoposide; Flow Cytometry; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Models, Biological; Neutrophils; Pyrimidinones; Rhodamines; Thymus Gland; Tumor Cells, Cultured

Toxicity of merocyanine 540 (MC540) was assessed in long-term (Dexter-type) bone marrow cultures and in a short-term in vitro clonal assay of fibroblast colony-forming cells (CFU-F). Exposure of freshly explanted mouse bone marrow cells to MC540 (15 micrograms/ml) and white light (fluence: 126 kJ/m2) reduced CFU-F by approximately 2 logs but did not abrogate the cells' capacity to establish and maintain long-term bone marrow cultures. Fat cells were rare or absence in adherent layers established with photosensitized bone marrow cells but the cultures' capacity to generate non-adherent cells, granulocyte/macrophage progenitors (CFU-GM), and early erythroid progenitors (BFU-E) was only moderately (28-36%) reduced.

Topics: Animals; Bone Marrow Purging; Bone Marrow Transplantation; Cells, Cultured; Hematopoietic Stem Cells; Mice; Photosensitizing Agents; Pyrimidinones; Stromal Cells; Transplantation, Autologous

The effect of merocyanine 540 (Mc 540) mediated photoirradiation on both neoplastic and normal hemopoietic progenitor cells was studied. Bone marrow (BM) cells from children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) at initial diagnosis, ALL in remission, neuroblastoma and normal children as well as cells of Reh-6 and HL-60 cell lines were incubated with Mc 540 in the presence of human albumin (HA) and exposed to different argon laser 514 nm doses. Cell survival was estimated using Trypan Blue supravital stain following a 24-h incubation and leukemic cell lines were studied in continuous cell cultures of 4 weeks duration. Our results showed that HA protects normal BM cells from Mc 540 mediated phototoxicity. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal BM cells was 77.4 +/- 12 and 70.3 +/- 10%, respectively. BM leukemic cells from children with ALL and AML were also very sensitive to Mc 540 photoirradiation in contrast to neuroblastoma cells where only a three-fold reduction was observed. Finally, the survival of normal BM progenitors was 38% for colony forming unit erythroid CFU-E, 37% for burst forming unit erythroid BFU-E, 55% for CFU-GM and 29% for CFU-GEMM. In conclusion it seems that Mc 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in BM.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Purging; Cell Line; Cell Survival; Child; Dose-Response Relationship, Radiation; Hematopoietic Stem Cells; HL-60 Cells; Humans; Lasers; Leukemia, Myeloid, Acute; Neuroblastoma; Photolysis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidinones; Radiation-Sensitizing Agents

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

Topics: Animals; Antineoplastic Agents; Clone Cells; Coloring Agents; Crystallography, X-Ray; Dequalinium; Humans; Mice; Mice, Nude; Models, Chemical; Models, Molecular; Neoplasm Transplantation; Pyrimidinones; Rhodanine; Structure-Activity Relationship; Tumor Cells, Cultured

Parameters of natural and probe fluorescence of peripheral blood lymphocytes have been studied in 25 normal persons and 72 patients suffering from extensive forms of pulmonary tuberculosis who live in various radioecological conditions. It was found that in cell suspension the intensity of fluorescence of protein and the membrane-bound merocyanine 540 dye was 2.0-3.2 and 3.9-7.1 times higher, respectively, for the lymphocytes of the examined patients. For lymphocytes of the patients who live in radionuclide-contaminated areas fluorescence of reduced pyridine nucleotides and some fluorescent probes (ANS, DPGT, the ratio of eximer to monomer fluorescence of pyrene) and the merocyanine-photosensitized cell death were also found to increase. These can indicate some changes of cell membranes and reduction of the oxidation resistance of lymphocytes. There was not any substantial dependence on the changes in the parameters determined in the patients calculated radiation doses was revealed. It was found that biophysical tests, in particular, photosensitized cell death, are informative for estimation of severity of the tuberculous process and prognosis of its outcome in patients who live in adverse radioecological conditions.

Topics: Adult; Anilino Naphthalenesulfonates; Diphenylhexatriene; Fluorescence; Fluorescent Dyes; Humans; Lymphocytes; Male; Middle Aged; Photosensitizing Agents; Power Plants; Pyrimidinones; Radioactive Hazard Release; Republic of Belarus; Tuberculosis, Pulmonary; Ukraine

Mechanism of merocyanine 540 (MC540) mediated photosensitization in glioblastoma (U-87MG) and neuroblastoma (Neuro 2a) cells was investigated. Photoinduced lipid peroxidation was measured in the presence of mechanistic probes-deuterium oxide (D2O), sodium azide, superoxide dismutase (SOD), mannitol and sodium benzoate. In both the types of cells, the photoinduced lipid peroxidation was enhanced in D2O whereas it showed inhibition in the presence of sodium azide. SOD also inhibited the lipid peroxidation while sodium benzoate and mannitol had no effect. These results suggest that photosensitization of U-87MG and Neuro 2a cells by MC 540 involves both type I (free radical mediated) and type II (singlet oxygen mediated) mechanisms.

Topics: Animals; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Lipid Peroxidation; Mice; Neuroblastoma; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

Alterations in phospholipid metabolism in blood elements have been proposed as the possible biochemical marker of schizophrenia. In the present study, we investigated the composition and membrane distribution of phospholipids in platelets of drug-free schizophrenic patients and controls. We have demonstrated that platelets of drug-free schizophrenics have significantly higher cytosolic Ca2+ levels in comparison with healthy controls. Platelets of drug-free schizophrenic patients have a lower content of phosphatidylinositol (PI). After thrombin activation, PI is the target of phospholipase C instead of phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed in platelets of controls. Alterations in the distribution of phospholipids were found in the plasma membrane of platelets of schizophrenic patients. We suggest that alterations in phospholipid metabolism might be evoked by a disturbance of calcium homeostasis in schizophrenic patients.

Topics: Adult; Blood Platelets; Calcium; Female; Flow Cytometry; Fluorescent Dyes; Homeostasis; Humans; Male; Phosphatidylinositol 4,5-Diphosphate; Phospholipids; Pyrimidinones; Schizophrenia; Thrombin

The interaction of the red cell membrane with merocyanine 540 or protoporphyrin led to four phenomena, most probably interrelated. (i) The morphology changed from the normal discoid to an echinocytic form. This morphological change persisted when followed over a period of 24 h. (ii) Simultaneously, cell deformability was decreased, as revealed by viscosity measurements and a cell-filtration technique. (iii) Both drugs caused swelling of the erythrocytes in isotonic medium, due to a very-short-term increased permeability of the membrane, also for larger molecules such as lactose. The pathway of this temporary leak seems to be unrelated to the Na+/K+ -ATPase, the K+/Cl- and the Na+/K+/Cl- cotransport systems, the Ca2+-activated Gardos pathway, the oxidation/deformation-activated leak pathway and the so-called residual transport route. Despite the morphological changes, K+-leakage induced by mechanical stress was not increased. (iv) During osmotic swelling, the critical hemolytic volume was found to be increased in the presence of either merocyanine 540 or protoporphyrin. The increase critical volume protected erythrocytes against osmotic hemolysis.

Topics: Antiviral Agents; Cell Membrane Permeability; Chemical Phenomena; Chemistry, Physical; Erythrocyte Deformability; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Osmosis; Phospholipids; Photosensitizing Agents; Protoporphyrins; Pyrimidinones; Viscosity

A procedure is described for the dual staining of lymphocytes with Hoechst 33342 (Ho342) to examine cell cycle position, and merocyanine 540 (MC540) that allows for the analysis of cells entering the early stages of apoptosis. Ho342 is a DNA specific dye and MC540 detects membrane phospholipid domain changes, some of which are associated with apoptotic cells. Flow analysis of B cells dually stained with Ho342 and MC540 allows for the discrimination of five distinct subpopulations. Two of these subpopulations represent viable, MC540 negative/dull cells with either 2n or 4n DNA. As 2n and 4n DNA B cells become MC540 bright they move into two distinct subpopulations representing cells entering and progressing through the early stages of apoptosis. As the apoptotic, MC540 bright cells move into the latter stages of apoptosis, they localize into a fifth subpopulation displaying reduced staining with Ho342 indicative of late stage apoptotic cells in the process of fragmenting their DNA. This experimental approach enables the characterization of lymphocyte populations for percentages of viable, early apoptotic, and late apoptotic cells. The cells are not fixed during this procedure, and since both dyes are viable dyes there is an additional opportunity to obtain sorted cells from any of the defined subpopulations for reculturing and functional analysis.

Topics: Animals; Apoptosis; B-Lymphocytes; Benzimidazoles; Cell Cycle; Cell Survival; Female; Fluorescent Dyes; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Pyrimidinones; Spleen; Staining and Labeling; Tumor Cells, Cultured

In order to evaluate the selective killing of merocyanine 540 (MC 540) mediated photoirradiation in neoplastic cells, bone narrow cells from children with leukaemia or neuroblastoma and normal children as well as peripheral blood cells and Reh-6 and HL-60 cell lines were studied. Cell suspensions were incubated with MC 540 and exposed to various argon laser 514 nm doses. Cell survival was estimated with trypan blue supravital stain following a 24 h incubation and has been followed in continuous cell cultures of 4 weeks duration. Our results showed that the inhibition of survival of neoplastic haemopoietic cells by laser in the presence of MC 540 is proportional to the MC 540 and photoirradiation doses. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal bone narrow cells was (33.6 +/- 15.5)% and (50.6 +/- 10.7)% respectively. Peripheral blood mononuclear cells were not sensitive to MC 540 mediated photoirradiation. The inhibition of survival of bone marrow metastatic neuroblastoma cells was (69.9 +/- 4.1)%. In conclusion, it seems that MC 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in the bone marrow.

Topics: Bone Marrow; Cell Line; Cell Survival; Child; Dose-Response Relationship, Radiation; Hematopoietic Stem Cells; HL-60 Cells; Humans; Kinetics; Leukemia; Light; Neuroblastoma; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

The antitumor activity of pMC540 has been shown to be mediated via its interaction with topoisomerase (Topo) II eventually leading cells into apoptosis. This agent was also found to down regulate the expression of the c-myc oncogene in L1210 leukemia cells. To investigate the possibility that damage within select genomic regions may contribute to the antiproliferative activity of pMC540, differential damage in regions surrounding the c-myc locus as well as other select genes was determined. Southern blot hybridization experiments show that pMC540 treatment induces in situ DNA cleavage products in the 5' end of the c-myc oncogene of L1210 leukemia cells. In cells pre-treated with 50 microM ethidium bromide, an inhibitor of the Topo II-dependent DNA cleavage, a subsequent treatment with pMC540 failed to induce DNA cleavage, suggesting that the cleavage activity of pMC540 was Topo II dependent. pMC540-induced cleavage does not appear to correlate with the over-expression of the c-myc oncogene in these cells as another over-expressed gene c-myb was not affected. Thus, it is proposed that the c-myc gene may be a preferred target for pMC540 may mediated antiproliferative activity.

Topics: Animals; Antineoplastic Agents; Blotting, Southern; DNA Topoisomerases, Type II; Ethidium; Genes, myc; Mice; Pyrimidinones; Tumor Cells, Cultured

Merocyanine 540 (MC540)-mediated photodynamic action is a novel approach for purging tumor cells from autologous remission bone marrow explants. The purpose of this study was to evaluate the effects of hemin (ferriprotoporphyrin IX), a potential source of pro-oxidant iron in bone marrow, on in vitro photodynamic inactivation of leukemia cells. Murine L1210 cells exhibited a progressive loss of clonogenicity when irradiated with broad-band visible light in the presence of MC540. Hemin had strikingly different effects on photokilling, depending on its contact time with cells, eliciting a sizable decrease in resistance after short-term (30-min) contact but a marked increase in resistance after long-term (24-h) contact. Similar trends were observed when cells were challenged with glucose/glucose oxidase, indicating that the responses apply to more than one type of oxidative stress. Immunoblot analyses revealed that the levels of inducible heme oxygenase (HO-1) and ferritin heavy (H) chain were substantially elevated 24 h after hemin addition. HO-1 increased relatively rapidly and maximized within 4 h after adding hemin, whereas H-ferritin increased more slowly in parallel with the development of hyperresistance, maximizing after 24-36 h. Desferrioxamine, an avid iron chelator, had no effect on HO-1 induction but inhibited both ferritin induction and the increase in cell resistance, suggesting that HO-mediated release of iron from hemin was necessary for triggering these responses. Spleen apoferritin was taken up by L1210 cells and strongly inhibited photokilling, further implicating ferritin involvement in hyperresistance. Photokilling was accompanied by free radical-mediated lipid peroxidation (thiobarbituric acid reactivity), which could be suppressed substantially by 24-h hemin preincubation. A plausible explanation for the long-term effects of hemin is that excess H-ferritin generated as a result of iron-regulatory protein deactivation sequesters toxic iron, which might otherwise catalyze damaging lipid peroxidation. Chronic oxidative release of hemin from bone marrow erythroid cells could compromise the efficacy of photopurging by making tumor cells more tolerant to photooxidative insult.

Topics: Animals; Antidotes; Apoferritins; Cell Survival; Deferoxamine; Drug Resistance; Enzyme Induction; Ferric Compounds; Heme Oxygenase (Decyclizing); Hemin; Hydrogen Peroxide; Hydroxyquinolines; Leukemia L1210; Lipid Peroxidation; Photochemotherapy; Photosensitizing Agents; Protoporphyrins; Pyrimidinones; Thiobarbituric Acid Reactive Substances

Merocyanine dyes with an oxygen in the electron donor heterocycle were rapidly degraded by plasma, serum and serum components. Replacement of the oxygen by a sulfur or selenium atom rendered the dyes refractory to degradation. The degradation of labile merocyanine dyes was temperature dependent and oxygen independent. The plasma component that was responsible for the degradation of merocyanine dyes was sensitive to heat and detergent, suggesting an enzymatic process. The identification of the structural requirements for sensitivity/resistance to degradation provides the experimenter with a simple means to manipulate the stability of merocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosensitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.

Topics: Blood Proteins; Fluorescent Dyes; Humans; Photosensitizing Agents; Pyrimidinones

The techniques of differential scanning calorimetry, fluorescence of merocyanine 540, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, proton permeability, and lipid peroxidation are used to compare the perturbations of cholesterol and alpha-tocopherol on lipid bilayer membranes composed of different phosphatidylcholines containing stearic acid in the sn-1 position and an unsaturated fatty acid (either oleic, alpha-linolenic, gamma-linolenic, or docosahexaenoic acid) in the sn-2 position. It is concluded that the structural roles of cholesterol and alpha-tocopherol may be similar with membranes composed of some phosphatidylcholines but are clearly different with membranes composed of other related phosphatidylcholines. alpha-Tocopherol exerts a much larger effect than cholesterol on membranes rich in polyunsaturated fatty acids that have their initial double bond before the delta 9 position. Cholesterol interacts more favorably with fatty acids that do not have an double bond before the delta 9 position. The membrane structural effects are explained in terms of the larger size of the sterol ring structure of cholesterol compared to the smaller chromanol ring of the alpha-tocopherol.

Topics: Calorimetry, Differential Scanning; Cholesterol; Diphenylhexatriene; Docosahexaenoic Acids; Fluorescence Polarization; Fluorescent Dyes; Lipid Bilayers; Lipid Peroxidation; Macromolecular Substances; Molecular Structure; Oleic Acid; Permeability; Phosphatidylcholines; Protons; Pyrimidinones; Spectrometry, Fluorescence; Thermodynamics; Vitamin E

Bicarbonate/CO2 is believed to be the key in vitro effector of sperm capacitation, a process which induces major changes in the sperm plasma membrane in preparation for fertilization. In a flow cytometric study, we examined the effect of bicarbonate on boar spermatozoa using merocyanine, an impermeant lipophilic probe which binds to plasma membranes with increasing affinity as their lipid components become more disordered. We found that bicarbonate causes a rapid increase in the ability of live boar spermatozoa to bind merocyanine. First detected about 100 sec after exposure to bicarbonate and largely complete by 300 sec, this increase appears to result from individual cells within the sperm population switching from a low merocyanine-binding state to a high binding state. The majority of live spermatozoa are capable of responding in this way, and do so in proportion to bicarbonate concentration, half-maximal response being induced by about 3 mM bicarbonate; however, overall population response varies greatly between ejaculates. Increased merocyanine stainability is observed over the whole surface area of the cell, and is reversible both with respect to temperature (it is only manifested above 30 degrees C) and with respect to presence of bicarbonate. A similar effect can be induced by phosphodiesterase inhibitors such as isobutylmethylxanthine, and enhanced by a permeant cyclic nucleotide analogue. We conclude that bicarbonate causes a major alteration in sperm plasma membrane lipid architecture, apparently by perturbing enzymic control processes. This novel action of bicarbonate may represent an initial permissive event in the capacitation sequence.

Topics: Animals; Bicarbonates; Calcium; Carbon Dioxide; Cell Membrane; Dose-Response Relationship, Drug; Lipid Metabolism; Male; Membrane Potentials; Nucleotides, Cyclic; Pyrimidinones; Semen; Serum Albumin, Bovine; Sperm Capacitation; Spermatozoa; Staining and Labeling; Swine

Osmotic shrinkage changes the surface properties of dipalmitoylphosphatidylcholine large unilamellar vesicles depending on the phase state of the bilayer. In the gel state, shrinkage produces an increase in the adsorption of hydrophobic dyes, such as Merocyanine 540 (MC540) monomers, toluidine and anilinonaphthalene sulfonic acid (TNS, ANS). In the fluid state, shrinkage does not affect the bilayer surface when gradients between the inner and the outer compartments below 0.2-0.25 M NaCl (higher concentration outside) are applied. Larger differences in concentrations produce an increase in packing as inferred from the desorption of the MC monomers. Kinetic experiments show that the surface changes correlate with the volume decrease produced by the water extrusion from the vesicle interior. It is interpreted that the decrease of water content compels the vesicles to a state in which defects at the membrane surface are likely to occur when the bilayer is in the gel state.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Coloring Agents; Diphenylhexatriene; Fluoresceins; Fluorescence Polarization; Fluorescent Dyes; Gels; Kinetics; Lipid Bilayers; Liposomes; Nephelometry and Turbidimetry; Osmosis; Particle Size; Pyrimidinones; Sodium Chloride; Spectrophotometry; Surface Properties; Temperature; Water

Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bone Marrow Transplantation; Drug Synergism; Female; Hematopoietic Stem Cells; Leukemia L1210; Light; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Oxygen; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Salicylates; Salicylic Acid; Serum Albumin, Bovine; Singlet Oxygen; Tumor Cells, Cultured

Previously, we have reported on the increase in procoagulant activity of human umbilical vein endothelial cells (HUVEC) after infection with human cytomegalovirus (HCMV). When using microvascular endothelial cells from foreskin (MVEC), we also observe a significant increase in membrane perturbation and a concomittant increase in procoagulant activity. This effect is both observed with a laboratory HCMV strain (AD169) with low pathogenicity for endothelium and a HUVEC adapted strain (VHL-E) that readily infects endothelial cells. We compared the membrane perturbation of two types of endothelial cells, HUVEC and MVEC with human embryonal fibroblasts (HEF), being fully permissive for both strains. A membrane effect was only found in endothelial cells. Our results suggest that HCMV induces in MVEC more merocyanine-540 incorporation in the membrane as in HUVEC. The increase in the procoagulant activity induced by HCMV was more pronounced in MVEC than in HUVEC. Inactivated virus, as well as virus pre-incubated with heparin was unable to evoke membrane perturbation. It therefore appears that HCMV induces a rapid membrane response in vascular endothelium and that physical interaction of the virion and the endothelial cell is required to elicit this response.

Topics: Blood Coagulation; Cell Membrane; Cells, Cultured; Cytomegalovirus; Endothelium, Vascular; Female; Humans; Male; Microcirculation; Pyrimidinones

The lateral organization of fluid cholesterol-dimyristoylphosphatidylcholine (DMPC) bilayers was studied by measuring the response of fluorescent membrane probes, dipyrenylphosphatidylcholines (diPyrxPCs) or merocyanine 540, to the variation of cholesterol concentration. Parallel absorbance and light-scattering measurements were also carried out. The excimer-to-monomer ratio of diPyrxPCs displayed abrupt deviations at particular cholesterol mole fractions (CMFs). The most notable of these occurred at CMFs of 0.15, 0.33, and 0.67. Deviations were also frequently observed at CMFs of 0.12, 0.20, 0.25, and 0.40. Merocyanine 540 reproducibly reported deviations at CMFs of 0.15 and 0.33 and frequently reported values close to 0.12, 0.20, and 0.25. In absorbance (turbidity) and light scattering versus CMF plots, well-defined kinks were observed at CMFs of 0.16, 0.33, 0.52, and 0.67. The occurrence of kinks or other deviations at those particular CMFs is most readily explained in terms of a superlattice model previously developed to explain the lateral distribution of pyrenylphospholipids in bilayers [Somerharju, et al. (1985) Biochemistry 24, 2773-2781; Virtanen, J. A., et al. (1988) J. Mol. Electron. 4, 233-236]. This model is based on the assumptions that (i) each cholesterol molecule replaces a single acyl chain in a hexagonal lattice, (ii) cholesterol molecules, because of their larger size, perturb the lattice, (iii) this perturbation is minimized when the cholesterol molecules are maximally separated from each other, and (iv) the maximal separation is achieved when the cholesterol molecules form a hexagonal or centered rectangular superlattice. All detected critical CMFs, except that at CMF 0.67, are predicted by the model, thus strongly supporting its validity. The critical CMF at 0.67 is a limiting case, which can be accounted for by assuming that cholesterol and phospholipid molecules form alternating rows, i.e., formation of a cholesterol superlattice with rectangular symmetry. As predicted by the superlattice model, composition-driven order-to-disorder transitions occur between the critical CMFs, as indicated by increased data scatter and sample fluctuations in those regions. Another important prediction of the superlattice model is that domains with different cholesterol superlattices should coexist at most cholesterol concentrations. Such domains do not have to be extensive to account for the critical events observed here; rather, they are expected

Topics: Binding Sites; Cholesterol; Dimyristoylphosphatidylcholine; Fluorescent Dyes; Light; Lipid Bilayers; Liposomes; Molecular Structure; Phosphatidylcholines; Pyrimidinones; Scattering, Radiation; Spectrum Analysis

In previous studies we have reported that preactivated merocyanine 540 (pMC540) and its chemically synthesized isolates merocil and merodantoin mediate their preferential cytotoxicity towards certain types of malignant cells including human breast cancer cells in vitro and in vivo. The mechanism of cytotoxic action appears to be, in part, via initial interaction with topoisomerase II leading to apoptosis. To further build upon these findings we now show that pMC540 and merodantoin disrupt mitochondrial morphology and function in intact MCF-7 human breast cancer cells as seen by their causing the release of rhodamine 123 from prestained cells, a rapid reduction in ATP levels, inhibition of succinate dehydrogenase activity and oxygen consumption. These data suggest that mitochondria may also be an important target for the cytotoxic action of pMC540 and merodantoin mediated through disruption of the energy balance.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Breast Neoplasms; Cell Survival; Ethylenethiourea; Fluorescent Dyes; Humans; Microscopy, Electron; Microscopy, Fluorescence; Mitochondria; Oxygen Consumption; Pyrimidinones; Rhodamine 123; Rhodamines; Succinate Dehydrogenase; Tumor Cells, Cultured

In the presence of albumin Merocyanine 540 (MC540) exhibits a very limited binding to the outer surface of the membrane of normal erythrocytes, whereas pronounced binding is observed to leukemia cells. To find out whether this difference is due to differences in the composition or structural organization of the cell membrane we analyzed effects of a number of covalent and non-covalent perturbations of the red cell membrane on the binding and fluorescence characteristics of membrane-bound MC540. It is shown that exposure of the cells to cationic chlorpromazine, neuraminidase or photodynamic treatment with AlPcS4 as sensitizer caused a limited increase (30-50%) of MC540 binding, together with a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield of membrane-bound MC540. Other forms of perturbation of the membrane structure, like hyperthermia (48 degrees C) and treatments that produce a decrease of phospholipid asymmetry in addition to accelerated flip-flop, did not result in increased MC540 binding, but did cause a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield. These changes in fluorescence properties indicate a penetration of the dye into more hydrophobic regions in the membrane. MC540, bound to Brown Norway myelocytic leukemia cells, exhibited a red shift of the fluorescence emission maximum and an increased relative fluorescence quantum yield as compared to MC540 bound to untreated erythrocytes. These changes were of the same order of magnitude as in photodynamically treated red blood cells. Dye binding per surface area, however, was about 3-times higher with these leukemia cells than with photodynamically treated red blood cells. This demonstrates that certain perturbations of the erythrocyte membrane evoked a MC540 binding that became qualitatively comparable to the dye binding to leukemia cells, although dye binding per surface area was still significantly lower.

Topics: Cell Membrane; Chlorpromazine; Cholesterol; Erythrocyte Membrane; Hot Temperature; Humans; Indoles; Leukemia, Myeloid; Light; Lipid Bilayers; Neuraminidase; Organometallic Compounds; Pyrimidinones; Radiation-Sensitizing Agents; Spectrometry, Fluorescence; Tumor Cells, Cultured

Mycoplasma infection can substantially affect the biological properties of cells in vitro. We have devised a method for the selective killing of mycoplasmas, e.g., A. laidlawii, M. fermentans, M. hyorhinis and M. arginini, from experimentally infected cell cultures. This approach is based on the differential binding of the lipophilic fluorescent probe Merocyanine 540 followed by illumination with visible light. The efficiency of the procedure depends on the Merocyanine 540 concentration, the intensity of illumination, and the presence of oxygen in the medium. When A. laidlawii contaminated corneal endothelial cell cultures were treated simultaneously with Merocyanine 540 and DNA-binding fluorochrome Hoechst 33258 and then illuminated, a significant degree of eradication was observed, even after one cycle of treatment. This combined treatment is therefore recommended as an effective method of purging mycoplasmas from contaminated cultures.

Topics: Animals; Bisbenzimidazole; Cells, Cultured; Culture Techniques; Endothelium, Corneal; Fluorescent Dyes; Microbiological Techniques; Mycoplasma; Photosensitizing Agents; Pyrimidinones; Vero Cells

Binding and photodynamic action of merocyanine 540 (MC540) has been studied in glioma (U-87MG) and neuroblastoma (Neuro 2A) cells as a function of dye concentration, incubation time of cells with MC540 and growth phase of cells. In the plateau phase, U-87MG cells accumulated more MC540 as compared to exponentially growing cells, whereas in Neuro 2A cells the opposite effect was observed. Exponentially growing U-87MG cells were more photosensitive than plateau phase cells. However, the photosensitivity of Neuro 2A cells was not dependent on the growth phase. Thus, MC540 mediated photosensitization may be useful for photodynamic therapy of brain tumours.

Topics: Fluorescent Dyes; Glioma; Neuroblastoma; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Staining and Labeling; Tumor Cells, Cultured

Merocyanine derivatives were prepared by structural alterations at the barbituric acid or chalcogenazole moieties. The photophysical properties of the dyes were markedly influenced by the presence of selenium rather than sulfur as a substituent at position 2 of the barbiturate. In methanol, quantum yields of both triplet state (phi T) and singlet oxygen sensitization (phi delta) were increased by over an order of magnitude, with a concomitant decrease in fluorescence, when selenium was present in the molecule. Photoisomerization, one of the dominant deactivation pathways in the sulfur- or oxygen-containing analogues, was completely absent in the selenium-containing derivatives. Efficient triplet state formation was observed for selenium-containing derivatives incorporated into L1210 cells by diffuse reflectance laser flash photolysis. Cytotoxicity studies, carried out using clonogenic assays on L1210 leukemia cells, showed a good correlation with phi T and phi delta, measured in solution. Experimental evidence provided by this paper supports a triplet state-, and probably singlet oxygen-, mediated phototoxic mechanism. Photoisomerization or singlet state mechanisms can be discounted.

Topics: Animals; Cell Survival; Lasers; Leukemia L1210; Mice; Photolysis; Photosensitizing Agents; Pyrimidinones; Quantum Theory; Selenium; Staining and Labeling; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Stem Cell Assay

Topics: Animals; Binding Sites; Bone Marrow Purging; Bone Marrow Transplantation; Hematopoietic Stem Cells; Humans; Neoplasms; Neoplastic Stem Cells; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Transplantation, Autologous

Absorption and fluorescence emission spectroscopy was applied to study the changes in albumin modified incorporation of merocyanine 540 into liposomes composed of different lecithins (DMPC, DPPC, POPC, and egg PC). Our results confirmed high affinity of merocyanine molecules toward albumin and revealed that albumin competed with all phospholipids used for binding merocyanine 540 molecules. However, the extent of this competition was determined by the kind of phospholipid. Albumin competed very successfully with lecithins containing saturated fatty acid chains (DPPC, DMPC) and weakly with unsaturated lecithins (POPC, egg PC) for binding merocyanine 540 molecules.

Topics: Albumins; Animals; Binding Sites; Binding, Competitive; Biophysical Phenomena; Biophysics; Cattle; Fluorescent Dyes; In Vitro Techniques; Liposomes; Phosphatidylcholines; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry

Two membrane-photosensitizing dyes were used to investigate whether selected sites in the plasma membrane vary in their sensitivity to damage by singlet oxygen (1O2*) and, if so, what factors are responsible for the variation. The relative ability of Rose bengal (RB) and merocyanine 540 (MC540), both of which localize in the plasma membrane and produce 1O2*, to photosensitize five plasma membrane functions in P388D1 cells was evaluated. The five membrane functions assessed were: plasma membrane potential, proline transport, facilitated glucose diffusion, 5'-nucleotidase activity, and dye exclusion. Photosensitization efficiency by RB varied by a factor of 188 for these membrane functions, whereas for MC540 a range of only 24 was found. RB was a more efficient photosensitizer than MC540 but the relative efficiencies varied with the membrane function. The wide range of P50 values for RB suggests that it binds selectively to membrane sites where it causes damage with high efficiency; possibly a non-1O2* mechanism is involved. In contrast, MC540 photosensitized the three membrane functions involving integral membrane proteins about equally suggesting that differences are due to small variations in the distribution of MC540 in the plasma membrane and/or variations in the inherent reactivity of the membrane targets with 1O2*. The results indicate that the lability of membrane sites to photosensitization depends both on their inherent reactivity with 1O2* and the relative location of specific protein and dye molecules.

Topics: 5'-Nucleotidase; Animals; Cell Line; Cell Membrane; Glucose; Light; Membrane Potentials; Mice; Photosensitizing Agents; Proline; Pyrimidinones; Rose Bengal

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits < 0.5 pmol for cholesterol hydroperoxides and < 50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12-15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (< 12 min) were assigned to the following species by virtue of comigration with authentic standards: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxycholest-5-ene-7 alpha/7 beta-hydroperoxide (7 alpha/7 beta-OOH). Formation of 5 alpha-OOH and 6 beta-OOH (single oxygen adducts) was confirmed by subjecting [14C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18-22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18-22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts of PCOOH and ChOOH at a light fluence that clonally inactivated < 10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.

Topics: Animals; Cholesterol; Chromatography, High Pressure Liquid; Fluorescent Dyes; Glutathione; Glutathione Peroxidase; Leukemia L1210; Lipid Peroxidation; Lipid Peroxides; Mice; Phosphatidylcholines; Phospholipases A; Photochemistry; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

Salicylate and several structurally analogous compounds enhance merocyanine 540 (MC540)-photosensitized killing of leukemia cells (M. A. Anderson, B. Kalyanaraman, and J. B. Feix, Cancer Res., 53: 806-809, 1993). In this work, we show that salicylic acid enhances the binding of MC540 prior to illumination, as well as the light-stimulated uptake of MC540 by target L1210 murine and K562 human leukemia cells. Acetylsalicylic acid, 2,3- and 2,5-dihydroxybenzoic acids, and sodium benzoate also enhance MC540 uptake. The irradiation dose responses for loss of cell survival and enhanced MC540 uptake are well correlated, both being shifted to earlier time points in the presence of salicylate. Salicylic acid also enhanced photodynamic cell killing of A549 lung carcinoma and NIH:OVCAR-3 ovarian carcinoma cells, two cell types which are relatively resistant to MC540-mediated photosensitization. Cellular uptake of the anionic, potential-sensitive oxonol dye, bis-(1,3-dibutylbarbituric acid)-trimethine oxonol, is also increased by salicylate in a dose-dependent fashion. In contrast, cellular uptake of the cationic cyanine dye, 3,3'-dihexyloxacarbocyanine, is unaffected by salicylate. These studies suggest that increased uptake of MC540 is the basis of salicylate enhancement and that changes in plasma membrane potentials may play a mechanistic role in the potentiation of MC540 binding and cell killing.

Topics: Animals; Benzoates; Benzoic Acid; Cell Survival; Drug Screening Assays, Antitumor; Drug Synergism; Fluorescence; Humans; Leukemia; Leukemia L1210; Mice; Neoplasms; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Salicylates; Salicylic Acid

Laser flash photolysis experiments were undertaken to investigate the interaction between stearic acid nitroxide spin probes and photoexcited merocyanine 540 (MC540) in dimyristoyl-L-alpha-phosphatidylcholine liposomes (membrane model). The measurements of the paramagnetic signal decay kinetics of four different spin-labelled stearic acids (n-DSA) show that the direct interaction between the dye and the probe is affected by the position of the nitroxyl group along the carbon chain. Laser flash photolysis results reveal a significant decrease in the MC540 triplet lifetime in the presence of n-DSA, the effect depending on the depth at which the nitroxyl moiety is localized in the bilayer. Previous results on the rate of disappearance of the electron spin resonance (ESR) nitroxide signal on continuous photolysis of the same systems do not show the same dependence on the localization of the nitroxyl moiety in the liposome. Although the MC540 triplet state seems to be implicated in the reaction process, the results suggest that ESR and laser flash experiments demonstrate two different kinds of mechanism.

Topics: Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Lasers; Liposomes; Photolysis; Photosensitizing Agents; Pyrimidinones; Spin Labels; Stearic Acids

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na+,K(+)-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na+,K(+)-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photo-inactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na+,K(+)-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na+,K(+)-ATPase, but not AChE. This is consistent with the fact that Na+,K(+)-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.

Topics: Animals; Glutathione; Glutathione Peroxidase; Leukemia L1210; Light; Lipid Peroxidation; Mice; Photochemistry; Photosensitizing Agents; Pyrimidinones; Selenium; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

Melittin fragments carrying spiropyran were synthesized, and their distribution in phospholipid bilayer membrane was studied by using spiropyran as a probe. Spiropyran was connected to the side chain of a Glu residue (Glu(OSp)), and the residue was replaced for the fourth position of melittin (1-7) fragment (M7Sp). M7Sp showed a high affinity for phospholipid membrane. The spiropyran group of M7Sp was converted to a merocyanin group by UV irradiation, which reduced the amount of the peptide bound to the membrane to the half of the initial amount. The location of the merocyanin group of M7Sp in the membrane was evaluated by the rate of thermal isomerization from merocyanin to spiropyran, which is sensitive to the microenvironment of merocyanin. A large fraction of the merocyanin group isomerized rapidly back to a spiropyran form, indicating that M7Sp is located in a relatively hydrophobic region of the membrane. Although the interaction of the peptide with phospholipid membrane is affected by photoisomerization of the spiropyran substituent, spiropyran was shown to be a useful tool to evaluate the location of the peptide in the lipid membrane.

Topics: Amino Acid Sequence; Benzopyrans; Chemical Phenomena; Chemistry, Physical; Fluoresceins; Fluorescent Dyes; Indoles; Isomerism; Kinetics; Melitten; Membranes, Artificial; Molecular Sequence Data; Nitro Compounds; Peptide Fragments; Phospholipids; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thermodynamics

Pretreatment of mammalian cells with certain genotoxic agents decreases the ability of the cell monolayers to support virus plaque formation but enhances repair of UV-irradiated virus. This study was made to determine whether these phenomena extend to pretreatments with light and photosensitizers, including one dye that primarily affects cell membranes. Confluent CV-1 monkey kidney fibroblast monolayers were pretreated with either gilvocarcin V (GV) or merocyanine 540 (MC540) and light of appropriate wavelengths and infected with control or UV-irradiated herpes simplex virus (HSV). GV phototreatment is known to affect cells at the DNA level, and MC540 at the membrane level. UV radiation served as a positive control pretreatment. Phototoxic concentrations of GV and MC540 were determined via the capacity of pretreated cell monolayers to support plaque formation by unirradiated HSV. Parallel monolayer pretreatment and subsequent infection by UV-irradiated HSV demonstrated that both types of phototreatments enhanced virus survival, but the dose responses and time courses were different. The DNA-damaging GV phototreatment mimicked the effect of UV-irradiating the cells and produced delayed enhanced repair of UV-irradiated virus. However, the MC540-phototreatment produced enhancement of virus survival with a bimodal dose response pattern for immediate infection, suggesting a different route for affecting repair of damaged virus.

Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Antiviral Agents; Cell Line; Chlorocebus aethiops; Coumarins; Dose-Response Relationship, Radiation; Glycosides; Kidney; Photosensitizing Agents; Pyrimidinones; Simplexvirus; Ultraviolet Rays; Viral Plaque Assay

The lipophilic dye merocyanine 540 (MC540) can photosensitize potentially lethal cell membrane damage as well as its own degradation (bleaching). Photobleaching in a test membrane, the human erythrocyte ghost has been examined. White light irradiation of MC540-sensitized ghosts resulted in lipid hydroperoxide (LOOH) formation, low-level thiobarbituric acid (TBA) reactivity, and dye bleaching (A568 decay). When the reaction was carried out in the presence of ascorbate (AH-), and added Fe3+, there was a large enhancement of TBA reactivity (indicative of free radical-mediated lipid peroxidation) and concomitant increase in the rate of photobleaching. Rapid bleaching also occurred when MC540 was incubated in the dark with ghosts that had been photoperoxidized with another dye (a phthalocyanine) and then exposed to AH-. The extent of bleaching in this system was found to be proportional to the starting level of LOOH. Like the wave of free radical lipid peroxidation that accompanied it, dye bleaching in AH(-)-treated, preperoxidized ghosts was stimulated by supplemental Fe3+, inhibited by desferrioxamine or butylated hydroxytoluene (BHT), but unaffected by catalase or superoxide dismutase. From this and related evidence, we deduce that: (1) in the absence of Fe3+/AH-, photoperoxidation and photobleaching occur independently and are nonradical, singlet oxygen-mediated processes; and (2) in the presence of Fe3+/AH-, 1-electron reduction of photogenerated LOOHs results in a surge of lipid peroxidation that amplifies dye loss via free radical processes. MC540 bleaching might be exploited as a relatively simple and sensitive indicator of lipid autoxidation in isolated membranes and cells.

Topics: Antioxidants; Ascorbic Acid; Erythrocyte Membrane; Ferric Compounds; Free Radicals; Humans; Lipid Peroxidation; Photochemistry; Photosensitizing Agents; Pyrimidinones; Thiobarbituric Acid Reactive Substances

Preactivation is a novel photochemical method for the production of chemotherapeutic compounds that exert their biologic effects independent of light. The compounds that are produced, preactivated merocyanine 540 (pMC540) and merodantoin, are cytotoxic to cultured human breast cancer cells but are only minimally cytotoxic toward normal cells. Their effects against breast cancer have not been studied in vivo.. Estrogen-stimulated human MCF-7 breast adenocarcinoma cells were grown as solid tumors in athymic carrier mice. Animals bearing defined sizes of subcutaneously transplanted solid breast tumors received injections of pMC540 (250 mg/kg) with or without concurrent treatment with tamoxifen. Growth inhibitory effects of merodantoin (N,N'-dibutyl-2-thio-4,5-imidazolidion) on the breast tumor growth were determined.. Direct injection of established tumors with eight doses of pMC540 (250 mg/kg) administered on alternate days resulted in significant tumor regression (P = 0.002). In three of seven animals, palpable tumors could not be detected after this treatment (16 days). Treatment through intramuscular injections (20 doses) with pMC540 (250 mg/kg) also caused a significant suppression of tumor area (P = 0.004; P = 0.0882; P = 0.0903) and a marginally significant suppression of tumor weight and volume, respectively. Combined treatment with tamoxifen and pMC540 (100 mg/kg) caused a 67% suppression of breast tumor growth. Treatment with 20 doses of merodantoin (75 mg/kg) suppressed the growth of breast tumors by 98%.. To the authors' knowledge, these results show for the first time that photochemically generated novel compounds in pMC540 alone and in combination with tamoxifen are effective in suppressing in vivo growth of xenografted human MCF-7 breast tumors.

Topics: Animals; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Ethylenethiourea; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Pyrimidinones; Tamoxifen; Transplantation, Heterologous

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) have been studied by visible absorption spectroscopy using native receptor-rich membranes from Discopyge tschudii electric tissue and liposomes obtained by aqueous dispersion of endogenous lipids extracted from the same tissue. The fact that merocyanine partitions into the membrane when this is in the liquid-crystalline state, exhibiting a characteristic peak at 567 nm, was exploited to obtain quantitative information about the physical state of the AChR-rich membrane. Spectra of MC540 revealed that this molecule was preferentially incorporated into AChR-rich membranes, with an affinity (Kdapp 30 microM) 10-fold higher than that in liposomes (Kdapp 290 microM). Changes were observed in the equilibrium dissociation constant of MC540 at different temperatures: the two-fold higher affinity at 8 degrees C than at 23 degrees C can be rationalized in terms of a higher value of the overall dimerization constant (Kdim) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher potency in AChR-rich native membranes than in liposomes made with endogenous lipids. This competition was found to be AChR concentration-dependent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent dissociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the benzocaine binding site is deduced from the competition experiments to be at the lipid annulus surrounding the nicotinic AChR protein.

Topics: Animals; Benzocaine; Binding, Competitive; Electric Organ; Liposomes; Membrane Proteins; Pyrimidinones; Receptors, Nicotinic; Temperature

Fluid shear stress is a ubiquitous stimulus of mammalian cell metabolism; however, its signal transduction pathway is unknown. We hypothesized that shear stress may alter some physical properties of the cell membrane. Using primary human umbilical vein endothelial cells (HUVECs), we investigated the effects of shear on the cell membrane by monitoring flow-induced changes in the uptake of the amphipath merocyanine 540 (MC540). Under static conditions, MC540 was rapidly internalized by HUVECs at 37 degrees C, and so was the membrane impermeant dye lucifer yellow, suggesting that the MC540 uptake was partly due to endocytosis. However, exposure to steady flow for 5 min at 37 degrees C induced an increase in MC540 uptake while that of lucifer yellow was unchanged, suggesting that the flow-induced increase in MC540 uptake was not endocytosis-related. The increase in MC540 uptake was significant for levels of steady shear of 6 dyne/cm2 and above. Pulsatile flow was more stimulatory than steady flow at 2 dyne/cm2, but no significant difference between the two was seen at higher shear stress levels. We conclude that fluid shear stress enhanced the uptake of MC540 by a mechanism other than endocytosis, suggesting an increase in plasma membrane permeability during exposure of the cells to shear stress.

Topics: Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Endothelium, Vascular; Flow Cytometry; Fluorescent Dyes; Humans; Pyrimidinones; Rheology; Stress, Mechanical

Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.

Topics: Animals; Apoptosis; B-Lymphocytes; Cycloheximide; Dextran Sulfate; DNA; Female; Membrane Lipids; Mice; Phospholipids; Pyrimidinones

The fluorescence lifetime, tau f, of merocyanine 540 (MC540) in small unilamellar vesicles was measured as a function of temperature and cholesterol content by using phase modulation fluorometry. These vesicles were formed by probe sonication of aqueous suspensions of egg phosphatidylcholine (PC) and cholesterol. The fluorescence lifetime of MC540 in these vesicles decreased with increasing temperature, but was independent of cholesterol. The decrease in tau f with temperature is attributed to trans-cis photoisomerization. At low temperatures, the inverse of tau f, or the fluorescence rate constant, kf, approaches a constant value of 0.45 +/- 0.02 ns-1, which corresponds to the value of the radiative rate constant, kr, of the dye. The photoisomerization rate constant, kiso, was obtained by subtracting kr from kf. The temperature dependence of kiso is well described by an Arrhenius equation, with an activation energy of 31.5 +/- 0.9 kJ mol-1. This Arrhenius behavior is rationalized in terms of the Smoluchowski limit for the Kramers theory for activated barrier crossing. The electronic spectra and kiso for MC540 in these vesicles are consistent with the dye being located in the polar headgroup region of the lipid bilayer.

Topics: Cholesterol; Lipid Bilayers; Photochemistry; Pyrimidinones; Stereoisomerism; Temperature

The transbilayer distribution of phospholipids in plasma membrane vesicles derived from BHK cells by treatment with iodoacetamide or fluoride and merocyanine 540 has been examined by exposing the vesicles to bee venom phospholipase A2 (PLA2) or to Bacillus cereus sphingomyelinase. The results show that almost all of the phosphatidylserine (PS) is on the inner lipid leaflet and most of the sphingomyelin is on the outer lipid leaflet. In contrast, about 50% of the phosphatidylcholine (PC) and 30-40% of the phosphatidylethanolamine (PE) is rapidly degraded by PLA2 and thus appears to be present on the surface of the vesicles. The pools of PC and PE which are accessible only slowly to PLA2 are degraded with halftimes of about 5 h and 2 h, respectively, and it is suggested that this rate reflects the rate of transbilayer migration of these lipids. We conclude that the profound energy depletion caused by treatment with iodoacetamide or fluoride does not alter the asymmetric distribution of PS across the plasma membrane but does have a marked effect on the transbilayer distribution of PE. Residual cells after treatment with fluoride and MC540 were also exposed to PLA2. The results were broadly in agreement with those obtained with vesicles, suggesting that the vesicles were representative of the BHK cell plasma membrane in terms of phospholipid asymmetry. Fluoride or MC540 added separately caused little vesicle release but did lead to significant loss of phospholipid asymmetry. When centrifuged on a sucrose density gradient, vesicles were separated into two major fractions accounting for about two thirds and about 20%, respectively, of total phospholipid but no significant differences were seen in the transbilayer phospholipid asymmetry of the two fractions.

Topics: Animals; Cell Line; Cell Membrane; Cricetinae; Iodoacetamide; Kidney; Lipid Bilayers; Phospholipases A; Phospholipases A2; Phospholipids; Pyrimidinones; Sphingomyelin Phosphodiesterase

By comparing the time-courses of rapid optical changes in the garfish olfactory nerve evoked by electric stimulation with those of mechanical changes observed at the site of optical recording, the origin of optical changes in the nerve has been investigated. Based on the finding that the time-course of the birefringence change accurately coincides with that of swelling of the nerve, optical changes are interpreted as being brought about by invasion of water into the superficial layer of the nerve fibers. A close relationship has also been demonstrated between nerve swelling and changes in light scattering and in dye absorbance.

Topics: Animals; Birefringence; Brachyura; Diffusion Chambers, Culture; Electric Stimulation; Electrophysiology; Fishes; Fluorescent Dyes; In Vitro Techniques; Light; Nerve Fibers; Olfactory Nerve; Pyrimidinones; Scattering, Radiation; Staining and Labeling; Thiobarbiturates

The surface properties of liposomes composed by saturated phosphatidylcholines and their mixtures with cholesterol in the gel state have been studied using merocyanine 540 as a fluorescent and optical probe. A new absorption peak at 450 nm and a new fluorescent band at 630 nm were observed when the dye was added to suspensions of DMPC multilamellar liposomes in the gel state. These peaks were also observed in membranes with different lipid compositions in conditions in which the P beta' and the L beta' phases were present. The increase of temperature above the main transition temperature of DMPC or the incorporation of 35% cholesterol into DMPC bilayers at 13 degrees C caused the disappearance of these peaks. The changes in the absorption and fluorescent spectra upon addition of cholesterol resembles very well the phase diagrams reported by Mortensen et al. ((1988) Biochim. Biophys. Acta 945, 221-245) indicating that the corrugated structures characteristic of the L beta' and the P beta' phases have different surface properties related to the partitioning of amphiphilic dies.

Topics: Cholesterol; Dimyristoylphosphatidylcholine; Fluorescent Dyes; Gels; Liposomes; Phosphatidylcholines; Pyrimidinones; Staining and Labeling; Surface Properties; Temperature

Photoactive compounds and drugs are used therapeutically as antibacterial, antiviral and antitumor agents. This report examines the use of a photoactive compound, preactivated merocyanine 540 (pMC540), in the treatment of stomatitis in two cats that are both feline immunodeficiency virus (FIV) positive. One of the cats was also feline leukemia virus (FeLV) positive. Dramatic short term improvement is reported with the dosage regimen and complications.

Topics: Animals; Antiviral Agents; Cat Diseases; Cats; Feline Acquired Immunodeficiency Syndrome; Immunodeficiency Virus, Feline; Leukemia Virus, Feline; Leukemia, Feline; Male; Photosensitizing Agents; Pyrimidinones; Stomatitis

Treatment of BHK or HL60 cell lines with merocyanine 540 in the presence of the sulphydryl blocker iodoacetamide caused budding of the cell surface to release vesicles about 50-100 nm in diameter which accounted for up to 25% of the total surface membrane lipid. Smaller amounts of vesicular material were released in the presence of fluoride and merocyanine 540. The vesicles had a membrane lipid composition which was characteristic of other purified plasma membranes, with large amounts of sphingomyelin, phosphatidylserine and cholesterol and low proportions of phosphatidylinositol, phosphatidylcholine, triacylglycerol and cholesterol ester. This procedure for the isolation of vesicles should be a general method for the purification of plasma membrane components from a wide range of different cell types.

Topics: Animals; Cell Line; Cell Membrane; Cholesterol; Cricetinae; Dose-Response Relationship, Drug; Humans; Iodoacetamide; Membrane Lipids; Mesocricetus; Phospholipids; Pyrimidinones; Tumor Cells, Cultured

The neuroblastoma cell lines SK-N-LO, SK-PN-DW and IMR 5 were stained with Merocyanine 540 and exposed to white light. 99% of clonogenic tumour cells were destroyed while 60% of bone marrow progenitor cells survived this treatment.

Topics: Bone Marrow; Bone Marrow Purging; Cell Survival; Hematopoietic Stem Cells; Humans; Light; Neuroblastoma; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

Photodynamic induced cytotoxicity by Victoria blue BO (VB-BO), merocyanine 540 (MC540), Nile blue A (NB) and 4-tetrasulfonatophenyl-porphyrin (4-TSPP) has been studied on two human leukemic cell lines: K-562 and TF-1. Cells were incubated with dyes and irradiated with different doses of white light. Cell survival was assessed by propidium iodide (PI) staining using flow cytometry analysis. Concentrations of 5 x 10(-8) M VB-BO were found to kill 75% of cells, and a concentration of 1 x 10(-7) M induced more than 99% of cell killing. To obtain the same cytotoxic level, the presence of 2.6 x 10(-5) M of MC540 during irradiation was needed. Under the conditions used, NB was ineffective as a photosensitizer, although uptake studies showed that this dye was taken by the cells in much greater amounts than any other studied dye. Cell cycle distribution of TF-1 cells, surviving MC540 or VB-BO photosensitization has been studied by flow cytometry analysis after staining with Hoechst 33342 and PI. It was found that cells in G1 phase were slightly more resistant toward MC540- and VB-BO-mediated photosensitization than cells in other phases of the cell cycle.

Topics: Antineoplastic Agents; Cell Cycle; Drug Screening Assays, Antitumor; Humans; Leukemia; Oxazines; Photochemotherapy; Photosensitizing Agents; Porphyrins; Pyrimidinones; Quaternary Ammonium Compounds; Tumor Cells, Cultured

This paper reports on the preclinical evaluation of merocyanine 540 (MC540) as an agent for the inactivation of tumour cells in BM grafts from non-Hodgkin's lymphoma (NHL) patients. The three cell lines used for this study, OCI-LY13.1, OCI-LY13.2 and OCI-LY9, originate from two patients with high-grade NHL. The OCI-LY13.1 and OCI-LY13.2 lines are derived from the same patient. The OCI-LY13.1 line was established at the time of diagnosis while the OCI-LY13.2 line was established after the tumour had become refractory to therapy. When used under conditions that are known to preserve about 50% of normal human pluripotent hematopoietic progenitor cells (CFU-GEMM), MC540-sensitized photoirradiation reduced in vitro clonogenic OCI-LY9 cells by 4 orders of magnitude and OCI-LY13.1 and OCI-LY13.2 cells by > or = 5. Survival curves for OCI-LY13.1 and OCI-LY13.2 cells were similar and followed first order kinetics, while those for OCI-LY9 cells were distinctly biphasic. Suspension cultures established with photoinactivated lymphoma cells confirmed that MC540-sensitized photoirradiation was cytotoxic and capable of eliminating > or = 4 log of tumour cells. These results encourage the further exploration of MC540-sensitized photoirradiation as a means to purge autologous marrow grafts from NHL patients.

Topics: Bone Marrow; Bone Marrow Transplantation; Humans; Light; Lymphoma, Large B-Cell, Diffuse; Neoplastic Stem Cells; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Transplantation, Autologous; Tumor Cells, Cultured

Merocyanine (MC 540) is a fluorescent probe whose optical properties depend on the environmental polarity. In the presence of lipid bilayers, MC 540 binds to the membrane surface while simultaneously changing its fluorescence properties. Previous studies have shown that the fluorescence of merocyanine depends upon the lipid packing in the membrane. We measured the partitioning of MC 540 and its fluorescence properties in the presence of phosphatidylcholine membranes. We found that the fluorescence of MC 540 shows, as expected, a major change around the main phase transition of phosphatidylcholine membranes. However, instead of a step-like increase of fluorescence, the maximum at phase transition was observed. We were able to explain our data by combining two effects; dependence of MC 540 fluorescence on temperature and lipid fluidity. In addition, we established that the increase of the fluorescence intensity in the presence of lipid bilayers in the fluid state is due to the elevated partitioning of the probe into the lipid phase. The partition of MC 540 into the fluid membrane does not depends on the dye concentration in the aqueous phase. When lipid was in the gel phase the partitioning of the dye increased with its bulk concentration, whereas the fluorescence intensity remained unchanged. We conclude, therefore, that MC 540 forms nonfluorescent complexes when in the gel lipid membrane.

Topics: Chemical Phenomena; Chemistry, Physical; Lipid Bilayers; Phosphatidylcholines; Pyrimidinones; Temperature

Planar lipid membranes were used to study photovoltage generation after the incorporation of various merocyanine dyes. These dyes undergo photochemical isomerization on illumination and can act as a photon-driven facilitated proton transport system under suitable conditions. This system has the advantage of preventing back recombination of the photodissociated charges, thus improving its storage capacity. A sufficiently high photovoltage, with a long storage time and good reproducibility, was obtained with these dyes. The spectral studies indicate the formation of a 1:1 complex between dye and lipid molecules. Our results show that the strength and stability of complex formation increase with the hydrophobicity of the dye which, in turn, increase the magnitude and storage of the photovoltage generated in the system.

Topics: Alkanes; Cholesterol; Fluorescent Dyes; Light; Lipid Bilayers; Models, Biological; Octanes; Photochemistry; Pyrimidinones; Spectrophotometry

Topics: Erythrocyte Membrane; Humans; Microscopy, Fluorescence; Phospholipids; Pyrimidinones

To test whether apoptotic thymocytes can be identified by altered packing of lipids in their plasma membranes, thymocytes were isolated from mice injected with hydrocortisone and stained with merocyanine 540 (MC540), a fluorescent probe sensitive to lipid packing. At 9-10 h after injection, a subpopulation of H2-Klo cells with increased MC540 staining was clearly discernable. When DNA degradation was assessed by staining fixed cells with propidium iodide (PI), the fraction of cells with increased MC540 staining corresponded to the fraction with reduced PI staining. In addition, enriching for the former by fluorescence-activated cell sorting enriched for the latter. These results indicate that living apoptotic thymocytes can be identified and separated on the basis of altered lipid packing and increased staining with MC540.

Topics: Animals; Apoptosis; Cell Death; Cell Separation; DNA Damage; Flow Cytometry; Fluorescent Dyes; Hydrocortisone; Male; Membrane Lipids; Mice; Mice, Inbred CBA; Propidium; Pyrimidinones; T-Lymphocytes; Thymus Gland

Merocyanine 540 (MC540) is a photosensitizing dye of potential use in the purging of cancer cells from autologous bone marrow explants. Treatment of marrow with MC540, followed by illumination with visible light, selectively kills neoplastic cells while sparing a sufficient number of stem cells to allow marrow engraftment. The photodynamic action of MC540 is thought to be mediated by reactive oxygen species, particularly singlet oxygen. We have previously shown that salicylic acid (SA) scavenges MC540-generated singlet oxygen. In this work, we sought to abrogate MC540-mediated cell killing of murine L1210 and human K562 leukemia cells with salicylate. Paradoxically, the presence of salicylate during illumination in the presence of MC540 appreciably enhanced cell killing. Enhancement was dependent on salicylate concentration in the range 0.1 to 10 mM, with 1.0 mM SA potentiating the MC540-mediated reduction in survival of L1210 and K562 cells by factors of 2.7 and 1.9, respectively. Neither preincubation with SA followed by washing prior to illumination nor addition of SA following illumination altered MC540-mediated cell killing, indicating that potentiation was dependent on the presence of SA during illumination. Illumination in the presence of salicylate alone did not diminish cell viability. In addition to SA, a number of structurally related compounds including dihydroxybenzoic acids, aspirin, and sodium benzoate also enhanced MC540-mediated cell killing. Potentiation of leukemic cell killing by salicylate could provide a basis for enhancing the clinical efficacy of MC540-mediated phototherapy.

Topics: Animals; Cell Survival; Drug Synergism; Gentisates; Humans; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Leukemia; Leukemia L1210; Oxygen; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Salicylates; Salicylic Acid

Fatty acid spin labels have been included into liposomes and micelles, in order to study the photochemical behavior of merocyanine 540 toward nitroxyl radicals situated at various depths in the bilayer or the surfactant layer. Visible illumination of the dye, either free in ethanol or bound to liposomes or micelles, leads to the reduction of the electron spin resonance signal of the label. The efficiency of the interaction between merocyanine 540 and spin labels depends on the depth at which the nitroxyl moiety is localized in the micelle or vesicle. Fluorescence measurements indicate that the first excited singlet state of merocyanine 540 is not directly implicated in the reaction mechanism. Flash photolysis experiments conducted in aqueous solutions of hexadecyltrimethylammonium bromide micelles show that the presence of nitroxyl radical decreases the rate constant of triplet decay in a concentration-dependent fashion. The corresponding quenching rate constant (kq) is determined for the different spin labels. The kq values and the reduction rates of ESR signal show the same dependence on the localization of the nitroxyl moiety in the micelles.

Topics: Cetrimonium; Cetrimonium Compounds; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Kinetics; Lipid Bilayers; Liposomes; Micelles; Photolysis; Photosensitizing Agents; Pyrimidinones; Spin Labels; Stearic Acids; Structure-Activity Relationship; Time Factors

EPR and fluorescence probes were used in this study to define the effects of L-carnitine and its short-chain esters, acetyl-L-carnitine and propionyl-L-carnitine, on the natural fluidity gradient and molecular packing of phospholipid headgroups of erythrocyte membrane in intact cells. Purified erythrocyte suspensions, labeled with different stearic acid derivatives containing a stable doxyl radical ring at the C-5, C-7, C-12 and C-16, were incubated with 0.5-5 mM L-carnitine and its esters for 60 min at 37 degrees C and washed twice with an isosmotic buffer. A decrease in the order parameter, calculated from the EPR spectra of the 5-doxylstearic acid derivative, was observed at all the concentrations of propionyl-L-carnitine and the extent of the decrease was dose and temperature dependent. An increase of the chain length between the doxyl ring and the carboxylic group of the spin label, resulted in a much lower efficacy of propionyl-L-carnitine in decreasing the order parameter. Acetyl-L-carnitine also showed a significant effect of decreasing the molecular order but only at the lower temperatures of red cells labeled with 5-doxyl and treated with the highest concentration of the drug. L-Carnitine did not modify the molecular dynamics at all the temperatures and concentrations used in this study. L-Carnitine and its short-chain derivatives did not alter significantly membrane fluidity of deeper regions of the erythrocyte membrane, measured by means of the excimer/monomer fluorescence intensity ratio of pyrene incorporated into the membrane of intact erythrocytes. However, these compounds were all capable of loosening the molecular packing of the polar head of erythrocyte membrane phospholipids evaluated by the membrane binding fluorescence properties of merocyanine-540. The binding of the fluorescent probe decreased in the order propionyl-L-carnitine > acetyl-L-carnitine > L-carnitine. Our findings suggest that this category of compounds affect the molecular dynamics of a membrane bilayer region close to the glycerol backbone of phospholipids, which might be relevant for the expression of membrane functions.

Topics: Acetylcarnitine; Carnitine; Diphenylhexatriene; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Esters; Humans; Membrane Fluidity; Membrane Proteins; Palmitoylcarnitine; Pyrimidinones; Spin Labels

Monoclonal antibodies (mAbs) directed against E2, a 32-kDa transmembrane protein encoded by the MIC2 gene located in the pseudoautosomal region, induce a transbilayer movement of phosphatidylserine and, to a lesser extent, phosphatidylethanolamine in human thymocytes and a Jurkat T lymphocytes. The translocation of phosphatidylserine has been evidenced by using either derivatization of anionic phospholipids with trinitrobenzenesulfonate (TNBS) or cytofluorimetry after labeling of cells with antiphosphatidylserine antibodies. The perturbation of membrane phospholipids induced by anti-E2 mAbs was further evidenced by labeling the cells with merocyanine 540. The specificity of anti-E2-induced perturbations of membrane asymmetry was tested by using a number of mAbs able to activate T cells, including CD3 and CD2. The results strongly suggest that anti-E2-induced changes in PtdSer are related to cell aggregation since the same mAbs specifically induce the aggregation of both thymocytes and Jurkat cells and since the E2 molecule has been previously implicated in the adhesive properties of human T cells with erythrocytes.

Topics: 12E7 Antigen; Antibodies, Monoclonal; Antigens, CD; Cell Adhesion Molecules; Cells, Cultured; Ethanolamine; Ethanolamines; Flow Cytometry; Fluorescent Dyes; Humans; Kinetics; Membrane Glycoproteins; Phosphatidylethanolamines; Phosphatidylserines; Pyrimidinones; Serine; T-Lymphocytes; Thymus Gland; Tritium; Tumor Cells, Cultured

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes.

Topics: Animals; Apoptosis; Benzimidazoles; Cell Membrane Permeability; Cell Nucleus; Cell Separation; Cells, Cultured; DNA; Flow Cytometry; Fluoresceins; Male; Pyrimidinones; Rats; Rats, Inbred F344; Thymus Gland

Merocyanine 540 (MC540) is a lipophilic photosensitizing dye of biomedical interest in connection with its ability to preferentially inactivate leukemia cells in bone marrow grafts and enveloped viruses in blood products. Evidence that iron plays a role in dye-mediated photokilling is presented in this report. When sensitized with MC540 and irradiated with visible light, cultured murine leukemia L1210 cells underwent lipid peroxidation (accumulation of iodometrically detectable lipid hydroperoxides) and photokilling (loss of clonogenic capacity). Selenium-deficient [Se(-)] cells, which expressed minimal selenoperoxidase activity, were found to be more sensitive to photoperoxidation and photokilling than selenium-replete [Se(+)] controls. Since redox active iron in the presence of electron donors has been shown to exacerbate photoperoxidative damage in isolated membrane systems, it was of interest to examine the possible role of iron in MC540/light-induced cytotoxicity. Involvement of iron was established by showing (i) that desferrioxamine (a high-affinity chelator and redox inhibitor of Fe3+) acted protectively on Se(-) and Se(+) cells and (ii) that treating these cells with sublethal concentrations of the lipophilic chelate ferric 8-hydroxyquinoline [Fe(HQ)2] made them much more sensitive to photokiling and thiobarbituric acid-detectable lipid peroxidation. Lehal damage induced by t-butyl hydroperoxide was also amplified by Fe(HQ)2. Fe(HQ)2-enhanced photoperoxidation and photokilling were suppressed by alpha-tocopherol, suggesting that iron-catalyzed free radical reactions were involved. A mechanism based on iron-mediated one-electron reduction of nascent photoperoxides is proposed. We believe that under the conditions used, toxic one-electron chemistry overwhelms two-electron detoxification catalyzed by GSH-dependent selenoperoxidase(s).

Topics: Animals; Cell Survival; Clone Cells; Deferoxamine; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Glutathione Peroxidase; Iron; Iron Chelating Agents; Kinetics; Leukemia L1210; Light; Lipid Peroxidation; Mice; Oxidants; Peroxides; Pyrimidinones; Radiation-Sensitizing Agents; Selenium; tert-Butylhydroperoxide; Tumor Cells, Cultured

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Cyclic N-Oxides; Docosahexaenoic Acids; Fluorescent Dyes; Gels; Lipid Bilayers; Mice; Phosphatidylcholines; Phospholipids; Pyrimidinones; Tumor Cells, Cultured

Light-activated merocyanine 540 (pMC540) has been shown in our earlier studies to be effective against certain types of tumor cells and viruses, including human immunodeficiency virus (HIV-1). To test the potential extracorporeal and systemic use of pMC540, its toxicity was investigated in DBA/2 mice, pigs, and dogs. The lethal dose in DBA/2 mice after an i.p. injection was 370 mg/kg, and the 50% lethal dose (LD50) was 320 mg/kg; however, following i.v. administration, the lethal dose and the LD50 dose were 240 and 160 mg/kg, respectively. Tritium-labeled MC540 was used to study the biodistribution of pMC540 in DBA/2 mice. Almost 70% of the injected radioactivity was excreted within 6 h of injection. After 1 week, the pMC540 was almost completely cleared, with only 1.89% of the activity remaining, and had a plasma half-life of 23 h. Pigs injected with an accumulated dose of 10 mg/kg and followed for a period of 30 days did not show adverse signs of toxicity as monitored by SMAC-28 analysis, CBC profile, and blood-coagulation studies. A dog injected with a single dose of 20 mg/kg showed induction of the hepatic enzymes glutamic oxaloacetic transaminase (AST) and glutamic pyruvic transaminase (AST); however, serum levels of gamma-glutamyl transpeptidase (GGT) remained unchanged. The data presented herein may serve to identify certain drug-dose limitations in the systemic use of pMC540.

Topics: Animals; Dogs; Female; Half-Life; Male; Mice; Mice, Inbred DBA; Photochemistry; Photosensitizing Agents; Pyrimidinones; Swine; Tissue Distribution

Many new photosensitizers and laser wavelengths are being tested to improve photodynamic therapy by enhancing specific tumor uptake and/or retention, lowering systemic toxicity, and increasing laser tissue penetration. In this study the potential synergistic effects of rhodamine-123 (Rh-123) and merocyanine-540 (MC-540) sensitization of human tumor cell lines after laser exposure were explored. In a first series of experiments, the kinetics of uptake of Rh-123 and M-540 were tested on three human leukemia cell lines (K562, RAJI, 729HF2), P3 squamous carcinoma, and M26 melanoma. Our results demonstrate a clear difference in the rate and amount of uptake of MC-540 (K562 > P3 > RAJI > 729HF2 > M26) and Rh-123 (P3 > RAJI > 729HF2 > K562 > M26) by these cell lines. In a second series of experiments, M26 tumor cells were sensitized with either Rh-123 (1 microgram/ml) or with MC-540 (20 micrograms/ml) alone or with a combination of the two dyes for 60 minutes, then exposed to the argon (514.5 nm) laser at nonthermal energy levels. Our results demonstrate a significant enhancement of the tumoricidal effects of the laser on M26 carcinoma cells after sensitization with both dyes together (MC-540 and Rh-123) when compared to each dye alone. As with combination antibiotherapy, the synergistic effects of two laser dyes that have different intracellular targeting sites appear to enhance tumoricidal effects significantly after exposure to a matching laser wavelength. The data provide evidence for effective laser phototherapy by dye synergy.

Topics: Antimetabolites, Antineoplastic; Burkitt Lymphoma; Carcinoma, Squamous Cell; Cell Survival; Drug Synergism; Fluorescent Dyes; Hot Temperature; Humans; Laser Therapy; Leukemia, B-Cell; Leukemia, Erythroblastic, Acute; Lung Neoplasms; Melanoma; Neoplasms; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Radiation Dosage; Rhodamine 123; Rhodamines; Tumor Cells, Cultured

Exposure of certain photoactive dyes to light prior to their use in biological systems (preactivation) has been shown to result in formation of long-lived cytotoxic photoproducts. The cytotoxic species responsible for the biological activity of preactivated merocyanine 540 (pMC540) appears to be a hydroperoxide generated by oxidation of ground-state dye by singlet molecular oxygen, formed via energy transfer from triplet excited-state dye to oxygen. A positive correlation (r = .93) exists between the levels of hydroperoxides and percent of tumor cells killed upon exposure to pMC540. Exposure of bovine serum albumin (BSA) (0.5 mg/mL) to pMC540 (0.2 mg/mL-1 mg/mL) results in loss of tryptophan fluorescence and 345 nm emission, suggesting a probable role of either hydroxyl (.OH) or .OH + superoxide (O2-). Polyacrylamide gel electrophoresis indicates fragmentation of treated BSA. Aggregation of pMC540-treated BSA is not detected. Bityrosine production is not observed. A dose-dependent decrease in BSA solubility is observed in treated samples, suggesting an increase in hydrophobicity. Amino acid analysis of BSA treated with pMC540 shows loss of some amino acids residues. The data presented here suggest that photoproducts of MC540 derived via the process of preactivation may mediate their effect (at least in part) by reactive oxygen species.

Topics: Amino Acids; Energy Transfer; Fluorescent Dyes; Hydrogen Peroxide; Hydrolysis; Hydroxides; Hydroxyl Radical; Light; Oxidation-Reduction; Oxygen; Peptide Fragments; Photochemistry; Protein Denaturation; Pyrimidinones; Serum Albumin, Bovine; Singlet Oxygen; Spectrometry, Fluorescence; Superoxides; Tryptophan

The amphipathic dye, merocyanine 540 (MC540), which preferentially photosensitizes enveloped viruses and virus-infected cells, is currently being evaluated in preclinical models as a blood sterilizing agent. In this communication, we report on an initial analysis of the site and nature of MC540-mediated photodynamic damages to human herpes simplex virus type 1 and human cytomegalovirus. The comigration of dye molecules and virions on a gel filtration column, the red-shift of the fluorescence emission spectrum of virus-containing fractions, and the distribution of MC540-treated virions in an aqueous two-phase partition system were indicative of MC540 binding to the enveloped viruses and localizing in a lipophilic environment (most likely the viral envelope). Fluorescence quenching and fluorescence resonance energy transfer experiments suggested that both dye monomers and dimers were capable of partitioning into the lipid bilayer of the viral envelope. Adsorption and penetration assays and immunohistochemical analyses of viral antigen expression showed that MC540-sensitized irradiation interfered with early phases of the infectious process, the adhesion to the host cell, the penetration of the host cell, and the translocation of the virus into the nucleus of the host cell. The inactivation of viruses was inhibited if oxygen in the medium was displaced by argon, enhanced if air was displaced by pure oxygen or if water was replaced by deuterium oxide. This suggested that the MC540-sensitized photoinactivation of enveloped viruses is an oxygen-dependent process and that singlet oxygen is one but not necessarily the only mediator of the antiviral effects of MC540.

Topics: Animals; Cell Membrane; Cells, Cultured; Cysteamine; Dithiothreitol; Glutathione; Humans; In Vitro Techniques; Oxidation-Reduction; Photochemotherapy; Pyrimidinones; Receptors, Virus; Simplexvirus; Vero Cells; Virus Diseases

Merocyanine 540 (MC540) is a photosensitizing dye that has been used in several preclinical models and in a phase I clinical trial for the extracorporeal purging of tumor cells from autologous bone marrow grafts. The mechanism of the cytotoxic activity of MC540 is not yet fully understood, and the subcellular targets of MC540-mediated photodynamic damage remain to be identified. The human neutrophil provides an attractive model with which to study the effects of photoactivated MC540 on several well-defined cellular functions. As we report in this paper, simultaneous exposure of neutrophils to MC540 and light inhibited phagocytosis, random migration, chemotaxis, hydrogen peroxide production, and oxygen consumption. By contrast, the ability of neutrophils to kill engulfed bacteria and to produce superoxide radical was not compromised. Intracellular ATP levels and the activities of the cytosolic enzymes superoxide dismutase, catalase, and myeloperoxidase were only slightly reduced. Even in HL-60 leukemia cells, which bind more dye and are more readily killed by MC540-mediated photodynamic therapy than neutrophils, superoxide dismutase, catalase, and myeloperoxidase activities remained at normal or near-normal levels. These results are compatible with the view that plasma membrane components are primary targets of MC540-mediated photodynamic damage.

Topics: Adenosine Triphosphate; Catalase; Chemotaxis, Leukocyte; Humans; Hydrogen Peroxide; Leukemia, Promyelocytic, Acute; Light; Neutrophils; Oxygen Consumption; Peroxidase; Phagocytosis; Photosensitizing Agents; Pyrimidinones; Superoxide Dismutase; Tumor Cells, Cultured

Spectral-data-processing and curve-fitting techniques have been applied to the decomposition of merocyanine-540 absorption spectra in aqueous, micellar and bilayer environments. The various resolved component bands have been assigned to dye monomers, dimers, or larger aggregates, either in polar or non-polar environments. The analysis of spectral parameters (lambda max and integrated intensity) of the overall spectra and of each component has revealed that merocyanine 540 is a useful probe in studies of membrane structure and dynamics using visible-absorption spectroscopy. In particular, the monomer lambda max and the integrated intensity, i.e. area, of the dimer population are very useful in this respect. The monomer lambda max is especially sensitive to polarity changes and is thus useful, e.g. in the precise determination of critical micellar concentrations. The fractional area of the dimer increases with the packing density of the phospholipid-hydrocarbon region near the interface and is thus very sensitive to changes in vesicle curvature and to the presence of sterols or intrinsic polypeptides in the bilayer.

Topics: Cholesterol; Detergents; Fluorescent Dyes; Gramicidin; Lipid Bilayers; Micelles; Octoxynol; Phospholipids; Polyethylene Glycols; Pyrimidinones; Spectrometry, Fluorescence

Photodynamic therapy with the lipophilic sensitizing dye merocyanine 540 (MC540) is a promising new approach for extracorporeal purging of neoplastic cells from autologous remission bone marrow grafts. Resistance-conferring cellular defenses against the cytotoxic effects of MC540/photodynamic therapy have not been well characterized. This study focuses on the cytoprotective effects of the glutathione-dependent selenoperoxidases GPX and PHGPX, which can detoxify a wide variety of hydroperoxides, including lipid-derived species (LOOHs). Murine leukemia L1210 cells were grown in 1% serum media without [L.Se(-)] and with [L.Se(+)] selenium supplementation. L.Se(-) cells expressed 10- to 20-fold lower GPX and PHGPX activities than L.Se(+) controls and were markedly more sensitive to MC540-mediated photoperoxidation (LOOH formation) and clonally assessed photokilling. Susceptibility of L.Se(-) cells to photoperoxidation and photokilling could be fully reversed to L.Se(+) levels by replenishing Se, and partially reversed by treating with Ebselen, a selenoperoxidase mimetic. Altered lipid composition, greater uptake of MC540, and defective catabolism of H2O2 were all ruled out as possible factors in the elevated photosensitivity of L.Se(-) cells. Human leukemia K562 cells (capable of expressing PHGPX but not GPX) exhibited 5- to 10-fold lower PHGPX activity under Se-deficient relative to Se-sufficient conditions. Although MC540 uptake (nmol/mg lipid) by K562 and L1210 cells was essentially the same, the former were more resistant to photoinactivation. However, like murine counterparts, Se-deficient cells were more susceptible to photoperoxidation and photokilling than Se-sufficient controls. These results clearly demonstrate that GPX and/or PHGPX in L1210 cells and PHGPX in K562 cells play an important cytoprotective role during photooxidative stress. Whether membrane damage due to lipid photoperoxidation is causally related to cell death is not certain; however, the parallel effects of Se deficiency on LOOH formation and cell killing are at least consistent with this possibility.

Topics: Animals; Azoles; Catalase; Cell Death; Glutathione; Glutathione Peroxidase; Humans; Isoindoles; Leukemia L1210; Leukemia, Experimental; Lipid Peroxidation; Mice; Organoselenium Compounds; Phospholipid Hydroperoxide Glutathione Peroxidase; Photochemotherapy; Pyrimidinones; Radiation Tolerance; Radiation-Sensitizing Agents; Selenium

Serum is known to inhibit the merocyanine 540 (MC540)-sensitized photoinactivation of cells and enveloped viruses in a concentration-dependent manner. In diagnostic applications of MC540, a moderate amount of serum or serum albumin is frequently added to the staining solution because it enhances the contrast between intensely staining cells (e.g., electrically excitable cells or leukemia cells) and cells with a lower affinity for the dye (e.g., nonexcitable cells, red cells, normal leukocytes). In this communication we report on a quantitative analysis of the interactions of MC540 with serum and serum components. Human serum inhibited the MC540-sensitized photoinactivation of K562 leukemia cells most effectively, followed in order of decreasing potency by calf, newborn calf, horse, and fetal bovine serum. The photoprotective capacity of these five sera was directly proportional to their albumin content. Gel filtration experiments and differential spectroscopy showed that MC540 bound to serum albumin and lipoproteins. Both delipidated and lipidated albumin were capable of binding MC540. However, lipidated albumin had a considerably higher binding capacity and affinity for dye molecules.

Topics: Animals; Cattle; Horses; Humans; Leukemia; Lipoproteins; Photosensitizing Agents; Plasma; Protein Binding; Pyrimidinones; Serum Albumin; Tumor Cells, Cultured

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.

Topics: Animals; Cell Survival; Dose-Response Relationship, Radiation; Humans; Kinetics; Leukemia L1210; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Light; Mice; Pyrimidinones; Radiation-Sensitizing Agents; Temperature; Time Factors; Tumor Cells, Cultured

In order to gain insight into the preactivation of merocyanine 540 (MC540) 1 for the photodynamic therapy (Gulliya et al., 1990a, Photochem. Photobiol. 52, 831-838) its photo-oxidation was investigated. After irradiation of MC540 1 on a preparative scale three main photodegradation products were isolated with 16-20% yields. They turned out to be derivatives of benzoxazole, thiouracil and thiohydantoin with the structures 4, 5 and 6, respectively. It may be possible that they contribute to the cytostatic and antiviral activity of preactivated MC540 1.

Topics: Humans; Magnetic Resonance Spectroscopy; Molecular Structure; Oxidation-Reduction; Photochemistry; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.

Topics: Animals; Cell Cycle; Leukemia L1210; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured

The characteristics of the fluorescent dye, merocyanine 540 (MC-540), incorporated in monolayers of 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied in different states of molecular packing. Conditions for phase separation in these monolayers were defined by their pressure/area (pi-A) isotherms. Within the liquid expanded (LE) and the liquid condensed (LC) coexisting phases of DPPC monolayers, low light level epifluorescence microscopy revealed 'dark' discoid domains embedded in a 'bright' matrix. Under the same conditions, and at temperatures as low as 12 degrees C, the pi-A isotherms of POPC demonstrate the existence of a single phase, and no fluorescent domains were observed. Fluorescence spectra of MC-540 labelled monolayers, recorded in different structural states, reveal three distinct emission peaks: a 572 nm peak, present for monolayer packing conditions at low surface pressures; a 585 nm peak, similar to that obtained from dye molecules in fluid phase lipid bilayers, and observed here within the respective area/molecule ranges of 54-62 A2 and 62-69 A2 for monolayers of DPPC and POPC with diminishing intensity at increasing surface pressure; and finally, a peak at 560 nm, which predominates in densely packed POPC monolayers. Our results are interpreted on the basis of dye partitioning between monolayer and subphase, and different orientations of the dye with respect to the monolayer in various structural states. The usefulness of MC-540 to differentiate lipid packing in cell membranes is discussed.

Topics: Antiviral Agents; Fluorescent Dyes; Microscopy, Fluorescence; Phosphatidylcholines; Pyrimidinones; Spectrometry, Fluorescence

The normal asymmetric distribution of phospholipids across the plasma membrane of erythrocytes can be abolished by lysing and resealing cells in the presence of Ca2+. In the present study, using flow cytometric analysis of the binding of merocyanine 540 to monitor transbilayer phospholipid distribution, Ca(2+)-induced loss of asymmetry is shown to be independent from the aminophospholipid translocase which catalyzes movement of normally internal phospholipids from the outer to the inner leaflet of the membrane. Loss of asymmetry is rapid, temperature-sensitive, and occurs in an uninterrupted, intact bilayer, rather than by diffusion of lipids through the hemolytic pore. Addition of ATP during lysis reverses loss of asymmetry, and this restoration can be blocked by inhibitors of the aminophospholipid translocase. These results suggest that the ATP-dependent translocase is essential for recovery of asymmetry, in turn suggesting that separate mechanisms mediate the loss and the recovery of lipid asymmetry in erythrocytes.

Topics: Adenosine Triphosphate; Calcium; Carrier Proteins; Erythrocyte Membrane; Flow Cytometry; Fluorescent Dyes; Kinetics; Membrane Lipids; Membrane Proteins; Phospholipid Transfer Proteins; Pyrimidinones; Vanadates

Singlet oxygen and fluorescence quantum yields of merocyanine 540 were measured in solution (methanol, ethanol, n-heptanol) and in model membrane systems (cationic micelles, unilamellar dimyristoyl- and dipalmitoylphosphatidylcholine vesicles). Both singlet oxygen quantum yields and fluorescence quantum yields increase with increasing viscosity/rigidity of the surrounding medium: the yield of singlet oxygen production (24 degrees C) goes from 0.002 in methanol to 0.04 in dipalmitoylphosphatidylcholine vesicles, and fluorescence yields (25 degrees C) change from 0.14 to 0.61 in the same media. The data are consistent with previous findings that photoisomerization is in direct competition with intersystem crossing and radiative relaxation. Therefore, a singlet oxygen yield close to the maximum value of 0.11 can only be achieved after both photoisomerization and internal conversion are prevented by a highly viscous environment.

Topics: Cell Membrane; Fluorescent Dyes; Liposomes; Models, Biological; Oxygen; Pyrimidinones; Solutions; Spectrometry, Fluorescence

Topics: Antineoplastic Agents; Antiviral Agents; Humans; Leukemia; Light; Molecular Structure; Pyrimidinones; Radiation-Sensitizing Agents; Structure-Activity Relationship; Tumor Cells, Cultured

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine-sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

Topics: Animals; Chloroquine; Drug Resistance; Erythrocytes; Humans; In Vitro Techniques; Malaria, Falciparum; Photochemotherapy; Plasmodium falciparum; Pyrimidinones

When triggered, cytolytic effector cells (cytolytic T-lymphocytes (CTL) and large granular lymphocytes (LGL)) release effector molecules from cytoplasmic granules, including the lytic protein perforin. This protein binds and incorporates into the plasma membrane of target cells, where it aggregates to form pores which cause target cell lysis and death. Phosphorylcholine, the headgroup of the ubiquitous phospholipids phosphatidylcholine (PC) and sphingomyelin, has been proposed as the specific receptor for perforin. We report here that any headgroup specificity is outweighed by phospholipid spacing in determining binding of perforin to liposomes. We also find that the spacing of outer leaflet lipids in a natural bilayer, the plasma membrane of the erythrocyte, influences susceptibility of the cell to perforin-mediated lysis. Finally, we demonstrate that the plasma membrane lipids in CTL are more closely spaced than in target cells, suggesting that lipid spacing contributes to the relative resistance of CTL to perforin-mediated lysis.

Topics: Animals; Binding Sites; Cell Line; Liposomes; Membrane Glycoproteins; Membrane Lipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Perforin; Pore Forming Cytotoxic Proteins; Pyrimidinones; Rats; Rats, Inbred F344

Two viral inactivation methods suggested for use with cellular blood products have been evaluated as to their effects on platelets. In the first study, it was proposed that pulsed laser-ultraviolet radiation (UVB) at 308 nm could favor photodamage to UVB-sensitive viral nucleic acid with minimal effects on blood platelets. A "window of efficacy" was observed with UVB doses of 10.5-21.5 J/cm2 at which 4-6 log10 poliovirus were inactivated while platelets were relatively tolerant. However, this "window" occurred only with low-intensity UVB radiation (less than or equal to 0.25 MW/cm2). Damage to platelet proteins, evident at high laser intensities, was probably due to multiple photon excitation of amino acids. In the second study, platelets and viruses were treated with the photosensitizer, merocyanine 540 (MC 540) (less than or equal to 24 micrograms/ml), and visible light (450-600 nm) (less than or equal to J/cm2). Activation of washed platelets by dye/light treatment resulted in a spontaneous release of serotonin, spontaneous aggregation, and marked morphological changes. Increasing concentrations of albumin in the suspension medium protected against dye-mediated photodamage to platelets, but also significantly reduced the antiviral activity of MC 540 and light. These results illustrate the relative sensitivities of platelets and viruses to two inactivation methods and the difficulty in optimizing inactivation of viruses and preservation of platelet function in a protein-rich medium.

Topics: Blood; Blood Platelets; Blood Proteins; DNA Damage; Humans; Lasers; Photochemistry; Platelet Activation; Pyrimidinones; Radiation-Sensitizing Agents; Ultraviolet Rays; Virus Diseases; Virus Physiological Phenomena; Virus Replication; Viruses

The purpose of this study was to assess the mechanism of merocyanine 540 (MC540) photobleaching in a liposomal system. Broad based visible irradiation of MC540 in unilamellar dilauroylphosphatidylcholine (DLPC) vesicles resulted in dye bleaching that was strictly O2 dependent. The rate of self-sensitized photobleaching was enhanced in D2O and inhibited by both azide and histidine, consistent with 1O2 intermediacy (Type II chemistry). Supportive evidence for this mechanism was obtained by using a Type II sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS lambda max = 678 nm). Irradiation of AlPcS and MC540 in DLPC with lambda greater than 630 nm (absorbed only by AlPcS) light resulted in rapid bleaching of MC540, which was stimulated by D2O and inhibited by azide. A rate constant of 10(7) M-1 s-1 was determined for the chemical quenching of 1O2 by MC540. The rate constant for physical quenching of 1O2 by MC540 was estimated to be ca 10(9) M-1 s-1.

Topics: Liposomes; Molecular Structure; Oxygen; Phosphatidylcholines; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Singlet Oxygen

Treatment of cultured BHK cells with merocyanine 540 caused the non-lytic release of vesicular material having the phospholipid composition characteristic of plasma membrane. The protein composition of the vesicles closely resembled that of the soluble fraction of the cell, as expected for exovesicles budding from the cell surface. Vesicles prepared from cells surface-iodinated with 125I contained no obvious iodinated membrane polypeptides, suggesting that no major proteins in the plasma membrane of the BHK cell are free to diffuse with lipids. The procedure described should represent a general method, applicable to a wide range of cell types, for isolating plasma membrane vesicles.

Topics: Cell Membrane; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Lipid Bilayers; Phospholipids; Pyrimidinones; Semliki forest virus

A novel photodynamic procedure employing "preactivated" merocyanine 540 (P-MC 540) was assessed for its effectiveness in inactivating human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Merocyanine 540 was preactivated by exposure to laser light at 514 nm prior to addition to viruses or infected cells. Treatment of cell-free HIV-1 and SIV with P-MC 540 significantly reduced their ability to infect and kill MT-4 cells in vitro. Preactivated MC 540 treatment of in vitro HIV-1-infected human peripheral blood mononuclear cells also decreased viral infection as assessed by a reduction in the amounts of HIV-1 p24 antigen produced and in the number of HIV-1 antigen-positive cells. Indirect immunofluorescence assays of target cell binding showed that treatment of cell-free HIV-1 and SIV with P-MC 540 interfered with their ability to bind to CD4+ target cells. Immunoprecipitation with a monoclonal anti-CD4 antibody of P-MC 540-treated and radiolabeled HIV-1 incubated with soluble recombinant CD4 (srCD4) resulted in coprecipitation of HIV-1 viral p17 and p24 core antigens with the envelope gp120/CD4 complex, suggesting cross-linking of viral components. However, no significant decrease in the binding of treated HIV-1 to srCD4 was observed. Because of the antitumor and antiviral properties of P-MC 540, this photopreactivation procedure may represent a promising therapeutic means for controlling systemic malignancies and viral infections, and for eliminating viral contaminants in biological fluids. Unlike conventional phototherapy, this procedure does not require the delivery of light energy at the target sites following binding of the photosensitizing compounds.

Topics: Antiviral Agents; Blood Banks; CD4 Antigens; Cell Line; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; HIV-1; Humans; Leukocytes, Mononuclear; Light; Pyrimidinones; Radiation-Sensitizing Agents; Simian Immunodeficiency Virus

Merocyanine 540 (MC 540) is a photoactive dye used to purge bone marrow of tumor cells in autologous bone marrow transplantation. The effects of MC 540 on the lymphoid components in the marrow are unknown. This study evaluates the treatment of lymphocytes by MC 540 (15 micrograms/mL) and light (70 W/m2) on: (1) phytohemagglutinin and Con A-induced proliferation; (2) allogeneic mixed lymphocyte cultures (MLC); (3) the regulation of Ig synthesis by T cells; and (4) the ability of B cells to produce polyclonal Igs as measured by an enzyme-linked immunosorbent assay-plaque assay. The results show that MC 540 and light treatment reduced Con A-stimulated T-cell proliferation greater than 50% after 30 minutes and greater than 80% after 60 minutes of MC 540-sensitized photoirradiation. Ninety minutes of MC 540 and light exposure (designated treatment) inhibited MLC greater than 90%. In polyclonal Ig synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited Epstein-Barr virus-stimulated Ig synthesis. These data show that T- and B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions.

Topics: B-Lymphocytes; Cells, Cultured; Concanavalin A; Fluorescent Dyes; Herpesvirus 4, Human; Humans; Immunoglobulins; Light; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Phytohemagglutinins; Pokeweed Mitogens; Pyrimidinones; T-Lymphocytes; T-Lymphocytes, Helper-Inducer

The effect of the photosensitizer merocyanine 540 (MC 540) on platelets and on three marker viruses was examined to assess its potential in reducing virus transmission by blood products. The results demonstrated several deleterious effects of MC 540 (4-24 micrograms/mL) on platelet morphology and function in both the absence and presence of light (450-600 nm). Treatment of washed platelets with MC 540 in the dark resulted in a significant release of serotonin in the absence of added agonist, as well as a diminished response to thrombin as measured in vitro. In addition, photosensitization caused spontaneous platelet aggregation and release of 92 percent of the releasable serotonin without the addition of an agonist. Because photo-treatment of blood products is likely to be performed in a protein-rich medium, the influence of albumin on the phototoxic effects on platelets was assessed. Albumin added to the suspension medium at concentrations greater than or equal to 1.0 percent protected the platelets against the effects of MC 540 in the dark, whereas 5-percent albumin was required for protection against the phototoxic effects of MC 540 on the platelet response to thrombin. The antiviral activity of MC 540 and light was examined by using the lipid-containing viruses herpes simplex virus (HSV) and bacteriophages phi 6 and PM2. Of the lipid-enveloped viruses, HSV was 25 times more photosensitive to MC 540 than was phi 6 (15 micrograms/mL). PM2, which has an internal lipid layer, was almost 300 times less sensitive to MC 540 and light than was HSV.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bacteriophages; Blood Platelets; Humans; In Vitro Techniques; Kinetics; L-Lactate Dehydrogenase; Light; Platelet Aggregation; Pseudomonas; Pyrimidinones; Radiation-Sensitizing Agents; Serotonin; Serum Albumin, Bovine; Simplexvirus

erocyanine 540 (MC540) is a membrane-directed photosensitizing dye with antileukemic and antiviral properties. In this study, biophysical and biochemical techniques have been used to examine MC540-sensitized photooxidative damage in the lipid and protein compartments of a test membrane, the human erythrocyte ghost. Irradiation of MC540-sensitized ghosts with white light resulted in oxidative damage to proteins, as manifested by (i) loss of sulfhydryl groups; (ii) intermolecular cross-linking of major polypeptides; and (iii) loss of Mg(2+)-ATPase and Na+,K(+)-ATPase activities. Photooxidation also produced a rapid and progressive increase in general protein motion, as measured by electron paramagnetic resonance spectrometry (EPR) with the sulfhydryl spin label MAL-6. In addition to these effects, ghosts exposed to MC540 and light underwent lipid peroxidation. EPR with two lipophilic spin probes, 5-doxylstearate and 16-doxylstearate, showed that lipid peroxidation is accompanied by a progressive decrease in bilayer fluidity (motional freedom). At a given dye concentration, structural perturbations of proteins were detected at much lower light fluences than those of lipids. When photoreactions were carried out in the presence of ascorbate and iron, there was a strong stimulation of lipid peroxidation (attributed to free radical chain reactions), with a concomitant greater decrease in lipid mobility. Thus, the deleterious effects of photoperoxidation on lipid structure and motional freedom were greatly exacerbated by ascorbate and iron. Membrane damage similar to that described here may play a role in the phototherapeutic activity of MC540.

Topics: Adenosine Triphosphatases; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Lipid Bilayers; Lipid Peroxidation; Membrane Fluidity; Membrane Lipids; Membrane Proteins; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Sulfhydryl Compounds

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.

Topics: Chromatography, High Pressure Liquid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Hydroxides; Hydroxyl Radical; Oxygen; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Salicylates

Bacteriophages may be useful as surrogates for animal viruses when the virucidal properties of different photosensitizing compounds are initially investigated. We studied photoinactivation of four bacteriophages, phi X174, T7, PRD1, and phi 6, by the dye merocyanine 540 (MC540) (15 micrograms/mL). Merocyanine 540 (MC540) should be most effective with lipid-containing viruses, since it is primarily lipophilic (but also binds to proteins). Two of the phages, PRD1 and phi 6 contain lipid, with only phi 6 having an external lipoprotein envelope. Filtered radiation (450-600 nm) from a 750 W projector was used at 16-100 W/m2. The survival curves of the different viruses clearly demonstrated different levels of sensitivity to photoinactivation by MC540, with phi 6 (Do = 1.5 kJ/m2) being the most sensitive, followed by T7 (21-fold less sensitive). While both PRD1 and phi 6 have lipid components, only phi 6 was photoinactivated by MC540. Thus the internal lipid components of PRD1 were not sufficient to allow photoinactivation by this dye, at fluences up to 300 kJ/m2. For comparison, we also photoinactivated Herpes simplex virus (Do = 0.053 kJ/m2) and found it to be 28-fold more sensitive than phi 6 to photoinactivation by the same concentration of MC540. Thus phi 6 may be used as a surrogate for enveloped human viruses for photoinactivation by lipophilic dyes, but the results may only be useful qualitatively.

Topics: Animals; Antiviral Agents; Bacteriophages; Cell Line; Dose-Response Relationship, Radiation; Light; Pyrimidinones; Radiation-Sensitizing Agents; Simplexvirus

Action spectra of the antileukemic and antiviral activities of merocyanine 540 (MC540) were determined using L1210 leukemia cells and human Herpes simplex virus type 1. The major peak of both action spectra aligned closely with the absorption spectrum of membrane-bound dye monomer, and by implication, the action spectrum of 1O2 generation. These results are compatible with the notion that the antileukemic and antiviral activities of MC540 are primarily attributable to membrane-bound monomer and at least in part mediated by 1O2.

Topics: Animals; Fluorescent Dyes; Herpes Simplex; Humans; Leukemia; Membranes; Mice; Pyrimidinones; Simplexvirus; Spectrophotometry; Tumor Cells, Cultured

The addition of oxygen to anaerobic rat liver mitochondria incubated at 15 degrees C in the absence of permeant cations produced negligible rapid H+ ejection, monitored spectroscopically with phenol red, which corresponded kinetically to the rise in delta psi, as monitored by merocyanine 540. Slow H+ translocation was observed under these conditions during the aerobic phase, the extent of which was proportional to the amount of oxygen added and the rate dependent on the rate of counter-ion movement. Measurement of H+ disappearance in the mitochondrial matrix, as monitored by neutral red, likewise showed little or no rapid H+ change in the absence of counter-ion movements. In the presence of permeant cations, the H+ disappearance in the matrix was readily measured. This observation argues against the importance of the mitochondrial outer membrane and intermembrane space in masking H+ movements. The H+ translocation required in the generation of maximal or static head delta psi was determined by following the spectral response of merocyanine to increasing oxygen additions. The amount of oxygen giving maximal Delta psi corresponds to an extrusion of 2-3 ng ions of H+ . mg of protein-1. The absence of H+ movement of near this magnitude during the development of the Delta psi argues against the Delta psi-driven backflow of H+ ions as the sole explanation of these observations.

Topics: Animals; Cytochromes; Depression, Chemical; Hydrogen; Membrane Potentials; Mitochondria, Liver; Oxygen; Phenolsulfonphthalein; Potassium; Protons; Pyrimidinones; Rats; Staining and Labeling; Valinomycin

Merocyanine binds extensively to rat liver mitochondria in spite of the presence of a sulfonic acid group which would suggest only limited penetration through the membrane. Passive binding shows both tight and weak binding components and is dependent on salt concentration and ionic strength in accord with the Gouy-Chapman theory. The binding of merocyanine to mitochondria is accompanied by both a fluorescence enhancement and a spectral shift. Induction of an electrical field by either respiration or K+ diffusion potential results in a partial reversal of the spectral shift seen on dye binding. At low temperature, the merocyanine spectral response to an electrical field is biphasic, consisting of a fast phase with a t1/2 of less than 1 sec at 15 degrees C and a slower phase which may vary considerably in rate and extent with conditions. The spectral shift during the two phases appears similar, but differ in sensitivity to ionic strength and temperature. The spectral shift during the fast phase at 15 degrees C indicates that the major component is a decrease in bound monomer and an increase in the aqueous dimer, indicating an "on-off" mechanism. It is suggested that the fast and slow phases of the merocyanine response may be due to two different populations of dye, possibly located at the outer and inner surfaces, respectively, of the mitochondrial membrane. The electrophoretic movement of the dye located in the membrane interior would result in the temperature-sensitive slow phase response. Demonstration of the proportionality of the fast phase response to the magnitude of the membrane potential suggests the usefulness of merocyanine in studies with mitochondrial systems.

Topics: Animals; Binding Sites; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Mitochondria, Liver; Osmolar Concentration; Polyethylene Glycols; Pyrimidinones; Rats; Spectrometry, Fluorescence

This paper examines the relationship between lipid composition, plasma membrane fluidity, expression of dye binding sites, and susceptibility to merocyanine 540 (MC540)-sensitized irradiation in L1210 leukemia cells. Reducing the cells' cholesterol content by exchange diffusion with phosphatidylcholine liposomes or by inhibiting its biosynthesis with 25-hydroxycholesterol enhanced plasma membrane fluidity, the expression of dye binding sites, and the cells' susceptibility to MC540-sensitized irradiation. Conversely, if the cholesterol content was enhanced by exchange diffusion with cholesterol:phosphatidylcholine liposomes, the cells' susceptibility to MC540-sensitized irradiation was decreased. However, contrary to expectations, dye-binding was slightly enhanced and plasma membrane fluidity remained unchanged. Growing the cells in fatty acid-supplemented medium had profound effects on their lipid composition. Cells enriched in polyunsaturated fatty acids had more fluid plasma membranes. However, dye-binding was not significantly affected and photosensitivity was slightly reduced. These results suggest that cholesterol is one, but probably not the only, determinant of the expression of cellular dye binding sites and, consequently, the cell's susceptibility to MC540-sensitized irradiation. By contrast, plasma membrane fluidity does not appear to play a major role in the regulation of dye-binding site expression.

Topics: Animals; Cell Membrane; Cholesterol; Fatty Acids; Fluorescent Dyes; Humans; Hydroxycholesterols; Leukemia L1210; Liposomes; Membrane Fluidity; Phosphatidylcholines; Photosensitivity Disorders; Pyrimidinones; Tumor Cells, Cultured

The merocyanine 540 (MC540)-mediated reduction of nitroxide spin labels in a liposomal system was examined using electron spin resonance (ESR) spectroscopy. Spin label reduction was light driven, and occurred in liposomes composed of both fully-saturated (dimyristoyl) and mono-unsaturated (1-palmitoyl-2-oleoyl) phosphatidylcholine. Loss of the nitroxide ESR signal was enhanced by the physiological electron donors glutathione, cysteine, and NADPH; and was strongly inhibited by the presence of molecular oxygen. Nitroxides reduced in the presence of MC540 alone could be regenerated either by purging the sample with air or by the addition of ferricyanide, indicating that the ESR signal loss was due to reduction to the corresponding hydroxylamines. Only partial regeneration was attained for nitroxides reduced in the presence of glutathione, cysteine, or NADPH. Reduction rates for the lipophilic spin labels, 5-, 12-, and 16-doxyl stearic acid, were not influenced by the position of the nitroxide moiety along the alkyl chain, however reduction of spin labels occupying primarily the aqueous phase was much slower. These studies demonstrate that MC540 can initiate oxidation/reduction (Type I) reactions. Such Type I processes may augment the effects of singlet oxygen in MC540-mediated photodynamic therapy.

Topics: Cysteine; Electron Spin Resonance Spectroscopy; Glutathione; Liposomes; NADP; Oxidation-Reduction; Pyrimidinones; Radiation-Sensitizing Agents; Structure-Activity Relationship

This paper reports on the role of endogenous and exogenous thiols in the merocyanine 540 (MC 540)-sensitized photoirradiation of L1210 leukemia cells, human erythrocytes, and human Herpes simplex virus type 1. Several measures taken to decrease the intracellular content of glutathione enhanced the cells' sensitivity to MC 540-sensitized photoirradiation while stimulation of glutathione biosynthesis or supplementation of the extracellular or extraviral thiol content decreased the photosensitivity of cells and viruses. Taken together, these data suggest that endogenous and exogenous thiols can modulate the sensitivity of cells and enveloped viruses to MC 540-sensitized photoirradiation. They also pose new questions as to the mechanism of MC 540-sensitized photolysis.

Topics: Animals; Cell Line; Erythrocytes; Glutathione; Humans; Leukemia L1210; Light; Mice; Pyrimidinones; Radiation-Sensitizing Agents; Simplexvirus; Sulfhydryl Compounds

The lipophilic photosensitizing dye merocyanine 540 (MC540) is being studied intensively as an antitumor and antiviral agent. Since plasma membranes are believed to be the principal cellular targets of MC540-mediated photodamage, we have studied membrane damage in a well characterized test system, the human erythrocyte ghost. When irradiated with white light, MC540-sensitized ghosts accumulated lipid hydroperoxides (LOOHs derived from phospholipids and cholesterol) at a rate dependent on initial dye concentration. Neither desferrioxamine nor butylated hydroxytoluene inhibited LOOH formation, suggesting that Type I (iron-mediated free radical) chemistry is not important. By contrast, azide inhibited the reaction in a dose-dependent fashion, implicating a Type II (singlet oxygen, 1O2) mechanism. Stern-Volmer analysis of the data gave a 1O2 quenching constant approximately 50 times lower than that determined for an extramembranous target, lactate dehydrogenase (the latter value agreeing with literature values). This suggests that 1O2 reacts primarily at its membrane sites of origin and that azide has limited access to these sites. Using [14C]cholesterol-labeled membranes and HPLC with radiodetection, we identified 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide as the major cholesterol photoproduct, thereby confirming 1O2 intermediacy. Irradiation of MC540-sensitized membranes in the presence of added iron and ascorbate resulted in a large burst of lipid peroxidation, as shown by thiobarbituric acid reactivity and appearance of 7-hydroperoxycholesterol and 7-hydroxycholesterol as major oxidation products. Amplification of MC540-initiated lipid peroxidation by iron/ascorbate (attributed to light-independent reduction of nascent photoperoxides, with ensuing free radical chain reactions) could prove useful in augmenting MC540's phototherapeutic effects.

Topics: Ascorbic Acid; Cholesterol; Erythrocyte Membrane; Free Radicals; Humans; In Vitro Techniques; L-Lactate Dehydrogenase; Lipid Peroxidation; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 +/- 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.

Topics: Cell Membrane; Erythrocyte Membrane; Humans; In Vitro Techniques; Liposomes; Oxygen Consumption; Photochemistry; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents

Merocyanine 540 (MC 540) is a photosensitizing dye that has been used in a phase I clinical trial for the purging of leukemia and lymphoma cells from autologous bone marrow grafts. In this paper we examine the role of plasma membrane negative charge, plasma membrane fluidity, and plasma membrane hydrophobicity in the regulation of a cell's susceptibility to MC 540-sensitized photoirradiation. Among solid tumor cells, we found an inverse correlation between surface electronegativity, affinity for dye molecules, and susceptibility to MC 540-sensitized photoinactivation. That is, the least electronegative cells bound the highest amount of dye and were the most susceptible to dye-sensitized photoirradiation. By contrast, no such correlations were found among leukemia/lymphoma cells. This suggested that dye binding and susceptibility to MC 540-mediated photodynamic damages are regulated differently in hematopoietic/lymphopoietic and solid tumor cells.

Topics: Bone Marrow; Bone Marrow Cells; Cell Membrane; Cell Separation; Humans; Leukemia; Light; Lymphoma; Membrane Fluidity; Neoplasms; Neuraminidase; Pyrimidinones; Radiation-Sensitizing Agents; Solubility; Surface Properties; Trypsin; Tumor Cells, Cultured

The production of singlet oxygen by merocyanine 540 was studied in dimyristoyl-phosphatidylcholine liposomes using two singlet oxygen probes: 9,10-anthracenedipropionic acid (water soluble) and 9,10-dimethylanthracene (liposoluble). Upper and lower limits of singlet oxygen quantum yield for bound merocyanine 540 were determined to be 0.055 and 0.015 respectively. The diffusion characteristics of singlet oxygen were examined using the isotropic enhancement effect of D2O and the inhibitory effect of sodium azide. It was shown that 1O2 spent more than 87% of its lifetime in a vesicle environment. When the singlet-reacting substrate and the dye were both located in the bilayer, approximately 40% of the singlet oxygen remained in the liposomes where it was originally generated.

Topics: Dimyristoylphosphatidylcholine; Kinetics; Light; Liposomes; Mathematics; Oxygen; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Rose Bengal; Singlet Oxygen; Spectrometry, Fluorescence; Spectrophotometry

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.

Topics: Diamide; Erythrocyte Membrane; Fluorescence; Humans; In Vitro Techniques; Membrane Fluidity; Microscopy, Electron; Oxidation-Reduction; p-Dimethylaminoazobenzene; Phenylhydrazines; Photochemistry; Pyrimidinones; Regression Analysis

The effects of primaquine (PQ), its enantiomers [(+)PQ,(-)PQ] and hydroxy metabolites [5-hydroxyprimaquine (5HPQ) and 6-desmethyl-5-hydroxyprimaquine (6D5HPQ)] on cell membranes of glucose-6-phosphate dehydrogenase (G-6-PD) deficient red cells were studied in vitro. There was no significant effect of PQ on the malonyldialdehyde (MDA) content of normal and heterozygous red cells, but it caused a significant increase in MDA in G-6-PD deficient red cells (P less than 0.05). There was no noticeable difference between the effects of the two enantiomers on this variable (P greater than 0.05). Compared to PQ, the hydroxy metabolites produced a significantly greater increase in MDA in all the groups studied (P less than 0.001). Of the two hydroxy metabolites, 6D5HPQ was more toxic than 5HPQ. Staining with MC540 showed that exposure to PQ, its enantiomers and two putative metabolites produced significant fluorescence, indicating that the drug produces marked alterations in membrane fluidity. Although the fluorescence was seen both in normal and heterozygous cells, the effect was marked in hemizygous deficient red cells (P less than 0.001). Scanning electron microscopic (SEM) studies revealed that PQ enantiomers had a stomatocytic effect on red cells of normal, heterozygous and hemizygous G-6-PD deficient red cells, whereas the putative metabolites had an echinocytic effect. The effects were most pronounced in G-6-PD deficient red cells.

Topics: Erythrocyte Membrane; Erythrocytes; Female; Glucosephosphate Dehydrogenase Deficiency; Humans; Lipid Peroxidation; Membrane Fluidity; Microscopy, Electron, Scanning; Primaquine; Pyrimidinones; Stereoisomerism

The photophysical properties of merocyanine 540 have been determined in methanol solution over a modest temperature range. Triplet state population is inefficient (the limiting triplet quantum yield being 0.25) due to rapid isomerization of the central double bond from the first excited singlet state. Activation energies have been measured for isomerization from the excited singlet state (20 kJ mol-1) and for conversion of the resultant cis-isomer back to the original trans-form (63 kJ mol-1), both processes involving formation of a twisted species. The dye is easily oxidized to give an unstable adduct which decomposes on the sub-ms timescale. Reversible redox chemistry occurs upon excitation in the presence of electron acceptors. These various observations are discussed in terms of the known chemotherapeutic activity of MC540 and it is concluded that the most probable mechanisms for cytotoxicity involve either local thermal disruption of cell membranes or in situ photogeneration of toxins derived from breakdown of the dye.

Topics: Molecular Structure; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Thermodynamics

The photosensitizing dye merocyanine 540 (MC 540) was evaluated as a means for purging malarially infected red cells from murine blood using the rodent malarial pathogens, Plasmodium yoelii and Plasmodium berghei, as models of human malaria. Malarially infected red cells bound more MC 540 and were more sensitive to MC 540-sensitized photoirradiation than were noninfected erythroid cells. Extracorporeal exposure of infected red cells to the dye and white light prevented the transmission of the disease in a transfusion model. P. berghei-infected red cells were more resistant to the antimalarial activity of MC 540 than were P. yoelii-infected cells, presumably because P. berghei preferentially infects reticulocytes whereas P. yoelii infects mature red cells. The possibility of using photoirradiation sensitized by MC 540 or related dyes to purge malarially infected donor blood is discussed.

Topics: Animals; Blood Transfusion; Disease Models, Animal; Erythrocytes; Female; Flow Cytometry; Malaria; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Microscopy, Fluorescence; Photochemotherapy; Plasmodium; Plasmodium berghei; Plasmodium yoelii; Pyrimidinones; Radiation-Sensitizing Agents

Liposomes of dipalmitoylphosphatidylcholine (DPPC) prepared in increasing glycerol/glucose ratios show an increase in the absorbance at 570 nm of merocyanine spectra at temperatures below the phase transition. Since this effect is not observed when liposomes are prepared in solutions containing solely glucose, it is attributed to specific interactions of glycerol with the membrane phase. The increase in the 570 nm absorbance is ascribed to a partial fluidification of the membrane interface and is dependent on the distribution of the dye between the inner and the outer compartments of the liposomes and on their osmotic state. The greatest differences in the absorbance ratio are obtained when merocyanine is added to the external media. In consequence, the changes in the spectra of MC are dependent on the surface state of the liposomes which can be modified by an increase of glycerol or glucose in the external media. The present results are examined in the light of the perturbations that glycerol can induce on the barrier properties of the bilayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cell Membrane; Glycerol; Lipid Bilayers; Liposomes; Osmotic Pressure; Pyrimidinones; Staining and Labeling

A novel photoelectrochemical cell using a proton pump mechanism in the aggregated planar structure of oxidised cholesterol incorporating merocyanine dyes is reported. Lipid dye binding, as verified from spectral studies and photoisomerisation of the dye, is responsible for this photovoltage generation whose magnitude and storage duration are related to the equilibrium constant of dye-lipid binding through an empirical formula.

Topics: Biological Transport; Cholesterol; Electrochemistry; Lipids; Membranes, Artificial; Protons; Pyrimidinones

Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light-exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light-exposed MC540 resulted in 70-90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte-macrophage colony forming cells (CFU-GM) survived the treatment. The observed cytotoxicity of light-exposed MC540 to the tumor cells was significantly greater (P less than 0.05) than the native MC540. Results show that tumor cell specificity and cytotoxicity in the light activated dye are retained for at least 30 days. Addition of catalase and mannitol decreased the cell kill by light-exposed compound, indicating that the observed effects may be due to reactive oxygen species. The electron micrographs of treated cells show a progression towards apoptosis in a majority of the cells. The life span of L1210 leukemia-bearing mice treated with light-exposed MC540 was prolonged compared to the untreated and native MC540 treated mice. High pressure liquid chromatography (HPLC) analysis of light-exposed material shows a completely different elution profile compared to the native compound. Results presented here show that light-exposed photoactive compounds can be used without further illumination and may have significant clinical applications. Photoactive mechanisms dependent on events other than short-lived transient elevations in energy or singlet oxygen must be invoked to explain the reported cytotoxicity.

Topics: Animals; Cell Survival; Light; Mice; Mice, Inbred DBA; Pyrimidinones; Tumor Cells, Cultured

This study demonstrates the binding of various fluorescent dyes (3,3' dihexyloxacarbocyanine iodide [DiOC6I], doxycycline [DOTC], rhodamine 123, and merocyanine 540) to infectious and intracellular forms of the Tulahuen strain of Trypanosoma cruzi. These dyes predominantly localize in mitochondria. Following treatment with DiOC6I and DOTC, both irradiated and nonirradiated samples showed dark toxicity to T. cruzi, whereas the other dyes effected toxicity only following irradiation with light. Under in vitro conditions, 91% protection was obtained 96 hr postinfection under dark conditions through the use of 0.573 micrograms/ml of DiOC6I. During in vivo studies, the onset of parasitemia was delayed by 7 days through the use of DiOC6I in ng/ml levels. Host deaths occurred in the infected control group on day 11 postexposure, whereas in the 5.7-ng/ml dye-treated group, no death had occurred after 20 days postexposure. This study demonstrates delay of onset of T. cruzi infections with the use of DiOC6I at concentrations well below the levels toxic to the host.

Topics: Animals; Carbocyanines; Cell Line; Chagas Disease; Cytosol; Doxycycline; Female; Fluorescent Dyes; Mice; Mice, Inbred ICR; Mitochondria; Pyrimidinones; Rhodamine 123; Rhodamines; Spectrometry, Fluorescence; Trypanosoma cruzi

The potential of various photoradiation therapy for the in vitro purging of residual tumor cells from autologous bone marrow (BM) transplants is discussed in this paper. The results with fluorescent dyes, Dihematoporphyrin Ether (DHE) and Merocyanine-540 (MC-540) are detailed. Following photoradiation of cells with white light, both DHE and MC-540 showed high cytocidal activity towards lymphoid and myeloid neoplastic cells, but had significantly less effect on normal granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and mixed colony-forming (CFU-GEMM) progenitor cells. Acute promyelocytic leukemia (HL-60), non-B, non-T, cALLa positive acute lymphoblastic leukemia (Reh), and diffuse histiocytic B-cell lymphoma (SK-DHL-2) cell lines were exposed to different drug concentrations in combination with white light at a constant illumination rate of 50,000 lux. With DHE doses varying from 2.0 to 2.5 ug/ml and MC-540 concentrations of 15 to 20 ug/ml, clonogenic tumor cells could be reduced by more than 4 logs, when treated alone or in mixtures with normal irradiated human marrow cells. However, preferential cytotoxicity towards neoplastic cells was highly dependent on the mode of light activation. MC-540 had no substantial effect on malignant lymphoid (SK-DHL-2) and myeloid (HL-60) cells, and on normal marrow myeloid (CFU-GM) precursors, when the drug incubation was performed in the dark and followed by light exposure of washed cells. Equal doses of MC-540 (15-20 ug/ml) could preferentially eliminate tumor cells under conditions of simultaneous light and drug treatment (30 minutes at 37 degrees C). Using DHE (2.5 ug/ml), 29.3%, 46.8%, and 27.5% of normal marrow CFU-GM, BFU-E, and CFU-GEMM, respectively, were spared, following sequential drug and light exposure of cells, while simultaneous treatment reduced both normal (CFU-GM) and neoplastic cells below the limits of detection. The data from various centers is briefly discussed with special emphasis on clinical trials. Our results provide a useful model for leukemia and lymphoma cells and suggest that these phototherapy experiments can be implemented into clinical trials.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Survival; Colony-Forming Units Assay; Dihematoporphyrin Ether; Hematoporphyrins; Humans; Leukemia, Lymphoid; Leukemia, Promyelocytic, Acute; Lymphoma; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous; Tumor Cells, Cultured

Merocyanine 540 was activated by exposure to 514 nm laser light. This preactivated merocyanine 540 was then mixed (in the dark) with tumour cells, normal cells and envelope viruses to assess its antiproliferative activity. This treatment resulted in 70-90% killing of tumour cells from different cell lines while 85% of normal human peripheral blood mononuclear cells survived the treatment. However, not all types of tumour cells were affected. Preactivated merocyanine 540 was also effective in virtually completely inactivating cell-free herpes simplex and human immunodeficiency viruses. Preactivated photoactive compounds can exert their toxic effects in the dark without further dependence on light and may have potential systemic use.

Topics: Cell Survival; HIV-1; Humans; Lasers; Leukocytes, Mononuclear; Mitosis; Pyrimidinones; Radiation-Sensitizing Agents; Simplexvirus; Tumor Cells, Cultured

Dry or wet heat, solvents, and detergents combined with ultraviolet irradiation provide effective means of sterilizing soluble blood products such as albumin or factor VIII. For obvious reasons, these procedures are not applicable to cellular blood components. We have recently shown that simultaneous exposure to the photosensitizer, merocyanine 540 (MC 540) and white light rapidly inactivates the Friend erythroleukemia virus complex and Friend virus-transformed cells, but causes relatively little damage to pluripotent hematopoietic stem cells. In this communication, we show that several lipid-enveloped human pathogenic viruses are also highly susceptible to MC 540-sensitized photoirradiation, and we report on an initial evaluation of the ability of MC 540-sensitized photoirradiation to sterilize blood products.

Topics: 2,3-Diphosphoglycerate; Adenosine Triphosphate; Adenoviruses, Human; Animals; Antiviral Agents; Blood Transfusion; Cell Line; Cytomegalovirus; Diphosphoglyceric Acids; Erythrocytes; Human T-lymphotropic virus 1; Humans; In Vitro Techniques; Kinetics; Light; Pyrimidinones; Radiation-Sensitizing Agents; Simplexvirus

The protein and phospholipid composition of microvesicles released from normal human erythrocytes after ATP depletion, on aging or by treatment with merocyanine 540, dimyristoyl phosphatidylcholine or Ca2+/ionophore A23187 has been compared with the composition of the original cell membrane. It has been shown that these microvesicles are depleted of band 3, glycophorin and phosphatidylinositol 4,5-bisphosphate relative to phospholipid by 40% or more. These data are interpreted to mean that less than half of these membrane components are free to diffuse laterally in the lipid bilayer. Acetylcholinesterase was found to be enriched 2-3-fold in microvesicles, possibly because the removal of non-diffusing proteins from the vesiculating region of the lipid bilayer allows more space for freely diffusing proteins like acetylcholinesterase to enter the microvesicle membrane.

Topics: Anion Exchange Protein 1, Erythrocyte; Calcimycin; Calcium; Diffusion; Dimyristoylphosphatidylcholine; Erythrocytes; Humans; Lipid Bilayers; Lipids; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pyrimidinones; Radiation-Sensitizing Agents

Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. Its mechanism of action is not yet well understood. This paper investigates the sites of MC 540-mediated photodamages in L1210 leukemia cells by examining the effects of MC 540-sensitized photoirradiation on several soluble and membrane-bound marker enzymes. When exposed to MC 540 and white light under a standard set of conditions, the activities of Na+/K(+)-ATPase, Mg2(+)-ATPase, and 5'-nucleotidase (three plasma membrane-bound enzymes) were reduced by 54, 49, and 55%, respectively. None of the intracellular enzymes included in this survey was affected by MC 540-sensitized photoirradiation as long as the plasma membrane remained intact. The two soluble enzymes, lactate dehydrogenase and malate dehydrogenase, remained refractory to MC 540-sensitized photoirradiation even after the plasma membrane had been disrupted. By contrast, the activities of the membrane-bound enzymes, NADPH-cytochrome c reductase and succinate dehydrogenase, were reduced in cell lysates by 55 and 81%, respectively. Purified NADPH-cytochrome c reductase was about 3 times less sensitive than the microsomal enzyme, suggesting that the membrane environment facilitated photoinactivation. The MC 540-sensitized photoinactivation of enzymes was accelerated in the presence of deuterium oxide and inhibited if oxygen in the medium was displaced by nitrogen or azide was added to the medium. Taken together, these data support the view that the plasma membrane is a major target of MC 540-mediated photodamages, that the inactivation of membrane-bound enzymes is an oxidative process, and that at least some photodynamic damages are mediated by type II chemistry.

Topics: 5'-Nucleotidase; Animals; Ca(2+) Mg(2+)-ATPase; Cell Membrane; Clone Cells; Dose-Response Relationship, Radiation; Free Radicals; Kinetics; Leukemia L1210; Light; Mice; NADPH-Ferrihemoprotein Reductase; Oxygen; Pyrimidinones; Radiation-Sensitizing Agents; Singlet Oxygen; Sodium-Potassium-Exchanging ATPase

Topics: Antiviral Agents; Blood Proteins; Blood Transfusion; HIV-1; Human T-lymphotropic virus 1; Humans; Light; Pyrimidinones; Radiation-Sensitizing Agents

Cytotoxic T lymphocytes (CTL) release from their granules a 70 kDa protein, called PFP, perforin or cytolysin, which inserts into the target cell plasma membrane in its monomeric form. Here it polymerizes into a macromolecular complex forming pores as large as 20 nm. Although purified PFP/perforin can effectively lyze all target cells tested. CTL are refractory to lysis. The mechanism underlying the resistance of CTL is currently unknown. This study represents a search for membrane structural properties that could confer resistance to CTL against PFP/perforin-mediated lysis. The fluorescent dye merocyanine 540 was used to measure the lipid head group packing of CTL and several target cells, and 1-[4-(trimethylamine)phenyl]-6-phenylhexa-1,3,5-triene was used to estimate the fluidity of the membrane hydrocarbon region. The resistance against PFP/perforin-mediated lysis was determined by the 51Cr release assay. A comparison of the membrane rigidity with cell resistance led to the conclusion that the membrane lipid structure cannot account for the unusually high resistance of CTL. In particular, the resistant CTL line CTLL-2 has a lipid head group packing that is looser than that of Yac-1, and the sensitive target cells Jy-25 and EL-4 have membrane acyl chains that are less fluid than those of the effector CTLL-R8.

Topics: Animals; Cell Line; Cell Membrane; Diphenylhexatriene; Erythrocyte Membrane; Fatty Alcohols; Fluorescent Dyes; Hemolysis; Membrane Fluidity; Membrane Glycoproteins; Membrane Lipids; Membrane Proteins; Mice; Perforin; Pore Forming Cytotoxic Proteins; Pyrimidinones; T-Lymphocytes, Cytotoxic

The effect of primaquine enantiomers on cell membranes of glucose-6-phosphate (G-6PD)-deficient erythrocytes was studied in vitro. Staining with merocyanine (Mc-540) showed that exposure to primaquine enantiomers produces significant fluorescence in G-6PD-deficient erythrocytes, indicating marked drug-induced alterations in membrane fluidity. Scanning electron microscopy (SEM) studies confirmed that primaquine enantiomers altered membrane morphology (by producing stomatocytes) in both normal and G-6PD-deficient cells. The concentration-dependent effect, however, was more pronounced with MC-540, a lipophylic dye, than with SEM (an expensive technique).

Topics: Erythrocyte Membrane; Fluorescent Dyes; Glucose-6-Phosphate; Glucosephosphates; Humans; Microscopy, Electron, Scanning; Primaquine; Pyrimidinones

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.

Topics: Actins; Animals; Erythrocyte Deformability; Erythrocyte Membrane; Fluorescamine; Hot Temperature; Hydrolysis; Macaca mulatta; Microscopy, Electron, Scanning; Phospholipases A; Phospholipids; Pyrimidinones; Radiation-Sensitizing Agents; Spectrin; Venoms

Monomers and aggregates of Merocyanine 540 (MC540) in water are able to photoisomerize. The shape of the photoisomer absorption spectrum is very similar to that of the ground state. Triplet state of MC540 in water has been produced by energy transfer from triplet anthracene and displays a broad absorption spectrum between 600 and 700 nm. The triplet state may also be produced by direct excitation of MC540 with UV light. However, when the dye is excited by visible light, no triplet state absorbance in the red could be detected so that the triplet yield of MC540 in water seems to be excitation wavelength dependent.

Topics: Kinetics; Photolysis; Pyrimidinones; Radiation-Sensitizing Agents; Water

Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. In this paper we present evidence that the MC 540-sensitized photoinactivation of leukemia cells is an oxygen-dependent process and that unsaturated plasma membrane lipids are substrates for singlet oxygen and/or other activated oxygen species generated by photoirradiated MC 540. A comparison of the inhibition of clonal growth, the inhibition of mitochondrial respiration, and the exclusion of trypan blue by the plasma membrane after exposure to MC 540 and graded doses of light showed that mitochondrial respiration is compromised relatively early in the course of the dye-mediated photoinactivation of cells, well before the plasma membrane loses its capacity to exclude trypan blue. It also showed that trypan blue exclusion assays can greatly underestimate the cytotoxic effects of MC 540-sensitized photoirradiation.

Topics: Animals; Cell Membrane; Fatty Acids, Unsaturated; Humans; Leukemia; Mice; Mitochondria; Oxygen; Oxygen Consumption; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Topics: Bone Marrow; Bone Marrow Transplantation; Breast Neoplasms; Cell Survival; Colony-Forming Units Assay; Combined Modality Therapy; Doxorubicin; Female; Humans; Laser Therapy; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous; Tumor Cells, Cultured

Peroxidation of erythrocyte membrane lipids by hydrogen peroxide perturbs the lipid bilayer and increases phagocytosis by macrophages. This study addresses the underlying mechanism of these processes, and in particular the role of malondialdehyde, a major byproduct of lipid peroxidation. When erythrocytes were treated with hydrogen peroxide or ascorbate/iron to generate malondialdehyde, or with malondialdehyde itself, only those cells treated with hydrogen peroxide showed increased phospholipid spacing and enhanced phagocytosis. This result indicates that the alterations observed are unique to hydrogen peroxide treatment, and that malondialdehyde does not play a role in inducing these changes in surface properties. Comparison of adherence to human umbilical vein endothelial cells and phagocytosis showed that increased phagocytosis was not mirrored by enhanced adherence. This result suggests that two different signals may mediate recognition of erythrocytes by macrophages and by endothelial cells.

Topics: Cell Adhesion; Cells, Cultured; Erythrocyte Membrane; Hemolysis; Humans; Hydrogen Peroxide; Lipid Bilayers; Lipid Peroxidation; Macrophages; Malonates; Malondialdehyde; Membrane Lipids; Phagocytosis; Phospholipids; Pyrimidinones; Spectrometry, Fluorescence

Merocyanine 540 (M540) is a potential-sensitive, hydrophobic dye that preferentially incorporates into the 'fluid' domains of cellular membranes, distinguishing between hemopoietic cells according to their differentiation state. A bright staining with M540 is usually achieved by UV illumination of the cells during staining. We show by flow cytometric analysis that: (1) staining is greatly enhanced by UV illumination of mouse spleen cells before addition of the dye; (2) UV treatment causes an increased permeability toward propidium iodide and intracellular fluorescein as well; (3) the increment in M540 fluorescence precedes permeabilization to propidium iodide, while the latter precedes leakage of fluorescein. We also describe an overshoot and accelerated recovery of M540 fluorescence after photobleaching by a 514 nm laser beam. It is suggested that penetration of M540 to the more fluid inner membrane structures explains the fluorescence increment in both experiments.

Topics: Animals; Cell Membrane; Cell Membrane Permeability; Flow Cytometry; Fluoresceins; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Photochemistry; Propidium; Pyrimidinones; Radiation-Sensitizing Agents; Rauscher Virus; Spleen; Staining and Labeling; Ultraviolet Rays

6-Hydroxydopamine(6-OHDA) and Merocyanine-540(MC-540) have been used clinically for purging of neuroblastoma cells prior to autologous bone marrow transplantation. Both substances were found to be more toxic against neuroblastoma cells than against hematopoietic stem cells. The more pronounced cytotoxic effects of 6-OHDA against neuroblastoma cells were not caused by its selective uptake; the rapid autooxidation at physiological pH leads to the formation of H2O2 already in the incubation medium. Cytotoxic effects were not detected in short-time test systems (4 hour chromium-51 release assay) but only after longer incubation periods. In contrast, MC-540 proved to be toxic almost equally in short- and long-time test systems. 4-Hydroxynonenal(4-HNE) that may be formed in the plasma membrane subsequently to photoactivation of MC-540 was only slightly more toxic to neuroblastoma cells than to hematopoietic cells. Although the use of 6-OHDA and MC-540 in bone marrow purging has some limitations, the sensitivity of neuroblastoma cells against reactive oxygen compounds may be exploited more generally for therapy of this tumor.

Topics: Aldehydes; Antineoplastic Agents; Cell Survival; Hematopoietic Stem Cells; Humans; Hydrogen Peroxide; Hydroxydopamines; Neuroblastoma; Oxidopamine; Pyrimidinones; Tumor Cells, Cultured

One limitation of autologous bone marrow transplantation for patients with cancer has been the presence of tumor cells in the bone marrow. Methods to eliminate tumor cells while preserving hematopoietic stem cells have been sought. The present study was performed to analyze the in vitro effectiveness of light-activated merocyanine 540 phototreatment (LAMP) and an aminothiol (ethiofos) as a marrow-purging regimen for small cell lung cancer (SCLC). Two human SCLC cell lines (ATCC HTB-119 and HTB-120) were treated with LAMP and exposed to light for varying periods of time up to 120 min. LAMP reduced SCLC cell proliferation and colony formation in a light exposure-dependent manner; colony formation was not totally inhibited until light exposure of 120 min was used. At this light exposure interval, multipotential hematopoietic progenitors, colony-forming units-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), were substantially reduced. In an attempt to diminish hematopoietic toxicity, SCLC cells were incubated with ethiofos (formerly WR-2721) for 1 hour before LAMP. SCLC colony formation was eliminated at light exposure intervals (90 min or less) which had no inhibitory effect on CFU-GEMM. Ethiofos did not protect CFU-GEMM from LAMP inhibition at 120 min. Ethiofos alone had no effect on the SCLC or hematopoietic cells. When normal bone marrow was contaminated with 1 or 5% SCLC cells, ethiofos plus 60 min of LAMP eliminated SCLC cells but had no effect on CFU-GEMM. The results suggest that ethiofos sensitized SCLC cells to LAMP; thus ethiofos-enhanced LAMP may be an effective method for removing metastatic SCLC cells from bone marrow used for autologous marrow transplantation after high dose chemotherapy.

Topics: Amifostine; Bone Marrow; Carcinoma, Small Cell; Cell Survival; Hematopoiesis; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Organothiophosphorus Compounds; Photochemotherapy; Pyrimidinones; Radiation-Protective Agents; Tumor Cells, Cultured

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.

Topics: Animals; Cell Differentiation; Cell Line; Chick Embryo; Drug Resistance; Light; Muscles; Pyrimidinones; Radiation-Sensitizing Agents; Stem Cells

The efficacy of photosensitization by merocyanine 540 (MC540), a lipophilic fluorescent dye, was investigated in the murine B cell leukemia (BCL1). Normal BALB/c mice were injected with BCL1 cells exposed to MC540, followed by photosensitization with white light for 15 min to 2 h. Mice injected with BCL1 cells exposed for 1 or 2 h showed no sign of leukemia. Lethally irradiated mice were successfully reconstituted with mixtures of syngeneic bone marrow (BM) and BCL1 cells treated with MC540 following exposure to white light. Exposure of BM/BCL1 mixtures for 2 h proved to be effective in purging all BCL1 cells without affecting BM viability, as documented by normal hemopoietic reconstitution of all recipients surviving without evidence of leukemia. All recipients of untreated BM/BCL1 cell mixtures developed leukemia within 42 days. Adoptive transfer of 10(6) spleen cells obtained from treated mice into secondary naive syngeneic recipients was carried out in order to test for dormant BCL1 cells in treated recipients. No leukemia developed in any of the secondary recipients. Previous studies indicate that as few as 10, or possibly less, BCL1 cells are sufficient to cause lethal disease in BALB/c recipients. Our results suggest that MC540 may be an extremely potent tool for in vitro elimination of residual tumor cells while leaving uncommitted progenitor hemopoietic cells intact for hemopoietic reconstitution following lethal marrow ablation.

Topics: Animals; Antineoplastic Agents; Bone Marrow; Bone Marrow Transplantation; Cell Line; Leukemia, B-Cell; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Phototherapy; Pyrimidinones; Radiation Chimera; Radiation-Sensitizing Agents

Topics: Animals; Bone Marrow; Bone Marrow Transplantation; Hematopoietic Stem Cells; Humans; Leukemia; Leukemia, Experimental; Mice; Neoplastic Stem Cells; Neuroblastoma; Photochemistry; Preoperative Care; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Tumor Stem Cell Assay

Human acute myelogenous or lymphoblastic leukemia cells of the K-562 and CCRF-SB lines were mixed with an excess of normal human bone marrow cells to simulate a leukemia remission marrow. The cell mixtures were then incubated in vitro with mafosfamide (AZ) followed by the photoreactive dye merocyanine-540 (MC). Treated cells (1 x 10(4] were seeded in microwell plates, and increasing numbers of the line used to contaminate the normal marrow were added. Treatment with AZ alone produced total elimination (i.e., 6 logs) of CCRF-SB cells, while addition of merocyanine-540 increased the cloning efficiency from 22% to 24.4%. After treatment of the K-562-contaminated cell mixtures with AZ, nearly 1.6 logs of K-562 acute myelogenous blasts were still present, whereas AZ purging followed by MC-mediated photosensitization resulted in 100% elimination of clonogenic cells. Moreover, the combined treatment caused an increase of the cloning efficiency from 37.3% to 62%, clearly indicating that cleansing by the two agents combined was more effective than treatment with one agent alone.

Topics: Antineoplastic Agents; Bone Marrow; Cell Line; Colony-Forming Units Assay; Cyclophosphamide; Dye Dilution Technique; Humans; Leukemia; Light; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Stem Cell Assay

Echinocytosis and release of microvesicles from human erythrocytes treated with the impermeant fluorescent dye merocyanine 540 (MC540) has been correlated with the extent of dye binding to intact cells and ghosts. At 20 degrees C binding appeared to saturate at about 9.3.10(6) molecules per cell (3.6 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 2.7%. Stage 3 echinocytes were formed upon binding of (3-4).10(6) molecules of MC540/cell (about 1.3 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 1.0%. Negligible release of microvesicles was observed with MC540 at 20 degrees C. Binding of MC540 to permeable ghosts was approximately twice that to cells suggesting that there was no selective binding to the unsaturated (more fluid) phospholipids which are concentrated in the inner lipid leaflet of the membrane. At 37 degrees C apparent maximal binding of MC540 was about 3.2 mol/100 mol phospholipid and correlated with the maximal release of microvesicles from the cells as measured by release of phospholipid and acetylcholinesterase. These results are discussed in relation to the bilayer couple hypothesis of Sheetz and Singer (Proc. Natl. Acad. Sci. USA 71 (1974) 4457-4461).

Topics: Erythrocyte Membrane; Erythrocytes; Fluorescent Dyes; Humans; Kinetics; Membrane Lipids; Phospholipids; Pyrimidinones

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus-transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.

Topics: Animals; Antiviral Agents; Cell Line, Transformed; Cell Transformation, Viral; Cell-Free System; Drug Resistance, Microbial; Female; Friend murine leukemia virus; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred DBA; Pyrimidinones; Radiation-Sensitizing Agents; Virion

Merocyanine 540 (MC 540) is a photosensitizing dye with antineoplastic and antiviral properties. Because photoexcited MC 540 generates singlet molecular oxygen and possibly other reactive oxygen species there is concern that MC 540-mediated photosensitization may be mutagenic. In this paper, we report on the induction of ouabain- and 6-thioguanine-resistant mutants in Chinese hamster ovary (CHO) cells by MC 540 and light. Incubation with merocyanine 540 in the dark was not mutagenic. Simultaneous exposure to dye and high-intensity (70-75 W/m2) white or green light caused a small but significant increase in the frequency of both ouabain- and 6-thioguanine-resistant mutants. Similar increases in mutation frequencies were observed when the cells were exposed to white or green light in the absence of dye. When the fluence rate of the white light source was reduced by 50% (i.e., to 35 W/m2) but the duration of exposure doubled to keep the fluence constant, dye-mediated photosensitization and exposure to light alone generated few if any mutants. These results indicated that MC 540-mediated photosensitization is not mutagenic (as defined by the employed assays) as long as a nonmutagenic light source is used to excite the photosensitizer. They caution against the use of very powerful light sources in clinical applications of MC 540-mediated photosensitization.

Topics: Animals; Cell Line; Cricetinae; Cricetulus; Drug Resistance; Female; Light; Mutagenicity Tests; Ouabain; Ovary; Pyrimidinones; Radiation-Sensitizing Agents; Thioguanine; Ultraviolet Rays

Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested. These results may have therapeutic relevance in patients undergoing interferon treatment who require bone marrow transplantation, as the complete elimination of tumor cells by marrow-purging procedures may be hampered by this increased survival in the presence of interferon.

Topics: Bone Marrow; Bone Marrow Cells; Burkitt Lymphoma; Cell Line; Humans; Interferon Type I; Laser Therapy; Lymphoma, Non-Hodgkin; Pyrimidinones; Radiation-Sensitizing Agents; Recombinant Proteins; Tumor Cells, Cultured

Atherosclerotic lesions have been reported to contain herpes simplex virus 1 (HSV-1) genomic material. This, and other previous evidence, suggests that latent viral infection may be an atherogenic trigger. Moreover, active HSV-1 lesions manifest marked fibrin deposition in microvessels. In this report we show that very early infection of human endothelial cells with HSV-1 appears to alter surface conformation as detected by merocyanine 540 staining. Concomitantly, the efficiency of prothrombinase complex assembly increases, resulting in a 2- to 3-fold accelerated rate of thrombin generation on the cell surface. Increased thrombin generation is probably doubly procoagulant, since we also demonstrate that thrombin-induced platelet accumulation on HSV-infected endothelium (50.7 +/- 9.3%) is increased compared to uninfected endothelium (9.5 +/- 2.1%; P less than 0.002). Associated with HSV infection, prostacyclin secretion in response to thrombin is diminished by a factor of 20, probably explaining the enhanced platelet attachment. We conclude that HSV infection shifts endothelial cell properties from anticoagulant to procoagulant, both by promoting prothrombinase complex formation and function and by increasing platelet binding, well before cell disruption takes place. Virus-induced changes in the endothelial plasma membrane and diminished prostacyclin secretion are suggested as the pathways for this pathophysiologic mechanism, which may be germane to atherosclerotic thrombosis as well as HSV-mediated tissue necrosis.

Topics: Arteriosclerosis; Blood Platelets; Cell Adhesion; Cell Aggregation; Endothelium; Herpes Simplex; Humans; Indomethacin; Pyrimidinones; Simplexvirus; Thrombin; Thromboplastin

In a model for ex-vivo purging of bone marrow grafts, leukemic cells and normal bone marrow cells were treated with merocyanine 540 and exposed to 514 nm laser light. With this treatment, 99.9999% of leukemic cells were killed while 55% of the normal bone marrow cells survived. The deleterious effects of laser light alone in the absence of photosensitizer were not observed as determined by cell viability, cell migration, and response of target cells to human migration inhibition factor. These results indicate that laser light induced photodynamic therapy can be useful for ex-vivo autologous bone marrow purging without regard to the deleterious effects of laser light alone.

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Cell Migration Inhibition; Cell Survival; Humans; Lasers; Leukemia, Myeloid, Acute; Macrophage Migration-Inhibitory Factors; Macrophages; Photochemotherapy; Pyrimidinones; Tumor Cells, Cultured

The quantum yields of singlet oxygen production by merocyanine 540 have been measured during visible light irradiation performed in methanol and ethanol. These appear to be one hundred times smaller than the quantum yield for rose bengal measured under the same conditions. Flash photolysis experiments demonstrate the ability of merocyanine 540 molecules to isomerize under visible light irradiation: the isomerization quantum yields are about 0.65 in both ethanol and methanol. This information combined with the fluorescence quantum yield data account for the low values for singlet oxygen production.

Topics: Isomerism; Light; Oxygen; Photochemistry; Photolysis; Pyrimidinones; Radiation-Sensitizing Agents; Singlet Oxygen; Spectrometry, Fluorescence

The orientation and morphology of the endothelium lining the cardiovascular system may result from hemodynamic forces acting on the endothelial cells. To investigate the flow effects at the membrane level, we have examined the variations of the fluorescence intensity of two membrane-sensitive dyes, merocyanine 540 and bis(1,3-diethylthiobarbiturate)trimethineoxonol, (i) as a function of flow shear stress and (ii) with the onset or cessation of the flow. We found a time-dependent decrease in fluorescence intensity with the onset of the flow with an exponential approach to steady state of the order of 1 min. The process is reversible; when the flow is stopped the fluorescence intensity returns to its original value. The polarization of the endothelial cell membranes or, more precisely, the amplitude of the fluorescence intensity responses is an increasing function of the shear stress (up to 120 dynes/cm2). Assuming the equilibrium potential for K+ is more hyperpolarized than the resting potential and using valinomycin, we have deduced from the sign of the ionophore effects that the flow hyperpolarizes the endothelial cell membrane.

Topics: Animals; Cattle; Endothelium, Vascular; Fluorescence Polarization; Hemodynamics; Kinetics; Membrane Potentials; Potassium; Pulmonary Artery; Pyrimidinones; Thiobarbiturates; Time Factors; Valinomycin

We studied the effects of 514-nm laser light-induced merocyanine 540 (MC540)-mediated toxicity on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) cells were incubated with MC540 (20 micrograms/ml) and exposed to 93.6 J/cm2 irradiation at a 514-nm wavelength. Normal bone marrow cells were treated under similar conditions. At this dose, 99.9999% of the leukemic cells were killed while 55% of the BM cell survived. Of the granulocyte-macrophage colony-forming cells (CFU-GM), 27% also survived this treatment. Photosensitization of a mixture of irradiated BM cells mixed with an equal number of nonirradiated HL-60 cell did not interfere with the killing of HL-60 cells. There was no significant reduction in the viability of cells when exposed to the laser light alone. In summary, laser light-induced photosensitization with MC540 has a selective cytotoxicity to leukemic cells; therefore, this procedure may be useful for purging neoplastic cells from autologous BM.

Topics: Bone Marrow; Bone Marrow Cells; Cell Survival; Colony-Forming Units Assay; Dose-Response Relationship, Radiation; Granulocytes; Humans; Laser Therapy; Leukemia, Myeloid, Acute; Macrophages; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Stem Cells; Tumor Cells, Cultured

Differential scanning calorimetry (DSC) has been employed to determine the effect of five commonly employed extrinsic potential-sensitive probes on phase transitions of multilamellar suspensions of L-alpha-dimyristoylphosphatidylcholine (DMPC). At mol% values of less than five, the effect of these probes on the excess heat capacity curve in the vicinity of the gel to liquid crystal phase transition can be described by an equation based on the formation of ideal solutions in both phases. Even at up to 4 mol%, these dyes only moderately reduce the enthalpy change associated with this transition, but cause a marked decrease in the size of the cooperative unit parameter. The excess heat capacity profile for diS-C3-(5) is represented by the ideal solution equation, even at 12 mol%, whereas the suspensions with the other probes present at this level have profiles covering large temperature ranges. Multiple peaks appear at the higher levels for the negatively charged oxonols V and VI, and merocyanine 540, a result consistent with the presence of well-defined microdomains or even phase separation. The enthalpy change associated with the transition near 15 degrees C involving packing in the headgroup region is decreased significantly, indicating that the probes probably affect the lipid headgroup conformation, even at low levels. The cyanine probe diS-C3-(5) causes the heat capacity profile of small unilamellar vesicles to be transformed very rapidly into one similar to that of the vortexed lipid preparations, presumably by a dye-mediated vesicle fusion process, enhanced by the surface location of this probe. All our results are consistent with diS-C3-(5) being located on the surface of the bilayer in both phases, but a penetration of the other probes into the hydrocarbon region, at least in the liquid crystal phase.

Topics: Algorithms; Calorimetry, Differential Scanning; Dimyristoylphosphatidylcholine; Isoxazoles; Mathematics; Molecular Probes; Pyrimidinones

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.

Topics: Dimyristoylphosphatidylcholine; Fluorescent Dyes; Kinetics; Liposomes; Luminescent Measurements; Oxygen; Photochemistry; Pyrimidinones; Singlet Oxygen

Merocyanine 540 (MC540) is a fluorescent probe that binds preferentially to membranes with loosely packed lipids. When combined with flow cytometry, it provides a novel methodology for rapidly and quantitatively assessing lipid organization in individual leukocytes. Analysis of cells stained simultaneously with MC540 and 1-[4-trimethylammoniumphenyl]-6-phenyl-1,3,5-hexatriene, to normalize for surface area, revealed that all leukocytes in peripheral blood bind equivalent amounts of dye per unit surface area. This result indicates that the lipids of the plasma membranes of all types of circulating cells are organized similarly. Upon activation with appropriate stimuli, lymphocytes, monocytes, and neutrophils all bound increased amounts of dye per unit surface area, indicating a change in lipid organization to a less-ordered state. Cells stained with MC540 were sorted on the basis of their fluorescence intensity yielding populations homogeneous with respect to lipid organization. Thus not only can MC540 and flow cytometry be combined for analyzing the organization of lipids in individual leukocytes, but sorted populations can be obtained for further biochemical, structural, and functional analyses.

Topics: Animals; Flow Cytometry; Leukocytes; Lymphocyte Activation; Lymphocytes; Membrane Lipids; Mice; Mice, Inbred C57BL; Pyrimidinones

Liposomes formulated to resemble the outer leaflet of the erythrocyte membrane were found to substantially avoid recognition and clearance by the reticuloendothelial system. When these models of the erythrocyte surface were modified by the incorporation of greater than 2 mol % of phosphatidylserine (PtdSer), their ability to remain in the circulation of mice was greatly reduced. To examine whether this altered behavior was the consequence of an alteration in bilayer organization induced by PtdSer, a method utilizing the fluorescent dye merocyanine 540 was used to assess the packing of external phospholipids. No significant difference in overall membrane lipid organization was detected between liposomes containing 2 or 3 mol % of PtdSer, at which dramatic differences in recognition and clearance occurred. These results exclude alterations in phospholipid packing as an indirect cause of increased clearance of PtdSer-containing liposomes and implicate PtdSer directly in recognition by the reticuloendothelial system.

Topics: Animals; Dosage Forms; Erythrocyte Membrane; Female; Liposomes; Membrane Lipids; Mice; Mononuclear Phagocyte System; Phosphatidylserines; Pyrimidinones; Structure-Activity Relationship; Surface Properties

Topics: Ethanol; Lipid Bilayers; Liposomes; Models, Biological; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents

Erythrocytes from patients with diabetes mellitus exhibit increased adherence to cultured human vascular endothelial cells. We investigated the alterations in erythrocyte surface characteristics that may contribute to their abnormal adherence. The organization of phospholipids in the lipid bilayer, as determined by phospholipase A2 treatment and chemical labeling with fluorescamine and trinitrobenzene sulfonic acid (TNBS), is altered in erythrocytes from diabetic patients. Specifically, 12-18% of phosphatidylserine in diabetic erythrocytes (n = 25) is accessible to phospholipase A2 hydrolysis and TNBS labeling, compared to none in normal subjects. These results suggest either a loss in lipid asymmetry or in vivo destabilization of erythrocyte membranes in diabetic patients, causing increased accessibility to phospholipase A2 degradation. The dye merocyanine 540 (MC-540), which is sensitive to the packing of lipids in the bilayer of the membrane, revealed more binding and fluorescence in erythrocytes from diabetic patients than in those from normal subjects. On flow cytometric analysis, 64.5 +/- 17.0% red blood cells (RBCs) in diabetic patients, compared to 35.1 +/- 25.9% RBCs in normal subjects, showed positive MC-540 binding, indicating significant (P less than .001) differences in the packing of lipids in the external leaflet of the bilayer. The results of our study suggest that a loss of lipid asymmetry and/or less ordered packing in the outer leaflet of the diabetic erythrocyte membrane may be responsible for the increased propensity of erythrocytes to adhere to vascular endothelium.

Topics: Adult; Blood Proteins; Cell Adhesion; Cells, Cultured; Diabetes Mellitus, Type 2; Endothelium, Vascular; Erythrocyte Membrane; Flow Cytometry; Fluorescamine; Fluorescent Dyes; Humans; Lipid Bilayers; Lysophosphatidylcholines; Membrane Lipids; Phosphatidylserines; Phospholipases A; Phospholipases A2; Phospholipids; Pyrimidinones; Spectrometry, Fluorescence

Merocyanin 540 (Mc540) is a fluorescent compound which is thought to bind to membranes in which there are substantial amounts of lipid in the lipid-crystalline phase. It is shown here to be of value in detecting the transformation by both mechanical and skin-penetration methods of the cercaria to the schistosomulum. The cercaria does not appear to bind Mc540, but the schistosomulum, binds Mc540 initially, in its anterior region, and at later times over the entire surface. The suggestion that transformation involves changes in the surface membrane lipid phase from gel to liquid-crystalline phase is supported by fluorescence recovery after photobleaching results with 5-N-(octadecanoyl)-amino fluorescein, a lipophilic dye which appears to be immobile in the cercaria, but fully mobile in the 40 min schistosomulum.

Topics: Animals; Diffusion; Fluorescent Dyes; Fluorometry; Larva; Membrane Lipids; Microscopy, Fluorescence; Pyrimidinones; Schistosoma mansoni

Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light. Results show that in the presence of 0.1%, 0.25% and 0.5% albumin, laser light doses of 62.4 J/cm2, 93.6 J/cm2 and 109.2 J/cm2, respectively, were required for a 5 log reduction in the survival of leukemic cells. Under identical conditions, 80% to 84% of the normal bone marrow cells and 41% of the granulocyte-macrophage colony forming cells survived. The number of surviving stromal cells was reduced (1+) compared to the untreated control (4+). Mixing of irradiated bone marrow cells with equal number of HL-60 cells did not interfere with the killing of HL-60 cells treated with MC540 and laser light. The non-specific cytotoxicity of laser light alone was less than 6% for normal bone marrow cells. These results suggest that the concentration of human albumin plays an important role in laser light-induced phototoxicity. This laser light-induced selective photolysis of leukemic cells can be used in ex vivo purging of tumor cell-contaminated bone marrow grafts to achieve very high survival rates of normal bone marrow cells and granulocyte-macrophage colony forming cells.

Topics: Albumins; Bone Marrow; Cell Survival; Colony-Forming Units Assay; Granulocytes; Humans; In Vitro Techniques; Lasers; Macrophages; Photochemotherapy; Pyrimidinones; Tumor Cells, Cultured

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.

Topics: Bone Marrow; Bone Marrow Cells; Cell Cycle; Cell Division; Cell Line; Cell Separation; Flow Cytometry; Hematopoietic Stem Cells; Humans; Leukemia, Experimental; Lymphocytes; Pyrimidinones

The changes of asymmetric distribution of anionic phospholipids of human platelets in diabetic patients were studied by fluorescent and freeze fracture cytochemistry, using merocyanine 540 (MC 540) and polymyxin B (PxB) as specific markers. The membrane anionic phospholipids were detected with PxB, a membrane nonpermeant probe, used either in native form for freeze fracture electron microscopy or as dansylated or iodinated derivative for fluorescence microscopy or gamma counting, respectively. MC 540 is a naturally fluorescent probe which reportedly inserts into less packed bilayer domains. Both in platelet rich plasma and in washed platelets obtained from diabetic patients, some small platelet aggregates were observed, their number being generally dependent on the level of hyperglycemia. In contrast with single platelets, the aggregated ones bind PxB as revealed by all assay methods. The fluorescence microscopic studies with dansyl PxB and MC 540 displayed a strong binding of the fluorescent markers to aggregated platelets. The electron microscopic examination of freeze fracture replicas showed the appearance of characteristic PxB-induced deformations in the plasmalemma of aggregated platelets. The gamma counting of 125I-PxB incubated samples indicates significant differences on the platelets of diabetic patients as compared to those obtained from healthy subjects. Our data provide evidence that in diabetic patients, the spontaneous aggregated platelets are a result of the appearance of the anionic phospholipids in the outer half of plasmalemma. These changes may enhance the procoagulant activity and should represent a determinant of activated platelet recognition and their removal from circulation by splenic macrophages.

Topics: Anions; Blood Platelets; Cell Membrane; Diabetes Mellitus, Type 1; Fluorescent Dyes; Freeze Fracturing; Humans; Microscopy, Electron; Microscopy, Fluorescence; Phospholipids; Platelet Aggregation; Pyrimidinones

Topics: Animals; Antiviral Agents; Friend murine leukemia virus; Hematopoietic Stem Cells; Humans; Photolysis; Pyrimidinones; Simplexvirus

To assess the potential of photoradiation therapy for the in vitro purging of residual tumor cells from autologous bone marrow (BM) transplants, we studied normal marrow and tumor cell clonogenicity in response to different light-activated compounds by using the fluorescent dyes dihematoporphyrin ether (DHE) and merocyanine-540 (MC-540). After photoradiation of cells with white light, both DHE and MC-540 showed high cytocidal activity toward lymphoid and myeloid neoplastic cells but had a significantly lesser effect on normal granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and mixed colony-forming (CFU-GEMM) progenitor cells. Acute promyelocytic leukemia (HL-60), non-B, non-T, CALLA-positive acute lymphoblastic leukemia (Reh), and diffuse histocytic B cell lymphoma (SK-DHL-2) cell lines were exposed to different drug concentrations in combination with white light at a constant illumination rate of 50,000 lux. With DHE doses varying from 2.0 to 2.5 micrograms/mL and MC-540 concentrations of 15 to 20 micrograms/mL, clonogenic tumor cells could be reduced by more than 4 logs when treated alone or in mixtures with normal irradiated human marrow cells. However, preferential cytotoxicity towards neoplastic cells was highly dependent on the mode of light activation. MC-540 had no substantial effect on malignant lymphoid (SK-DHL-2) and myeloid (HL-60) cells and on normal marrow myeloid (CFU-GM) precursors when drug incubation was performed in the dark and followed by light exposure of washed cells. Equal doses of MC-540 (15 to 20 micrograms/mL) could preferentially eliminate tumor cells under conditions of simultaneous light and drug treatment (30 minutes at 37 degrees C). When using DHE (2.5 micrograms/mL), 29.3%, 46.8%, and 27.5% of normal marrow CFU-GM, BFU-E, and CFU-GEMM, respectively, were spared after sequential drug and light exposure of cells, whereas simultaneous treatment reduced both normal (CFU-GM) and neoplastic cells below the limits of detection. In summary, our results indicate the usefulness of various photoradiation models for the ex vivo treatment of leukemic and lymphomatous bone marrow autografts.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Line; Dihematoporphyrin Ether; Hematopoietic Stem Cells; Hematoporphyrins; Humans; Leukemia; Lymphoma; Phototherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous

The photochemistry of merocyanine 540 (MC 540), a sensitizing dye that binds preferentially to leukemia and electrically excitable cells, has been investigated. MC 540-mediated photooxidation of histidine, arachidonate, and unsaturated phospholipid vesicles was assessed by spin label oximetry and shown to involve type II (singlet oxygen) chemistry. The dye was also shown to be a potent sensitizer of lipid peroxidation in a natural cell membrane, the erythrocyte ghost. Inhibition by azide, stimulation by 2H2O, and identification of the cholesterol product 5 alpha-cholest-6-ene-3 beta,5-diol in this system, all were consistent with singlet oxygen intermediacy. Finally, MC 540 was found to be considerably more phototoxic to K-562 leukemia cells in 2H2O than in H2O. We conclude that singlet oxygen plays a major role in the phototherapeutic effects of this dye.

Topics: Cell Line; Cell Membrane; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Fluorescent Dyes; Humans; Leukemia, Myeloid; Liposomes; Oxygen; Phosphatidylcholines; Photochemistry; Pyrimidinones; Singlet Oxygen

MC540-mediated photolysis has several features that make it potentially attractive as a clinical purging procedure. (1) The experience with experimental tumors suggests that MC540-mediated photolysis is effective against a broad range of leukemias and solid tumors, including drug-resistant tumors (Sieber et al., 1984b). Drug-resistant tumor cells are likely to occur in heavily pretreated patients. (2) MC540-mediated photolysis is not cell-cycle dependent (Manna and Sieber, 1985). It kills both resting and cycling cells. In this regard, MC540-mediated photolysis is a valuable complement to cell-cycle specific cytotoxic drugs. (3) There is a large differential in sensitivity between normal pluripotent hematopoietic stem cells and leukemia and neuroblastoma cells. (4) The mechanism of action of MC540-mediated photolysis is different from that of lectins, antibodies and most cytotoxic drugs. MC540 binds to the lipid portion of the plasma membrane and membrane lipids are probably a primary target of the toxic photoproducts. Antibodies and lectins react with proteins and carbohydrates and most drugs have intracellular targets (e.g., nuclear DNA). We would therefore expect little cross-resistance if MC540-mediated photolysis were used in combination with other purging procedures.(5) The small amounts of dye that remain associated with the marrow graft and are infused into the patient are approximately 100,000-fold less than the LD(10) (in mice) and therefore unlikely to cause any harm. The outcome of the first clinical application of the technique supports this view (Sieber et al., 1986c). A better understanding of the underlying molecular mechanisms will undoubtedly lead to more effective applications of the technique and perhaps to the identification of more potent analogs of MC540.

Topics: Bone Marrow; Bone Marrow Transplantation; Humans; Neoplasms; Photolysis; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous

Topics: Hemolysis; Humans; Light; Oxygen; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Rose Bengal; Singlet Oxygen

Topics: Animals; Female; Hematoporphyrins; Indoles; Isoindoles; Mice; Mice, Inbred DBA; Neoplasms, Experimental; Photochemotherapy; Photolysis; Pyrimidinones; Radiation-Sensitizing Agents; Spectrometry, Fluorescence

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Cyclophosphamide; Humans; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Pyrimidinones; Radiation-Sensitizing Agents

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.

Topics: Bee Venoms; Calcium; Erythrocyte Membrane; Humans; Membrane Lipids; Membrane Proteins; Phospholipases A; Phospholipids; Pyrimidinones

The physicochemical mechanism for merocyanine 540 (M540) binding to unilamellar phosphatidylcholine (PC) vesicles was examined by steady-state and dynamic fluorescence and fluorescence stopped-flow methods. At 530-nm excitation, aqueous M540 has an emission peak at 565 nm, which red shifts to 580 nm with formation of membrane-bound monomers (M); bound dimers (D) are nonfluorescent. Equilibrium fluorescence titrations show that 50% of total M540 partitions into the membrane to form D at [M540]/[PC] (Rm/p)_approximately 0.6. M and D concentrations are equal at Rm/p approximately 0.05. For Rm/p less than 0.1, M540 has a single fluorescence lifetime (tau), which decreases with Rm/p [tau-1 (ns-1) = 0.48 + 3.3Rm/p], indicating a rapid collisional rate between M to form D. Dynamic depolarization studies show that hindered rotation of M (r infinity = 0.13 at Rm/p = 0.006) becomes more rapid (rotational rate 0.2-1.9 ns-1) with increasing Rm/p (0.006-0.075). The efficiencies of energy transfer between n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12, 16) and bound M540 suggest that M is oriented parallel to the phospholipids near the membrane surface; studies of efficiencies of n-AF quenching by D are consistent with an orientation of D perpendicular to the phospholipids. In stopped-flow fluorescence measurements in which M540 is mixed with PC vesicles, there is a rapid (1 ms) followed by a slower (10-50 ms) concentration-dependent fluorescence increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Fluorometry; Kinetics; Phospholipids; Pyrimidinones; Temperature; Time Factors

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.

Topics: Animals; Azo Compounds; Calcium; Cattle; Cell Adhesion; Cells, Cultured; Diamide; Endothelium, Vascular; Erythrocytes; Flow Cytometry; Humans; Hydrogen Peroxide; Lysophosphatidylcholines; Membrane Proteins; Oxidation-Reduction; Phospholipids; Pyrimidinones

Topics: Cell Separation; Cells, Cultured; Drug Carriers; Erythrocyte Membrane; Fluorescent Dyes; Humans; Macrophages; Monocytes; Phagocytosis; Phospholipids; Pyrimidinones

The effect of a number of commonly employed potential-sensitive molecular probes on the 31P-NMR properties of dimyristoylphosphatidylcholine vesicles at two field strengths has been investigated in order to obtain information on the location and effect of these probes on the membrane bilayer. In comparison to the control dye-free vesicle spectrum, the probes diS-C3-(5) and diS-C4-(5), when added to a vesicle suspension, cause a substantial broadening of the 31P resonance with no detectable chemical shift within an uncertainty of +/- 0.05 ppm at 24 MHz. The spin-lattice and spin-spin relaxation times are also reduced when the cyanines are present by well over 20% relative to those of the control vesicle preparation. The addition of anionic probes, including several oxonol derivatives and merocyanine 540, causes no chemical shift, line broadening, or changes in the relaxation times. Possible explanations for the failure of the anionic probes to alter the vesicle 31P-NMR properties include charge repulsion between these dyes and the phosphate group that prevents the probes from penetrating the bilayer to a depth sufficient to alter the local motion of the phosphate moiety. The 31P resonance broadening and reduction in the relaxation times caused by the two cyanines is at least in part due to an increase in vesicle size as judged by electron microscopy measurements, although an inhibition of the local phosphate motion as well cannot be completely eliminated. The cyanine-mediated increase in vesicle size appears to be due to an irreversible vesicle-fusion process possibly initiated by the screening of surface charge by these probes. The implications of these observations in relation to functional energy-transducing preparations is discussed.

Topics: Benzothiazoles; Carbocyanines; Dimyristoylphosphatidylcholine; Indicators and Reagents; Isoxazoles; Kinetics; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Potentials; Microscopy, Electron; Oxazoles; Pyrimidinones; Quinolines

The spectral modifications in the absorption and emission properties of merocyanine 540 have been evaluated in solvents of varying dielectric constants. The fluorescence behavior of dye in solutions of low dielectric constant has offered a possibility for monitoring the micropolarity of sialoganglioside micelles in aqueous solutions. Our results demonstrate that sialic acid residues markedly influence the aggregation properties of gangliosides in solution as well as the nature of dye binding to the micellar structures.

Topics: Electrochemistry; Fluorescence Polarization; Fluorescent Dyes; Gangliosides; Micelles; Pyrimidinones; Solutions; Spectrometry, Fluorescence

The purpose of this study was to determine the sensitivity to merocyanine 540 (MC 540)-mediated photolysis of normal human hematopoietic progenitor cells and four leukemia cell lines (Daudi, Raji, K562 and HL-60). Late erythroid progenitors were the most sensitive normal cells. Early erythroid progenitors were of intermediate sensitivity. Granulocyte/macrophage progenitors and multipotent progenitors were the least sensitive normal marrow cells. A combination of dye concentration, serum concentration, and illumination that eliminated 50% of multipotent progenitor cells reduced the concentration of leukemic cells by greater than or equal to 4.5 log. It is conceivable that this difference in photosensitivity can be exploited for the extracorporeal purging of autologous remission marrow grafts.

Topics: Bone Marrow Cells; Cells, Cultured; Hematopoietic Stem Cells; Humans; Kinetics; Leukemia; Photolysis; Pyrimidinones; Time Factors

Voltage-sensitive dyes were used to stain intact perfused hearts and to simultaneously measure optical action potentials (APs) from 124 sites on the epicardium. Patterns of electrical depolarization (activation) and repolarization (recovery) along the surface of the heart were determined from the upstrokes and repolarization phases of optical APs. Standard surface extracellular techniques can detect electrical activation but not the recovery or the duration of APs. The optical recordings were previously shown to be equivalent to intracellular electrode measurements (Salama and Morad, Science Wash. DC 191: 485-487, 1976) and now reveal that AP durations are heterogeneous throughout the epicardium, with durations increasing from the base to the apex of the ventricles. In hearts beating under normal sinus rhythm, the direction and conduction velocity of the activation waves could be altered by electrical stimulation. The normal heterogeneities in AP durations became more pronounced in the presence of the Ca2+-entry blocker, verapamil. The local metabolic state of the tissue was also monitored optically through its intrinsic NADH fluorescence measured from 124 separate regions on the heart. The time course and extent of metabolic injury caused by general anoxia or by a local ischemia induced by a coronary ligation was monitored through maps of NADH fluorescence. The present technique makes it possible to correlate changes in the metabolic state of the muscle with detailed changes in patterns of electrical activity and thus provides a powerful new tool to study fundamental aspects of normal and abnormal cardiac rhythm.

Topics: Action Potentials; Algorithms; Animals; Coronary Vessels; Fluorescence; Guinea Pigs; Heart; Heart Rate; Hypoxia; In Vitro Techniques; Ligation; Myocardium; NAD; Pyrimidinones; Verapamil

We have evaluated the effect of the perfluorinated fatty acids pentadecafluoro-n-octanoic acid (PFOA) and nonadecafluoro-n-decanoic acid (NDFDA) on the ability of a human B-lymphoblastoid cell line to bind the lipid-binding, membrane-impermeant, fluorescent dye merocyanine 540 (MC540). Subtoxic concentrations of perfluorinated fatty acids (0.9 mM PFOA; 0.5 mM NDFDA) greatly diminish binding of MC540 by normal plasma membranes, as determined by fluorescence flow cytometry. When perfluorinated fatty acids are added to cells at toxic or lethal concentrations (1.2 mM PFOA; 0.75 mM NDFDA), MC540 binding increases dramatically, with entrance of dye to internal membrane domains. Neither perfluorinated fatty acid molecule reduces the ability of surface immunoglobulin to migrate laterally and cap on cells. Our data suggest that perfluorinated fatty acids either interact directly with lipid binding sites for MC540, and thereby inhibit dye intercalation, or alter membrane lipid architecture and lipid packing to diminish MC540 binding. Both possibilities support a direct, physical, membrane-altering mechanism for perfluorinated fatty acid toxicity on mammalian cells.

Topics: Blood Physiological Phenomena; Caprylates; Cell Membrane; Cells, Cultured; Decanoic Acids; Fluorocarbons; Immunologic Capping; Membrane Lipids; Pyrimidinones

Based on the previous finding that erythrocytes from patients with chronic myelogenous leukemia stain with the fluorescent dye merocyanine 540, erythrocytes from patients with other myeloproliferative disorders were examined for their ability to bind the membrane probe. As assessed by both fluorescence staining and a quantitative dye removal assay, all samples of erythrocytes from patients with chronic myelogenous leukemia, polycythemia vera, myelofibrosis with myeloid metaplasia and essential thrombocythemia bound more dye than did erythrocytes from normal, healthy individuals. Erythrocytes from three of six patients with acute myelogenous leukemia also showed increased affinity for the dye. In contrast, erythrocytes from three patients with acute lymphocytic leukemia and one with unclassifiable leukemia bound only normal amounts of dye. The procedures described may be useful as a supplemental aid to diagnosis of myeloproliferative disorders or for investigation of hematological diseases where multilineage involvement is suspected.

Topics: Cell Differentiation; Erythrocytes; Fluorescent Dyes; Humans; Myeloproliferative Disorders; Pyrimidinones

The fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen-induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial system.

Topics: Age Factors; Animals; Cell Differentiation; Concanavalin A; Cortisone; Humans; Lipopolysaccharides; Lymphocytes; Membrane Lipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Fluorescence; Pokeweed Mitogens; Pyrimidinones; Spleen; Staining and Labeling; Thymus Gland

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.

Topics: Bone Marrow; Bone Marrow Cells; Granulocytes; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Pyrimidinones; Reference Values; Staining and Labeling

To assess the role of transbilayer phospholipid distribution in the recognition and phagocytosis of erythrocytes by macrophages, human erythrocytes with either a symmetric or asymmetric distribution of membrane phospholipids were prepared by hypotonic hemolysis and then incubated with cultures of human monocyte-derived macrophages. Erythrocytes with an abnormal, symmetric distribution were phagocytosed 4 times more readily than their counterparts with an asymmetric distribution or than normal, asymmetric intact erythrocytes. This enhanced phagocytosis correlated with two biophysical properties of the membrane: the spacing of phospholipids, as assessed by binding of the dye merocyanine 540, and the relative hydrophobicity, as measured by aqueous two-phase polymer partitioning. These results suggest a mechanism by which loss of membrane asymmetry is translated into recognition by macrophages and provide guidelines in loading erythrocytes that may be useful in manipulating the mode of delivery when erythrocytes are used as drug carriers in vivo.

Topics: Calcium; Erythrocyte Membrane; Humans; Macrophages; Magnesium; Membrane Lipids; Phagocytosis; Phospholipids; Pyrimidinones; Solubility; Surface Properties

The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

Topics: Animals; Bone Marrow; Bone Marrow Transplantation; Cell Line; Cell Survival; Female; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Neuroblastoma; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous; Trypsin

Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.

Topics: Bone Marrow; Bone Marrow Transplantation; Cell Line; Cell Survival; Hematopoietic Stem Cells; Humans; Neoplastic Stem Cells; Neuroblastoma; Photolysis; Pyrimidinones; Transplantation, Autologous

Topics: Cholesterol; Light; Mathematics; Membrane Potentials; Membranes, Artificial; Microscopy, Fluorescence; Models, Molecular; Molecular Conformation; Pyrimidinones; Spectrometry, Fluorescence; Spectrum Analysis

Bee venom phospholipase A2 and the fluorescent probe merocyanine 540 were used to examine plasma membrane phospholipid organization in the spicules released by deoxygenation and reoxygenation of sickle red cells, as well as in reversibly and irreversibly sickled erythrocytes. Digestion of phosphatidyl ethanolamine in spicules was comparable to that of phosphatidyl choline, and these structures were stained by the fluorescent probe. Both assays suggest that membrane lipid asymmetry is disrupted in spicules. The residual cells, from which the spicules were derived, retain the normal asymmetry in phospholipid distribution between the outer and inner leaflets of the plasma membrane bilayer. Comparable experiments with cell fractions enriched in irreversibly sickled cells revealed a partial enhancement of phosphatidyl ethanolamine digestion, confirming the similar experiments of Lubin et al (1981). Staining of these cells with merocyanine 540, however, did not reveal a subfraction of stainable cells, indicating that this increase in phosphatidyl ethanolamine digestion is not due to the presence of a small fraction of cells which have completely lost their membrane asymmetry.

Topics: Anemia, Sickle Cell; Erythrocyte Membrane; Fluorescent Dyes; Hemolysis; Histocytochemistry; Humans; Membrane Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A; Phospholipases A2; Phospholipids; Pyrimidinones

Various chemical compounds have been described to induce photosensitization of tumor cells resulting in cell death. We studied the effect of merocyanine-540 (MC-540) on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) and common acute lymphoblastic leukemia antigen-positive non-T, non-B acute lymphoblastic leukemia (Reh) cell lines were incubated with MC-540 and simultaneously exposed to white light. Normal human BM and mixtures of leukemic cells with BM cells were treated under similar conditions. At constant illumination rates of 50,000 lx, significant (at least 4 to 5 logs) tumor cell destruction was obtained with MC-540 concentrations of 20 micrograms/ml or more for HL-60, and 10 micrograms/ml or more for Reh cells. Incubation of BM under equivalent conditions preserved 18.0% of granulocyte-macrophage colony-forming units and 14.2% of erythroid burst-forming units. Similar results were obtained when tumor cells were mixed with irradiated BM and then treated with MC-540. In summary, cell photosensitization with MC-540 has a selective cytotoxic effect towards leukemic cells and therefore may be useful for purging tumor cells from autologous BM.

Topics: Bone Marrow; Cell Line; Cell Survival; Hematopoietic Stem Cells; Humans; Leukemia; Light; Pyrimidinones; Radiation-Sensitizing Agents

Phenylhydrazine-induced oxidative damage in red cells results in increased binding of merocyanine 540, a fluorescence probe sensitive to changes in lipid packing. Fluorescence polarization studies with diphenylhexatriene did not reveal major changes in order parameters both in intact red cells and lysates treated with phenylhydrazine. These fluorescence studies indicate that major changes are observed in membrane lipids. Analytical studies of membrane phospholipids revealed a significant decrease in phosphatidylethanolamine. The results of the fluorescence and lipid studies, taken in association with our previously reported findings on spectrin and other cytoskeletal protein degradation in red cells exposed to phenylhydrazine, suggests that degradation of cytoskeleton membrane proteins is also responsible for changes in the lipid bilayer surface of the red cell membrane.

Topics: Diphenylhexatriene; Erythrocyte Membrane; Fluorescence; Humans; In Vitro Techniques; Lipid Bilayers; Membrane Lipids; Phenylhydrazines; Phospholipids; Pyrimidinones; Spectrin

Ram, bull, and mouse sperm cells were stained with several fluorescent membrane probes. In contrast to nonspecific probes, merocyanine 540 (MC540), which displays preferential binding to loosely packed phospholipids in model membranes, was specifically localized to the anterior portion of the head and the midpiece of mature sperm. To establish when during development this distinctive staining pattern was acquired, germ cells from prepubescent and adult mouse testes as well as sperm from the caput, corpus, and cauda epididymides were isolated and examined. Localized staining with MC540 was not observed until sperm reached the corpus epididymidis, where those cells with a completely translocated (i.e., distally located) cytoplasmic droplet fluoresced. Likewise, when sperm were stained with fluoresceinated concanavalin A (fl-ConA), a localized pattern of fluorescence with lectin restricted to the anterior portion of the head was not observed until the corpus epididymidis was reached. However, in contrast to MC540 staining, only a fraction of sperm with completely translocated droplets exhibited this localized staining with fl-ConA, the remainder exhibiting diffuse fluorescence over the entire cell as seen on caput epididymal sperm. These developmental changes in staining patterns are specific to murine cells, since no change in the pattern of staining by either MC540 or fl-ConA was seen on epididymal sperm of the ram. These results are discussed with respect to: 1) species-to-species differences in sperm membrane features; and 2) the hypothesis that domains of loosely packed lipids may be involved in the regionalization of membrane proteins that occurs during sperm development.

Topics: Animals; Cattle; Cell Membrane; Epididymis; Fluorescent Dyes; Lipid Bilayers; Male; Membrane Lipids; Mice; Polymyxin B; Pyrimidinones; Receptors, Concanavalin A; Sheep; Sperm Maturation; Spermatocytes; Spermatogenesis; Spermatogonia; Spermatozoa

Purified lipoteichoic acids (LTAs) from several gram-positive organisms have been shown, by methods involving spectral changes of an added merocyanine dye probe, to have critical micelle concentrations in the range of 1 to 10 micrograms/ml, suggesting that acylated LTAs in their monomer forms may represent the major configuration of extracellular LTAs in bacterial culture fluids. The critical micelle concentrations obtained did not differ markedly with degree of carbohydrate substitution of the polymers. The significance of these findings in relation to the biological properties of LTA is discussed.

Topics: Acridine Orange; Colloids; Fluorescence; Lipid Bilayers; Lipopolysaccharides; Micelles; Phosphatidic Acids; Pyrimidinones; Spectrophotometry; Teichoic Acids; Tolonium Chloride

Previous work based on fluorescence microscopic observation has indicated that leukemic leukocytes and immature hematopoietic precursor cells show a greater permeability to the membrane stain, merocyanine 540 (MC) than normal, mature cells and that changes in MC permeability seem to be correlated with failure in membrane maturation during leukemic cell differentiation. In the interest of addressing questions concerning the efficacy of the MC staining reaction as a diagnostic tool in clinical contexts relevant to leukemia, we have looked for any correlations which might exist between the MC staining patterns displayed by circulating leukocytes, cellular morphology and the clinical status of 53 patients with leukemia and non-Hodgkin's lymphoma, using fluorescence activated cell sorting. In 85% of cases, MC staining was found to be correlated with blood status while in 15% of the cases discrepancies were found. These results are discussed in light of changes in the hematologic profiles of the patients during the clinical course.

Topics: Diagnosis, Differential; Diagnostic Errors; Humans; Leukemia; Leukocytes; Lymphoma; Microscopy, Fluorescence; Neoplasm Staging; Pyrimidinones; Staining and Labeling

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.

Topics: Calcium; Erythrocyte Membrane; Humans; Lipid Bilayers; Magnesium; Phospholipases A; Phospholipases A2; Phospholipids; Pyrimidinones; Staining and Labeling

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.

Topics: Animals; Cell Line; Cricetinae; Erythrocyte Membrane; Erythrocytes; Fibroblasts; Fluorescein; Fluoresceins; Fluorescent Dyes; Humans; Kidney; Membrane Lipids; Microscopy, Fluorescence; Parainfluenza Virus 1, Human; Polyethylene Glycols; Pyrimidinones; Serum Albumin, Bovine

Merocyanine 540 is a fluorescent dye which stains erythrocytes that have lost their normal membrane phospholipid asymmetry. Because erythrocytes from patients with chronic myelogenous leukemia have been reported to display this abnormal phenotype, peripheral blood erythrocytes from such patients were examined for their ability to stain with the dye. Erythrocytes from all patients with active disease states stained, whereas neither erythrocytes from normal, healthy individuals nor from a patient whose disease symptoms were eliminated by chemotherapy stained. These results suggest that merocyanine 540 may have utility in the clinical evaluation of chronic myelogenous leukemia.

Topics: Adult; Erythrocyte Membrane; Fluorescent Dyes; Humans; Leukemia, Myeloid; Membrane Lipids; Middle Aged; Phospholipids; Pyrimidinones

Topics: Animals; Bone Marrow Transplantation; Graft vs Host Disease; Hematopoietic Stem Cells; Humans; Leukemia; Light; Neoplasms; Pyrimidinones; Transplantation, Autologous

The temperature-jump technique was used to study the rapid kinetics of merocyanine 540 (M-540) interactions with single-walled phosphatidylcholine (PC) vesicles. The absorption spectrum of M-540 in PC vesicles has an isosbestic point at 560 nm at low [PC]/[M-540], where solution M-540 and membrane-bound M-540 dimers are present, and an isosbestic point at 548 nm at high [PC]/[M-540], where membrane-bound M-540 monomers and dimers are present. In response to a 15-kV discharge across a solution containing M-540 and PC vesicles (2.5 degrees C temperature increment), there was a rapid increase in absorbance at 575 nm (less than 5 microseconds) followed by a slower (approximately 1 ms), monoexponential relaxation process of opposite sign and approximately equal amplitude to the initial rise. The amplitude of the slower process was wavelength-dependent and reversed sign at approximately 540 nm. The slower relaxation time constant decreased as [PC] was increased at constant [M-540]. A proposed model for the potential sensitivity of M-540 involves intramembrane reorientation of dye molecules and dimerization. The results obtained here suggest that reorientation of dye molecules is the rate-limiting step, with a rate constant for reorientation from parallel to perpendicular to the plane of the membrane of 1340 +/- 200 s-1 at 23 degrees C.

Topics: Indicators and Reagents; Kinetics; Lipid Bilayers; Mathematics; Models, Biological; Phosphatidylcholines; Pyrimidinones; Thermodynamics

The plasma membranes of human or duckling erythrocytes infected with malarial parasites (Plasmodium falciparum and P. lophurae respectively) were stained by the fluorescent dye merocyanine 540 in the presence of serum. Unparasitized erythrocytes from infected ducklings or from in vitro cultures remained unstained in the presence of serum. Because merocyanine 540 has a greater affinity for fluid phased or disordered lipid bilayers the results suggest that upon infection of the red blood cell the erythrocyte plasma membrane becomes disordered or is increased in its fluidity. Such alterations of the host erythrocyte are probably due to parasite-induced modifications in the underlying spectrin network (required for lipid leaflet asymmetry) as well as changes in erythrocyte membrane lipid composition.

Topics: Animals; Cell Survival; Cells, Cultured; Ducks; Erythrocyte Membrane; Erythrocytes; Humans; Membrane Fluidity; Plasmodium; Plasmodium falciparum; Pyrimidinones

Topics: Animals; Erythrocyte Membrane; Erythrocytes, Abnormal; Humans; Mice; Phospholipids; Pyrimidinones; Spectrin; Spherocytes

Sickle cell erythrocytes exhibit reduced carboxyl methylation of membrane proteins compared to normal erythrocytes. This altered methylation in sickle membrane proteins is also observable when extracted membranes, both intact and alkali treated, were used as substrates for the homologous protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24). However, when glycophorin A, one of the major methyl acceptors in both membranes, was extracted by lithium diiodosalicylate and used as the methyl acceptor, the proteins from both membranes were methylated equally, suggesting an involvement of membrane structure in membrane-bound protein methylation. Merocyanine 540 (MC-540), a fluorescent probe, was used to determine if the membranes differed in organization. Incubation of both normal and sickle erythrocytes membranes with MC-540 produced a marked increase in extrinsic fluorescence, reflecting a relatively nonpolar environment for the dye bound to the membranes. The fluorescence from sickle cell ghosts was only 87% as intense as that from normal ghosts, while the actual amount of MC-540 associated with sickle cell membranes was only 62% of normal. These data suggest that differences exist in the distribution of surface charges on these plasma membranes. These results are consistent with the hypothesis that abnormal levels of membrane protein methylation observed in sickle erythrocytes may be a result of abnormal membrane organization characteristic to sickle cell anemia.

Topics: Anemia, Sickle Cell; Blood Proteins; Erythrocyte Membrane; Erythrocytes, Abnormal; Fluorescent Dyes; Glycophorins; Humans; Membrane Proteins; Methylation; Pyrimidinones; Spectrometry, Fluorescence

In vitro incubation of leukemic bone marrow with the lipophilic fluorescent dye merocyanine 540 and simultaneous exposure to light reduced the concentration of L1210 leukemia cells by about 5 orders of magnitude but spared enough normal pluripotent hematopoietic stem cells to allow hematopoietic reconstitution of lethally irradiated syngeneic mice. This simple and rapid procedure may find an application in the purging of tumor cells from autologous bone marrow grafts.

Topics: Animals; Bone Marrow; Cell Survival; Colony-Forming Units Assay; Hematopoietic Stem Cells; Leukemia L1210; Light; Mice; Pyrimidinones; Radiation-Sensitizing Agents

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.

Topics: Ammonia; Animals; Cell Membrane; Female; Fertilization; Fluorescent Dyes; Membrane Lipids; Microscopy, Fluorescence; Ovum; Pyrimidinones; Sea Urchins

Phospholipase A2 from bee venom and Naja naja has been used to study the orientation of phospholipids present in the membrane of intact human erythrocytes and in spectrin-free microvesicles derived from the cells by treatment with Ca2+ and A23187. Little difference between the cells and microvesicles was observed in the apparent accessibility of phospholipids to the enzyme, suggesting that the original lipid asymmetry was maintained in the absence of spectrin. However, incubation of the microvesicles for 16 h at 37 degrees C did lead to partial loss of asymmetry in the transmembrane distribution of phosphatidylcholine and phosphatidylethanolamine but not of phosphatidylserine. Despite the similarity of lipid asymmetry in cells and fresh microvesicles, the latter were about 40-fold more sensitive to phospholipase treatment than were cells. Although they retained the lipid asymmetry of intact cells, the microvesicles resembled ghosts in their great sensitivity to phospholipase A2 attack, suggesting that the lipid packing in microvesicles and ghosts was similar. This conclusion was supported by the results of experiments with a fluorescent probe Merocyanine 540.

Topics: Animals; Bee Venoms; Elapid Venoms; Erythrocyte Membrane; Fluorescent Dyes; Humans; Kinetics; Membrane Lipids; Phospholipases A; Phospholipases A2; Phospholipids; Pyrimidinones; Spectrin

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 ( M540 ) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540 -membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 M NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membrane-binding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence

Topics: Adenosine Triphosphate; Animals; Binding Sites; Cattle; Electrochemistry; Energy Transfer; Fluorescent Dyes; In Vitro Techniques; Kinetics; Mitochondria, Heart; Models, Chemical; Phosphorylation; Pyrimidinones; Spectrometry, Fluorescence

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.

Topics: Animals; Fluorescent Dyes; Humans; Leukemia; Lysine; Membrane Lipids; Mice; Mice, Inbred BALB C; Phospholipids; Pyrimidinones

Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane-bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540-mediated photosensitization. Late erythroid progenitors (CFU-E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU-E), megakaryocyte progenitors (CFU-Meg), day 7-spleen colony forming cells (day 7-CFU-S), granulocyte/macrophage progenitors (CFU-GM), and day 11-spleen colony forming cells (day 11-CFU-S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies ("clusters"). We conclude that sensitivity to MC 540-mediated photosensitization is develop-mentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late-appearing spleen colonies are the progeny of two distinct classes of progenitor cells.

Topics: Animals; Bone Marrow Cells; Colony-Forming Units Assay; Colony-Stimulating Factors; Erythropoiesis; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Light; Macrophages; Megakaryocytes; Mice; Pyrimidinones; Spleen

Merocyanine 540 (MC 540), an impermeant photoreactive dye with a high affinity for the plasma membrane of hemopoietic precursors, was examined for effects on the self-replicative capacity (SR) of CFUS from normal mouse marrow, and on various CFUS subpopulations fractionated by velocity sedimentation. Brief exposure (30 s) of whole marrow to MC 540 resulted in a reduction in overall CFUS self-renewal in primary recipient spleens. Reduced self-renewal was also observed when fractionated CFUS subpopulations were used. Reduced self-renewal was not accompanied by obvious changes in primary spleen colony number or composition. MC 540 may interact with specific superficial membrane sites relevant to the SR process. Upon longer exposure, further MC 540 effects appear restricted to a reactive subpopulation of large (low SR) CFUS. Self-renewal was enhanced in this fractionated pool, without evidence of primary colony reduction, by intermediate staining. This strongly suggests that under certain conditions the dye can alter a fundamental functional property of susceptible stem cells, without their inactivation. Still more prolonged staining apparently leads to the selective elimination of this reactive low SR CFUS subset. A marked reduction in whole marrow-derived spleen colonies was accompanied by an enhanced self-renewal capacity among the survivors. These two MC 540 effects--stem cell modification, and stem cell elimination--may reflect different stages in an ongoing membrane photo-oxidation process.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Membrane; Cell Survival; Colony-Forming Units Assay; Female; Hematopoiesis; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Pyrimidinones

The effects of merocyanine 540 on the electrical properties of lipid bilayer membranes have been investigated. The alterations this dye was found to produce in the intrinsic conductances of these membranes were minimal, but it profoundly altered the conductances produced by extrinsic permeant species. These alterations were much larger for neutral membranes than for negatively charged ones. The dye increased the conductances mediated by positively charged permeant species and decreased those by negatively charged permeant species, suggesting that it produces a negative electrostatic potential on the membrane; it also altered the kinetics and the voltage dependencies of permeation by these charge carriers. The magnitudes of dye-mediated conductance changes were much larger for positively charged permeants than for negatively charged ones; also, changes in ionic strength altered these dye effects in opposite directions from those predicted by the Stern equation, and the dependence of the conductance alteration on dye concentration was steeper than that predicted by this equation. Finally, only very small changes in liposome zeta potentials were induced by the dye. Calculations show that a large fraction of these effects can be accounted for by the dipole potential produced by merocyanine at the membrane surface, but that additional effects of the dye must be postulated as well.

Topics: Coloring Agents; Electric Conductivity; Electricity; Electrophoresis; Membrane Potentials; Membranes, Artificial; Pyrimidinones

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-A unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200--300-A unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-A unilamellar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.

Topics: Cholesterol; Fluorescent Dyes; Kinetics; Lipid Bilayers; Microscopy, Electron; Models, Biological; Phosphatidylcholines; Pyrimidinones; Structure-Activity Relationship

Experiments were carried out to examine the possible physiological role of disordered membrane domains in hematopoietic cell surface differentiation. The hematopoietic stem cell line 416B has been shown to bind the dye merocyanine 540 (MC540), a fluorescent probe which may be specific for disordered regions of lipid bilayers (Williamson et al., 1981). The surface receptors for the lectins Concanavalin A (Con A) and wheat germ agglutinin (WGA) exhibit patchy distributions on the surface of 416B cells which correspond to the distribution of MC540 binding regions. Appropriate incubation of these cells with either of the two lectins results in the induced formation of a cap. The binding regions for MC540 and the receptors for the other lectin become localized to the same region of the membrane by this process. Such coordinated rearrangements of surface glycoproteins associated with disordered lipid domains may play a role in the cocapping of disparate surface molecules (Raz and Bucana, 1980) or in differentiation-related cell surface rearrangements (Schlegel et al., 1980).

Topics: Animals; Cell Line; Concanavalin A; Glycoproteins; Hematopoietic Stem Cells; Lectins; Membrane Proteins; Mice; Pyrimidinones; Receptors, Concanavalin A; Receptors, Mitogen; Wheat Germ Agglutinins

When human erythroleukemic cells are induced to differentiate in vitro, the lipids in the plasma membrane that bind the fluorescent dye merocyanine 540 are redistributed into a cap at one pole of the cell. This capping phenomenon can also be observed in uninduced cells that have been incubated with cytochalasin B, an agent which disrupts actin-containing microfilaments or with local anesthetics which act on both microfilaments and microtubules. Colchicine which acts on microtubules, however, has no effect. This suggests that the uniform distribution seen in uninduced cells is maintained by the cytoskeletal microfilaments and that loss of these structures leads to spontaneous redistribution of merocyanine 540-binding sites.

Topics: Benzoxazoles; Binding Sites; Cell Line; Cell Membrane; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Pyrimidinones

Topics: Animals; Cell Differentiation; Cell Nucleus; Cells, Cultured; Erythrocyte Membrane; Erythrocytes; Erythropoiesis; Lectins; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Pyrimidinones; Receptors, Concanavalin A; Receptors, Drug; Receptors, Mitogen; Spleen; Surface Properties

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Erythrocytes; Female; Fluorescent Dyes; Granulocytes; Hematopoietic Stem Cells; Light; Macrophages; Mice; Microscopy, Fluorescence; Pyrimidinones; Spleen

The fluorescent probe merocyanine 540 does not stain the plasma membrane of normal human or murine erythrocytes, nor of genetically abnormal human spherocytic erythrocytes. It does, however, stain erythrocyte membranes in several systems in which the underlying spectrin network is altered or missing. Because of the greater affinity of merocyanine 540 for fluid--phase lipid bilayers, these results suggest that the external leaflet of erythrocyte membranes becomes more disordered upon alteration or loss of the internal spectrin network. Analysis of the transbilayer arrangement of membrane phospholipids by digestion with phospholipase A2 suggests that lipid compositional asymmetry of the erythrocyte membrane is responsible for a phase-state asymmetry between the two lipid leaflets, and that spectrin is required to maintain this asymmetry and the gel-like state of the external leaflet.

Topics: Animals; Erythrocyte Aging; Erythrocyte Membrane; Erythrocytes; Humans; Membrane Fluidity; Membrane Lipids; Membrane Proteins; Phospholipases A; Phospholipases A2; Pyrimidinones; Spectrin; Spherocytosis, Hereditary; Tetrathionic Acid

Transformed murine hematopoietic cells of several lineages bound the fluorescent membrane probe merocyanine 540, whereas their normal counterparts did not. Similar selective binding was reproduced in artificial liposomes which bound this probe above their phase transition temperature, but not below it. The regions of the membrane to which merocyanine 540 binds along with the receptors for the lectin concanavalin A, but not the receptors for the lectin wheat germ agglutinin, were rearranged during the course of induced differentiation of erythroleukemia cells. Based on these findings, we propose a model of hematopoietic cell surface differentiation in which proteins such as concanavalin A receptors, which are destined for removal from the plasma membrane, are specifically associated with disordered, liquid-like lipid domains which can be visualized with merocyanine 540. For the specific case of erythroid differentiation, these domains and their associated proteins are collected at the region of the membrane where nuclear extrusion occurs and are eliminated from the reticulocyte plasma membrane by the enucleation event.

Topics: Animals; Cell Line, Transformed; Cell Membrane; Hematopoiesis; Hematopoietic System; Leukemia, Erythroblastic, Acute; Liposomes; Membrane Fluidity; Mice; Pyrimidinones; Receptors, Concanavalin A; Receptors, Mitogen

Topics: Action Potentials; Animals; Anura; Benzoxazoles; Heart; In Vitro Techniques; Pyrimidinones; Spectrum Analysis

Partition of merocyanine dyes, which have a negative charge, onto photosynthetic membranes of chloroplasts and bacteria was analyzed by measuring the fluorescence intensity change, absorbance change, and amount of dye in the supernatant after centrifugation. The partition depended on the surface potential, which is a function of valence and concentration of ions in the medium. The distribution of dyes between the membrane and aqueous phase was determined after centrifugation. The logarithm of the ratio of distribution was linearly related to the logarithm of salt concentration as predicted from the Gouy-Chapman theory and the Boltzmann distribution. Plots of the logarithm of fluorescence intensity against the logarithm of KCl and MgSO4 concentrations gave two straight lines with a slope ratio of about two. The absorbance change upon salt addition was also explained by the Gouy-Chapman theory. The use of these dyes as probes of the surface potential of membranes is discussed.

Topics: Benzoxazoles; Chloroplasts; Chromatophores; Fluorescent Dyes; Intracellular Membranes; Membrane Potentials; Models, Biological; Pyrimidinones; Salts; Spectrum Analysis

In vivo and in vitro clonal assays of immature mouse blood cells showed that different populations of hematopoietic progenitor cells differ considerably with respect to their sensitivity to photodynamic damages caused by the fluorescent dye Merocyanine 540. Late erythroid progenitors were the most sensitive cells followed in order of decreasing sensitivity by pluripotent stem cells, early erythroid progenitors, and granulocyte/macrophage progenitors. Only about 2%-4% of all nucleated marrow cells were stained with Merocyanine 540 which correlated well with current frequency estimates of progenitor cells in mouse bone marrow. Our findings indicate that the expression of Merocyanine binding sites is developmentally regulated and might, therefore, provide a useful molecular marker for blood cell differentiation and a basis for an effective purification of hematopoietic progenitor cells.

Topics: Animals; Benzoxazoles; Cell Differentiation; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Female; Fluorescent Dyes; Hematopoietic Stem Cells; Mice; Pyrimidinones

Normal erythroid cells and both uninduced and induced erythroleukemia cells were stained with the leukemia-specific fluorescent probe merocyanine 540 and its analogs. The external membranes of normal intact cells bound the dye, but this general low-affinity binding was completely abolished by the addition of competing serum. In contrast, erythroleukemia cells bound the dye even in the presence of serum; binding was not affected by reversing the sign of the charge carried by MC540, but was abolished upon removal of certain hydrophobic side chains. When the erythroleukemia cells were induced to differentiate, the distribution of dye-binding regions was altered by the cell such that staining became localized to one region of the membrane. Concomitantly, conconavalin A binding sites were redistributed and became localized in the same region of the membrane as the merocyanine binding sites. Merocyanine 540 is thus shown to bind to a hematopoietic surface feature whose topological distribution is subject to cellular control during differentiation. This leukemia-specific marker may be one of several eliminated during enucleation of mammalian erythroid cells.

Topics: Animals; Benzoxazoles; Cell Line; Chickens; Dimethyl Sulfoxide; Erythrocyte Membrane; Erythrocytes; Erythropoiesis; Female; Fluorescent Dyes; Leukemia, Erythroblastic, Acute; Mice; Pyrimidinones; Receptors, Concanavalin A; Staining and Labeling; Structure-Activity Relationship

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.

Topics: Animals; Benzoxazoles; Cell Membrane; Cell Membrane Permeability; Cell Survival; Chick Embryo; Microscopy, Fluorescence; Muscles; Pyrimidinones; Staining and Labeling

Topics: Benzoxazoles; Fluorescent Dyes; Lipid Bilayers; Liposomes; Phosphatidylcholines; Phosphatidylserines; Pulmonary Surfactants; Pyrimidinones; Solvents; Spectrophotometry; Temperature

Differential scanning calorimetry of multilamellar liposomes, interacting with the optical probe Merocyanine 540, yields quantitative information about perturbances of the bilayer structure induced by this dye. At low dye: lipid ratios, the dye perturbs primarily its own microenvironment, which is laterally separated from the unmodified lipid domain and exhibits modified thermotropic properties. A further increase in the dye concentration results in a perturbance of the whole lipid bilayer. The degree of perturbance is sensitive to structural modifications in the head-group region of the lipids. It is concluded that Merocyanine 540 reports in every case, even at infinite dilution, on localized events originating from a perturbed microenvironment.

Topics: Benzoxazoles; Calorimetry, Differential Scanning; Liposomes; Molecular Conformation; Pulmonary Surfactants; Pyrimidinones; Structure-Activity Relationship; Thermodynamics

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid. 2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K+ concentration gradient. 3. It was estimated that chromatophore membrane became 40--60 mV and 110--170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid. 4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl2) and between BChl2 and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response. 5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response. 6. The dye response was decreased under phosphorylating conditions. 7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.

Topics: Antimycin A; Bacterial Chromatophores; Bacteriochlorophylls; Benzoxazoles; Carotenoids; Chromatium; Cytochrome b Group; Cytochromes; Electron Transport; Intracellular Membranes; Membrane Potentials; Oxidation-Reduction; Photosynthesis; Pyrimidinones; Spectrophotometry; Valinomycin

Topics: Benzoxazoles; Circular Dichroism; Erythrocytes; Humans; Male; Potassium; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry; Valinomycin

Topics: Alkenes; Animals; Benzoxazoles; Cattle; Coloring Agents; Glycerides; Isoxazoles; Kinetics; Mathematics; Membrane Potentials; Membranes, Artificial; Mitochondria; Mitochondria, Heart; Polyenes; Pyrimidinones; Structure-Activity Relationship; Submitochondrial Particles

Topics: Animals; Benzoxazoles; Biological Transport, Active; Calcium; Fluorescent Dyes; In Vitro Techniques; Membrane Potentials; Muscles; Pyrimidinones; Rabbits; Sarcoplasmic Reticulum; Valinomycin

Topics: Animals; Animals, Newborn; Benzoxazoles; Cerebellum; Culture Techniques; Fluorescent Dyes; Mice; Mice, Inbred Strains; Neuroglia; Neurons; Pyrimidinones; Staining and Labeling

The fluorescence and absorbance of merocyanine 540 in suspensions of skeletal muscle microsomes is altered by the binding of Ca2+ and other cations to the membrane. The order of effectiveness of various cations in causing this effect is La greater than Ca congruent to Mg greater than K. Competition between Ca2+, Mg2+, and K+ suggests the involvement of low affinity, relatively nonspecific cation binding sites in the process. Changes in the fluorescence and absorbance of merocyanine were also observed during ATP-dependent accumulation of calcium into sarcoplasmic reticulum. These changes are satisfactorily explained by the binding of accumulated calcium to binding sites on the interior of sarcoplasmic reticulum membrane. The small absorbance response of the oxonol dye bis[1,3-dibutylbarbituric acid-(5)]trimethinoxonol to Ca2+ and ATP is qualitatively similar to that of merocyanine 540 and can be readily explained by the same mechanism. We have found no clear evidence that any of the observed dye responses are due to changes in the diffusion potential across the sarcoplasmic reticulum membrane generated by an electrogenic transport mechanism. The possibility is considered that merocyanine and oxonol dyes respond to changes in electrostatic surface potential caused by the binding of cations.

Topics: Adenosine Triphosphate; Animals; Barbiturates; Binding, Competitive; Biological Transport; Calcium; Coloring Agents; Intracellular Membranes; Lanthanum; Magnesium; Membrane Potentials; Potassium; Protein Binding; Pyrimidinones; Rabbits; Sarcoplasmic Reticulum; Spectrometry, Fluorescence

The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantiatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.

Topics: Animals; Benzoxazoles; Fluorescent Dyes; In Vitro Techniques; Intracellular Membranes; Liposomes; Membrane Potentials; Mitochondria; Pyrimidinones; Rats; Spectrometry, Fluorescence

With the exception of certain blood cells considered in the accompanying paper (Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively strains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca2+-independent when staining proceeds at high ionic strength, inhibited by La3+ and sodium Suramin, enhanced by controlled, low level photosensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.

Topics: Benzoxazoles; Calcium; Cell Membrane; Cells, Cultured; Electrophysiology; Fluorescent Dyes; Lanthanum; Muscles; Osmolar Concentration; Phospholipids; Pyrimidinones; Staining and Labeling; Suramin

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.

Topics: Benzoxazoles; Blood Platelets; Calcium; Erythrocytes; Fluorescent Dyes; Hematopoietic Stem Cells; Humans; Ionophores; Kinetics; Leukemia; Leukocytes; Light; Oxygen; Pyrimidinones; Staining and Labeling

Changes in the fluorescence intensity of merocyanine-540 were measured in suspensions of synaptic plasma membrane ghosts isolated from rat brain cortex. With preincubation of the membrane ghosts in isotonic KCl or NaCl solution, K- and Na-enriched ghosts samples were prepared. In suspensions of both sort of synaptic membrane ghosts, merocyanine-540 showed a fluorescnece emission peak at a wavelength of 590 nm. Under a fixed total concentration of NaCl and KCl, high external K+ induced an increase in the fluorescence intensity, such an increase being proportional to logarithm of K+ concentration. Replacing K+ by Rb+, NH4+ or Cs+, a similar effect was observed. Rb+ was about as effective as K+ ; NH4 is about 2/3 and Cs+ 1/4 as effective. But the changes in fluorescence with increasing K+ concentration were larger in K-ghosts than in Na-ghosts. K+-induced fluorescence changes were very small when gramicidin D was added to the suspension. K+-induced fluorescence changes were not observed in ultrasonicated ghost suspensions. Such findings seem to indicates that the K+-ions induced fluorescence increase reflect the depolarization in the isolated synaptic plasma membrane ghosts. Furthermore, the permeability ratio PNa/PK (PNa, PK: the permeability constant for Na+ and K+) was estimated to be smaller than 0.03.

Topics: Animals; Benzoxazoles; Cell Fractionation; Cerebral Cortex; Fluorescence; Fluorescent Dyes; Gramicidin; In Vitro Techniques; Membrane Potentials; Potassium; Pyrimidinones; Rats; Synaptic Membranes

The fluorescence and optical absorption of the membrane-staining dye merocyanine 540 (M-540) have been widely used to measure cellular transmembrane potentials. We have studied the molecular mechanisms of these optical changes by measuring the fluorescence polarization of M-540 and its response to membrane potential changes in hemispherical lipid bilayer membranes. The fluorescence responds to a potential step in two distinct time scales: a fast response with a rise time less than the instrumental capability of 6 micromilligram and a slow response with a time constant around 10(-1) s. Both response amplitudes are proportional to the amplitude of the membrane potential change and both require an asymmetrical distribution of M-540 across the membrane. The slow response is ascribed to a net change of the dye concentration in the membrane. The fast response appears to be dominated by a change in the distribution of orientations of the dye molecules in the membrane, with a concomitant perturbation of a monomer-dimer equilibrium, due to interaction of the applied electric field with the permanent molecular dipol moment of M-540. The amplitude of the fast fluorescence response is concentration dependent and can be modeled by including membrane saturation effects and the presence of a nonfluorescent dimer species in the membrane at high dye concentrations. Absorbance changes reported by other investigators are consistent with this model mechanism.

Topics: Chemical Phenomena; Chemistry, Physical; Cholesterol; Fluorescence Polarization; Fluorescent Dyes; Membrane Potentials; Membranes, Artificial; Pyrimidinones