carbocyanines and 3-3--diethyloxacarbocyanine

We demonstrated the monolithic integration of reusable and wavelength reconfigurable ring resonator lasers and waveguides of arbitrary shapes to out-couple and guide laser emission on the same fused-silica chip. The ring resonator hosts were patterned by a single-mask standard lithography, whereas the waveguides were inscribed in the proximity of the ring resonator by using 3-dimensional femtosecond laser inscription technology. Reusability of the integrated ring resonator - waveguide system was examined by depositing, removing, and re-depositing dye-doped SU-8 solid polymer, SU-8 liquid polymer, and liquid solvent (toluene). The wavelength reconfigurability was validated by employing Rhodamine 6G (R6G) and 3,3'-Diethyloxacarbocyanine iodide (CY3) as exemplary gain media. In all above cases, the waveguide was able to couple out and guide the laser emission. This work opens a door to reconfigurable active and passive photonic devices for on-chip coherent light sources, optical signal processing, and the investigation of new optical phenomena.

Topics: Carbocyanines; Lasers; Photons; Polymers; Rhodamines; Solvents

This paper presents wavelength configurable on-chip solid-state ring lasers fabricated by a single-mask standard lithography. The single- and coupled-ring resonator hosts were fabricated on a fused-silica wafer and filled with 3,3'-Diethyloxacarbocyanine iodide (CY3), Rhodamine 6G (R6G), and 3,3'-Diethylthiadicarbocyanine iodide (CY5)-doped polymer as the reconfigurable gain media. The recorded lasing threshold was ~220 nJ/mm(2) per pulse for the single-ring resonator laser with R6G, marking the lowest threshold shown by solid-state dye-doped polymer lasers fabricated with a standard lithography process on a chip. A single-mode lasing from a coupled-ring resonator system with the lasing threshold of ~360 nJ/mm(2) per pulse was also demonstrated through the Vernier effect. The renewability of the dye-doped polymer was examined by removing and redepositing the dye-doped polymer on the same resonator hosts for multiple cycles. We recorded consistent emissions from the devices for all trials, suggesting the feasibility of employing this technology for numerous photonic and biochemical sensing applications that entail for sustainable, reconfigurable, and low lasing threshold coherent light sources on a chip.

Topics: Benzothiazoles; Biosensing Techniques; Carbocyanines; Equipment Design; Fluorescent Dyes; Lasers, Solid-State; Optics and Photonics; Polymers; Reproducibility of Results; Rhodamines; Silicon Dioxide

Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carbocyanines; Cell Line; Gene Expression; Gene Expression Profiling; Hepacivirus; Hepatocytes; Humans; Proto-Oncogene Proteins c-bcr; Real-Time Polymerase Chain Reaction; Rhodamine 123; Viral Nonstructural Proteins; Virus Replication

Ponatinib is a novel tyrosine kinase inhibitor with potent activity against BCR-ABL with mutations, including T315I, and also against fms-like tyrosine kinase 3. We tested interactions between ponatinib at pharmacologically relevant concentrations of 50 to 200 nmol/L and the MDR-associated ATP-binding cassette (ABC) proteins ABCB1, ABCC1, and ABCG2. Ponatinib enhanced uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins, with a greater effect on ABCG2 than on ABCB1. Ponatinib potently inhibited [(125)I]-IAAP binding to ABCG2 and ABCB1, indicating binding to their drug substrate sites, with IC(50) values of 0.04 and 0.63 μmol/L, respectively. Ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner and stimulated ABCB1 ATPase activity at low concentrations, consistent with it being a substrate of both proteins at pharmacologically relevant concentrations. The ponatinib IC(50) values of BCR-ABL-expressing K562 cells transfected with ABCB1 and ABCG2 were approximately the same as and 2-fold higher than that of K562, respectively, consistent with ponatinib being a substrate of both proteins, but inhibiting its own transport, and resistance was also attenuated to a small degree by ponatinib-induced downregulation of ABCB1 and ABCG2 cell-surface expression on resistant K562 cells. Ponatinib at pharmacologically relevant concentrations produced synergistic cytotoxicity with ABCB1 and ABCG2 substrate chemotherapy drugs and enhanced apoptosis induced by these drugs, including daunorubicin, mitoxantrone, topotecan, and flavopiridol, in cells overexpressing these transport proteins. Combinations of ponatinib and chemotherapy drugs warrant further testing.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Carbocyanines; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; fms-Like Tyrosine Kinase 3; Fusion Proteins, bcr-abl; Humans; Imidazoles; Inhibitory Concentration 50; Mitoxantrone; Neoplasm Proteins; Protein Binding; Pyridazines; Rhodamine 123; Topotecan

Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.

Topics: Annexin A5; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Ethylenediamines; Humans; Liver Neoplasms; Molecular Weight; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Polyethylene Glycols; Polymers; Propidium; Propylene Glycols; Rhodamine 123; Vinblastine

We developed a homogenous microtiter based assay using the cationic dye 3, 3'-Diethyloxacarbocyanine iodide, DiOC2(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.

Topics: Anti-Bacterial Agents; Bacteriological Techniques; Carbocyanines; Escherichia coli; High-Throughput Screening Assays; Inhibitory Concentration 50; Membrane Potentials; Staphylococcus aureus

The large interspecies differences of hepatobiliary transport present a challenge for the allometric prediction of human biliary excretion for drug candidates primarily cleared via hepatobiliary secretion. In the present study, we determined the metabolic stabilities of common fluorescent substrates of hepatobiliary efflux transporters and developed a rapid efflux assay to determine the functional activities of MRP/Mrp, BCRP/Bcrp and P-gp in hepatocytes of four species. The specificities of transporter-mediated dye efflux were confirmed by selective transporter inhibitors. Among tested species, transporter-specific dye efflux kinetics was consistent between freshly isolated and cryopreserved hepatocytes. Hepatocyte elimination half-lives of MRP/Mrp substrates GS-MF and calcein were observed in the rank order of human>monkey>dog>rat. The fourfold higher MRP/Mrp substrate efflux rate of rat hepatocytes compared to human is likely due to the species-specific functional differences of MRP2/Mrp2 expressed on the canalicular membrane. We also observed efficient BCRP-mediated pheophorbide A (PhA) efflux by human and dog hepatocytes, while PhA extrusion in monkey and rat hepatocytes appeared limited. P-gp function measured by DiOC2(3) efflux was minimal in hepatocytes of all origins and no significant species differences were detected. Our results demonstrated marked differences in hepatocyte MRP/Mrp and BCRP/Bcrp activities across species, indicating that they may contribute to the species differences of in vivo hepatobiliary excretion. These results also suggest the potential utility of primary hepatocytes, either fresh or cryopreserved, as an in vitro model to predict interspecies differences in the biliary transport of MRP/Mrp and BCRP/Bcrp substrates.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bile; Carbocyanines; Carrier Proteins; Cell Separation; Cryopreservation; Dogs; Female; Fluoresceins; Fluorescent Dyes; Half-Life; Hepatocytes; Humans; In Vitro Techniques; Macaca fascicularis; Male; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Species Specificity

This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms.

Topics: Anti-Bacterial Agents; Bacterial Physiological Phenomena; Carbocyanines; Cell Membrane Permeability; Flow Cytometry; Fluorescent Dyes; Membrane Potentials; Mycobacterium smegmatis; Staphylococcus aureus

We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found that the mitochondrial membrane potential was oscillating, and that these oscillations displayed the same frequency and duration as the NADH oscillations. It was confirmed that DiOC(2)(3) localizes itself in the mitochondrial membrane and thus reports qualitative changes solely in mitochondrial membrane potential. Our studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolarization of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane potential oscillations and delocalized DiOC(2)(3), while glycolysis continued to oscillate unaffected.

Topics: Biological Clocks; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; Glycolysis; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Microscopy, Phase-Contrast; NAD; Saccharomyces cerevisiae; Uncoupling Agents

During transport-associated adenosine triphosphate hydrolysis, P-glycoprotein (Pgp) undergoes conformation transitions detected by UIC2, a functional anti-Pgp monoclonal antibody. A newly developed UIC2 shift assay is based on increased UIC2 reactivity in the presence of Pgp substrates. All peripheral blood leukocytes express low Pgp levels. The existing antibody-based detection methods are limited in their sensitivity and require additional techniques to simultaneously analyze Pgp expression and efflux, making it difficult to ascertain the physiologic role of Pgp-mediated transport.. We validated the UIC2 shift assay against UIC2 immunostaining and DiOC(2) efflux. The UIC2 shift assay was then used to characterize Pgp functional expression and its physiologic substrates in peripheral blood leukocytes.. A strong correlation was observed between the UIC2 shift assay versus immunostaining and dye efflux tests. The UIC2 shift assay showed improved sensitivity (compared with conventional UIC2 staining) and allowed for simultaneous detection of Pgp expression and function. Using this assay, we identified several new Pgp substrates, including monensin and retinol, and confirmed that interleukin-2 and interferon-gamma can be transported by Pgp.. Our findings validate the use of the UIC2 shift assay in MDR1 detection and support the idea that Pgp plays a physiologic role in immunoregulation.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Antigens, Surface; ATP Binding Cassette Transporter, Subfamily B, Member 1; B-Lymphocytes; Carbocyanines; Cell Membrane; Flow Cytometry; Humans; Immunoassay; Immunohistochemistry; Interferon-gamma; Interleukin-2; Killer Cells, Natural; Lymphocytes; Mice; Monensin; Protein Transport; Reproducibility of Results; T-Lymphocytes; Vitamin A

The multidrug resistance (MDR) transporter-proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP) and lung resistance protein (LRP) have been associated with treatment failure. The aim of this study was to investigate prospectively the clinical significance of expression and function of the MDR proteins, considering other prognostic factors, such as age, immunophenotype, and cytogenetics. Mononuclear cells of peripheral blood or bone marrow from 61 patients with de novo acute myelogenous leukemia (AML) were analyzed. The monoclonal antibodies JSB1, MRPm6 and LRP56 were used for expression studies. Accumulation and retention studies were performed using the substrates Daunorubicin, Calcein-AM, Rhodamine-123 and DiOC(2) in the presence or absence of the modifiers Verapamil, Genistein, Probenecid, BIBW22S and PSC833. Induction treatment consisted of a 3+7 combination of Ida/Ara-C for patients < or = 60 years of age and a 3+5 Ida/VP-16 combination per OS for patients >60. MDR function was expressed as the ratio of mean fluorescence intensity substrate in the presence of modifier over the substrate alone (resistance index, RI). Patients with advanced age, low CD15 expression and high RI for accumulation of DiOC(2) in the presence of BIBW22S had significantly lower complete remission (CR) rates. No factor was prognostic for event-free survival analysis, which was limited to remitters only. Overall survival was shorter in patients with advanced age, poor prognosis cytogenetics, high CD7 expression, and high RI for Daunorubicin efflux modulated by Verapamil. These results suggest that MDR transporter-proteins have a limited role in the treatment failure of patients treated with Idarubicin-based regimens.

Topics: Acute Disease; Adolescent; Adult; Age Factors; Aged; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bone Marrow Transplantation; Calcium Channel Blockers; Carbocyanines; Combined Modality Therapy; Cytarabine; Daunorubicin; Disease-Free Survival; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Fluoresceins; Fluorescent Dyes; Genistein; Humans; Idarubicin; Immunophenotyping; Leukemia, Myeloid; Male; Middle Aged; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Probenecid; Prognosis; Prospective Studies; Rhodamine 123; Survival Analysis; Tumor Cells, Cultured; Vault Ribonucleoprotein Particles; Verapamil

Surface charge in track-etched polyethylene terephthalate (PET) membranes with narrow pores has been probed with a fluorescent cationic dye (3,3'-diethyloxacarbocyanine iodide (diO-C2-(3))) using confocal microscopy. Staining of negatively charged PET membranes with diO-C2-(3) is a useful measure of surface charge for the following reasons: 1) the dye inhibits K(+) currents through the pores and reduces their selectivity for cations; 2) it inhibits [3H]-choline+ transport and promotes 36Cl- transport across the membrane in a pH- and ionic-strength-dependent fashion; and 3) staining of pores by diO-C2-(3) is reduced by low pH and by the presence of divalent cations such as Ca2+ and Zn2+. Measurement of the time dependence of cyanine staining of pores shows fluctuations of fluorescence intensity that occur on the same time scale as do fluctuations of ionic current in such pores. These data support our earlier proposal that fluctuations in ionic current across pores in synthetic and biological membranes reflect fluctuations in the surface charge of the pore walls in addition to molecular changes in pore proteins.

Topics: Biophysical Phenomena; Biophysics; Calcium; Carbocyanines; Cell Membrane; Fluorescent Dyes; Hydrogen-Ion Concentration; Ions; Microscopy, Confocal; Models, Chemical; Time Factors; Zinc

Expression of multidrug resistance (MDR) features by acute myeloid leukemia (AML) cells predicts a poor response to many treatments. The MDR phenotype often correlates with expression of P-glycoprotein (Pgp), and Pgp antagonists such as cyclosporine (CSA) have been used as chemosensitizing agents in AML. Gemtuzumab ozogamicin, an immunoconjugate of an anti-CD33 antibody linked to calicheamicin, is effective monotherapy for CD33(+) relapsed AML. However, the contribution of Pgp to gemtuzumab ozogamicin resistance is poorly defined. In this study, blast cell samples from relapsed AML patients eligible for gemtuzumab ozogamicin clinical trials were assayed for Pgp surface expression and Pgp function using a dye efflux assay. In most cases, surface expression of Pgp correlated with Pgp function, as indicated by elevated dye efflux that was inhibited by CSA. Among samples from patients who either failed to clear marrow blasts or failed to achieve remission, 72% or 52%, respectively, exhibited CSA-sensitive dye efflux compared with 29% (P =.003) or 24% (P <.001) among samples from responders. In vitro gemtuzumab ozogamicin--induced apoptosis was also evaluated using an annexin V--based assay. Low levels of drug-induced apoptosis were associated with CSA-sensitive dye efflux, whereas higher levels correlated strongly with achievement of remission and marrow blast clearance. In vitro drug-induced apoptosis could be increased by CSA in 14 (29%) of 49 samples exhibiting low apoptosis in the absence of CSA. Together, these findings indicate that Pgp plays a role in clinical resistance to gemtuzumab ozogamicin and suggest that treatment trials combining gemtuzumab ozogamicin with MDR reversal agents are warranted. (Blood. 2001;98:988-994)

Topics: Acute Disease; Aminoglycosides; Anti-Bacterial Agents; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bone Marrow; Carbocyanines; Clinical Trials, Phase II as Topic; Cyclosporine; Drug Resistance, Multiple; Drug Synergism; Fluorescent Dyes; Gemtuzumab; Humans; Immunotoxins; Leukemia, Myeloid; Leukocytes, Mononuclear; Phenotype; Regression Analysis; Remission Induction; Treatment Outcome; Tumor Cells, Cultured

Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC(2)(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC(2)(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 microg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 microg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 microg/ml did not change permeability, while a tetracycline concentration of 4 microg/ml permeabilized 50% of the bacteria; 4 microg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

Topics: Anti-Bacterial Agents; Carbocyanines; Cell Membrane Permeability; Colony Count, Microbial; Flow Cytometry; Fluorescent Dyes; Ionophores; Membrane Potentials; Micrococcus luteus; Staphylococcus aureus

Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP.. Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent.. In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium.. The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.

Topics: Calibration; Carbocyanines; Colony Count, Microbial; Escherichia coli; Flow Cytometry; Fluorescent Dyes; Membrane Potentials; Spectrometry, Fluorescence; Valinomycin

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC2(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein beta-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.

Topics: Carbocyanines; Fluorescence; Fluorescent Dyes; Molecular Structure; Solutions; Solvents; Staining and Labeling

The present study aimed to determine the kinetics of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in Opossum kidney (OK) cells and to define the type of inhibition produced by L-5-hydroxytryptophan (L-5-HTP), cyanine 863 and 3,3'-diethyloxacarbocyanine (3,3'-DOC). Non-linear analysis of the saturation curves revealed for L-DOPA a Km (in microM) of 129 (114, 145) and a Vmax (in nmol/mg protein per 6 min) of 30.0 +/- 0.4 IC50 values for L-5-HTP (1454 microM) obtained in the presence of a nearly saturating (250 microM) concentration of L-DOPA were almost 4-fold those obtained when non-saturating (25 microM) concentrations of L-DOPA were used (330). IC50 values for cyanine 863 and 3,3'-DOC (638 and 353 microM) obtained in the presence of a nearly saturating (250 microM) concentration of L-DOPA were similar to those obtained when non-saturating (25 microM) concentrations of L-DOPA were used (654 and 339 microM). Vmax values (in nmol/mg protein per 6 min) for L-DOPA uptake were identical in the absence (36.4 +/- 0.7) and the presence of L-5-HTP (39.2 +/- 1.3), but Km values (microM) were significantly greater (P < 0.05) when L-DOPA uptake was studied in the presence of L-5-HTP (121 (100, 142) versus 318 (237, 399)). In contrast, the effect of cyanine 863 and 3,3'-DOC was to cause a significant reduction in Vmax values without significant changes in Km values. It is concluded that L-5-HTP exerts a competitive type of inhibition of L-DOPA uptake in cultured OK cells, whereas both cyanine 863, an organic cation transport inhibitor and 3,3'-DOC behave as non-competitive inhibitors.

Topics: Animals; Binding, Competitive; Carbocyanines; Cells, Cultured; Coloring Agents; Kidney; Kinetics; Levodopa; Opossums; Quinolinium Compounds; Serotonin

Dynamic and steady-state fluorescence spectroscopic properties of a dye probe measured in a multicomponent biological system are often required to be separated into the spectra and lifetimes of individual spectroscopically distinct species. The conventional method of obtaining decay-associated spectra fails when the lifetimes of the fluorophore in the membrane phase and in the aqueous phase are very close to each other. This paper describes a global analysis method which takes advantage of the known spectrum and lifetime of the dye in the aqueous phase. This method is used to identify the spectra for two fluorescent species (lifetimes, 0.84 and 1.97 ns) of the dye DODCI in EggPC vesicle membranes by keeping the spectrum and lifetime (0.68 ns) of the dye in the aqueous phase as fixed parameters. The structural identity of the two membrane-bound dye species was established by the effect of refractive index and/or viscosity of the aqueous medium on the lifetimes.

Topics: Carbocyanines; Fluorescence Polarization; Fluorescent Dyes; Kinetics; Liposomes; Models, Biological; Models, Chemical; Phosphatidylcholines; Spectrometry, Fluorescence

Synaptic transmission from single preganglionic hypogastric nerves innervating monopolar pelvic ganglion neurones has been studied with intracellular electrodes to record transmission from all the boutons and with extracellular electrodes placed over boutons visualized with DiOC2(5) in order to record transmission from selected boutons. Intracellular electrodes revealed spontaneous excitatory postsynaptic potentials (EPSPs) with amplitude histograms that show increasing numbers of large EPSPs as the external calcium ([Ca2+]o) was increased from 0.15 to 1.0 mM. These histograms were in general well fitted by a Poisson mixture of gamma distributions. Extracellular electrodes placed over visualized boutons revealed evoked excitatory postsynaptic potentials (extracellular EPSPs) with amplitude histograms that were best described by single gamma distributions in most cases in low [Ca2+]o (less than 0.5 mM). The standard deviation of these gammas was not much larger than that of the electrical noise. In a minority of extracellular recordings the amplitude histogram of evoked extracellular EPSPs was best described by a gamma distribution in which the standard deviation was much greater than that of the noise. Confocal microscopy of boutons orthogradely labelled with dextran-rhodamine showed that about 30% of these formed closely apposing pairs on the surface of the neurones. These observations are discussed in terms of the hypothesis that multiquantal release at boutons occurs as a consequence of the coupled secretion from closely apposed boutons.

Topics: Animals; Carbocyanines; Electrophysiology; Extracellular Space; Fluorescent Dyes; Ganglia, Autonomic; In Vitro Techniques; Microscopy, Confocal; Nerve Endings; Neurons; Neurotransmitter Agents; Rats; Rats, Inbred Strains; Synaptic Transmission

Multidrug resistance (MDR) is often related to expression of P-glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp-mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine-123 (Rh-123), and 3,3'-diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP-expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh-123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug-free medium), no retention difference of the three agents was detected between parental and MRP-expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh-123 and Dox in the resistant lines became apparent and even more pronounced after 24h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK-104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh-123 can be used to evaluate MRP-function, but DiOC2(3) can not.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Carbocyanines; Cyclic P-Oxides; Doxorubicin; Drug Resistance, Multiple; Female; Flow Cytometry; Fluorescent Dyes; Humans; Neoplasm Proteins; Nicotinic Acids; Rhodamine 123; Rhodamines; Tumor Cells, Cultured

Resistance to chemotherapy is a major factor limiting successful treatment of acute myeloid leukemia (AML); one of the best characterized drug resistance mechanisms is extrusion of drugs by the energy-dependent multidrug resistance (MDR1) transport protein. Expression of MDR1 is common in AML and has been linked to lower remission induction rates and decreased remission durations. Because MDR1 efflux function may be modified by drugs such as cyclosporin A, accurate identification of MDR1+/efflux+ AML cases will be critical to identify patients who may benefit from therapies that contain such MDR1 modulators. We have optimized single and multiparameter flow cytometric assays to detect efflux of drugs or fluorescent dyes by previously cryopreserved AML blasts. These assays allowed precise identification of efflux by leukemic blasts, and correlation with CD34 and MDR1 expression. We subsequently studied a series of 60 previously untreated AML cases. Functional efflux was identified in 39 cases and was significantly correlated with MDR1 expression (P = .0002). However, discrepant cases were identified; 10 cases were efflux+ without significant MDR1 expression, whereas 6 MDR1+ cases were efflux-. There was also a highly significant correlation of efflux with CD34; 31 (79%) of the 39 efflux+ cases were CD34+ in comparison with only 5 (24%) of the 21 efflux- cases (P < .0001). Multivariate analysis showed that efflux was significantly associated with independent effects of both CD34 (P = .0011) and MDR1 expression (P = .034); the majority of efflux+ cases were CD34+, whereas 5 of the 6 MDR1+ efflux- cases lacked CD34 expression. Cyclosporin A blocked efflux in all but 2 cases regardless of MDR1 expression. Functional efflux in AML is frequently detected without the classic MDR1+ phenotype indicating that alternate non-MDR1-mediated efflux mechanisms may be important. Efflux assays may better identify patients who would benefit from therapies that include efflux modulators.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, CD34; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Carbocyanines; Cryopreservation; Cyclosporine; Drug Resistance, Multiple; Female; Flow Cytometry; Fluorescent Dyes; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid; Male; Middle Aged; Multicenter Studies as Topic; Neoplasm Proteins; Neoplastic Stem Cells

The distribution of axons and axon varicosities on the surface of the mouse vas deferens has been determined following fluorescence of these structures with 3,3-diethyloxardicarbocyanide (DiOC2 (5)) to locate varicosities and FAGLU to detect catecholamine containing nerves. Small bundles of fluorescent axons treated with DiOC2(5), were shown to give rise to single or very small bundles of 2 or 3 varicose axons that passed over the surface of the muscle bundles. Varicosities had the same average diameter of 0.9 microns, length of 1.1 micron and spacing apart of 4.6 microns whether identified following DiOC2(5) fluorescence or the FAGLU method for catecholamines and this was shown statistically to imply that they came from the same population of varicosities. Serial thin sections through small axon bundles and single axons, viewed with the electron microscope, confirmed the dimensions of varicosities along axons observed following DiOC2(5) staining or use of the FAGLU method.

Topics: Animals; Axons; Carbocyanines; Catecholamines; Electrophysiology; Fluorescent Dyes; Formiminoglutamic Acid; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron; Sympathetic Nervous System; Vas Deferens

In this study, we determined that three structurally related oxacarbocyanine dyes, 3,3'-diethyloxacarbocyanine (DiOC2(3)), 3,3'-dipentyloxacarbocyanine (DiOC5(3)), and 3,3'-dihexyloxacarbocyanine (DiOC6(3)), and one oxadicarbocyanine, 3,3'-diethyloxadicarbocyanine (DiOC2(4)), inhibit bovine heart mitochondrial NADH oxidase activity and one of them, DiOC6(3), inhibits Paracoccus denitrificans NADH oxidase activity. The mitochondrial I50 values were 9 microM (DiOC2(3)), approximately 1 microM (DiOC5(3)) and DiOC6(3)), and approximately 3 microM (DiOC2(4)), whereas the I50 value for P. denitrificans was approximately 2 microM (DiOC6(3)). Neither succinate nor cytochrome oxidase (EC 1.9.3.1) activity was inhibited significantly by any of the compounds in either electron transport chain, localizing the inhibitory site of the oxacarbocyanine dyes to the respiratory chain segment between NADH and ubiquinone. With submitochondrial particles (SMP), NADH-dependent reduction of duroquinone and coenzyme Q1 was inhibited markedly by all four compounds with DiOC6(3) being the most potent inhibitor, and the reduction of menadione was inhibited substantially by DiOC6(3). When purified complex I was used, NADH-dependent reduction of ferricyanide was inhibited by DiOC5(3) and coenzyme Q1 reduction was inhibited by all oxacarbocyanines. With P. denitrificans membrane vesicles, DiOC6(3) substantially inhibited NADH-dependent reduction of coenzyme Q1. All the oxacarbocyanines were more effective inhibitors with membrane preparations than with complex I, suggesting that membrane interactions play a role in inhibition. The mechanism of inhibition of the oxacarbocyanines appears to be similar to that of rotenone since (a) essentially only electron acceptors affected by rotenone were affected by the compounds, (b) inhibition of menadione reduction was diminished drastically with rotenone-saturated SMP, and (c) inhibition of coenzyme Q1 was largely eliminated with rotenone-insensitive complex I, and P. denitrificans membrane vesicles.

Topics: Animals; Carbocyanines; Cattle; Coloring Agents; Electron Transport; Electron Transport Complex I; Kinetics; Mitochondria, Heart; NAD; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Oxidoreductases; Paracoccus denitrificans; Stimulation, Chemical; Structure-Activity Relationship