carbocyanines and 3-3--dioctadecyloxacarbocyanine

Specific interactions between viruses and host cells provide essential insights into material science-based strategies to combat emerging viral diseases. pH-triggered viral fusion is ubiquitous to multiple viral families and is important for understanding the viral infection cycle. Inspired by this process, virus detection has been achieved using nanomaterials with host-mimetic membranes, enabling interactions with amphiphilic hemagglutinin fusion peptides of viruses. Most research has been on designing functional nanoparticles with fusogenic capability for virus detection, and there has been little exploitation of the kinetic stability to alter the ability of nanoparticles to interact with viral membranes and improve their sensing performance. In this study, a homogeneous fluorescent assay using self-assembled polymeric nanoparticles (PNPs) with tunable responsiveness to external stimuli is developed for rapid and straightforward detection of an activated influenza A virus. Dissociation of PNPs induced by virus insertion can be readily controlled by varying the fraction of hydrophilic segments in copolymers constituting PNPs, giving rise to fluorescence signals within 30 min and detection of various influenza viruses, including H9N2, CA04(H1N1), H4N6, and H6N8. Therefore, the designs demonstrated in this study propose underlying approaches for utilizing engineered PNPs through modulation of their kinetic stability for direct and sensitive identification of infectious viruses.

Topics: Animals; Carbocyanines; Chickens; Eggs; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Influenza A virus; Limit of Detection; Membrane Fusion; Membranes, Artificial; Nanoparticles; Peptides; Polyethylene Glycols; Viral Fusion Proteins

A novel and versatile design of DNA-lipid conjugates is presented. The assembly of the DNA headgroups into G-quadruplex structures is essential for the formation of micelles and their stability. By hybridization with a complementary oligonucleotide the micelles were destabilized, resulting in cargo release. In combination with a hairpin DNA aptamer as complementary strand, the release is obtained selectively by the presence of ATP.

Topics: Adenosine Triphosphate; Animals; Aptamers, Nucleotide; Carbocyanines; Cattle; DNA; Drug Carriers; Drug Liberation; Fluorescent Dyes; G-Quadruplexes; Lipids; Micelles; Nucleic Acid Hybridization; Oxazines; Serum Albumin, Bovine

Capsaicin has been used in clinical applications for the treatment of pain disorders and inflammatory diseases. Given the strong pungency and high oil/water partition coefficient of capsaicin, capsaicin-loaded nanolipoidal carriers (NLCs) were designed to increase permeation and achieve the analgesic, anti-inflammatory effect with lower skin irritation. Capsaicin-loaded NLCs were prepared and later optimized by the Box-Behnken design. The physicochemical characterizations, morphology, and encapsulation of the capsaicin-loaded NLCs were subsequently confirmed. Capsaicin-loaded NLCs and capsaicin-loaded NLCs gel exhibited sustained release and no cytotoxicity properties. Also, they could significantly enhance the penetration amount, permeation flux, and skin retention amounts of capsaicin due to the application of NLCs. To study the topical permeation mechanism of capsaicin, 3,3'-dioctadecyloxacarbocyanine perchlorate (Dio) was used as a fluorescent dye. Dio-loaded NLCs and Dio-loaded NLCs gel could effectively deliver Dio up to a skin depth of 260 and 210 μm, respectively, primarily through the appendage route on the basis of version skin sections compared with Dio solution, which only delivered Dio up to 150 μm. In vivo therapeutic experiments demonstrated that capsaicin-loaded NLCs and capsaicin-loaded NLCs gel could improve the pain threshold in a dose-dependent manner and inhibit inflammation, primarily by reducing the prostaglandin E2 levels in the tissue compared with capsaicin cream and capsaicin solution. Meanwhile, skin irritation was reduced, indicating that application of NLCs could decrease the irritation caused by capsaicin. Overall, NLCs may be a potential carrier for topical delivery of capsaicin for useful pain and inflammation therapy.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Capsaicin; Carbocyanines; Dermatitis; Dinoprostone; Dose-Response Relationship, Drug; Drug Carriers; Female; Fluorescent Dyes; Lipids; Male; Mice, Inbred ICR; Nanocomposites; Particle Size; Rabbits; Rats; Rats, Sprague-Dawley; Skin Absorption

Acinetobacter baumannii is an important nosocomial pathogen that is resistant to many commonly-used antibiotics. One strategy for treatment is the use of aromatic compounds (carvacrol, cinnamaldehyde) against A. baumannii. The aim of this study was to determine the interactions between bacteria and lipid nanocapsules (LNCs) over time based on the fluorescence of 3,3'-Dioctadecyloxacarbocyanine Perchlorate-LNCs (DiO-LNCs) and the properties of trypan blue to analyse the physicochemical mechanisms occurring at the level of the biological membrane. The results demonstrated the capacity of carvacrol-loaded LNCs to interact with and penetrate the bacterial membrane in comparison with cinnamaldehyde-loaded LNCs and unloaded LNCs. Modifications of carvacrol after substitution of hydroxyl functional groups by fatty acids demonstrated the crucial role of hydroxyl functions in antibacterial activity. Finally, after contact with the efflux pump inhibitor, carbonylcyanide-3-chlorophenyl hydrazine (CCCP), the results indicated the total synergistic antibacterial effect with Car-LNCs, showing that CCCP is associated with the action mechanism of carvacrol, especially at the level of the efflux pump mechanism.

Topics: Acinetobacter baumannii; Acinetobacter Infections; Acrolein; Anti-Bacterial Agents; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cymenes; Lipids; Monoterpenes; Nanocapsules

Increased permeability of blood vessels is an indicator for various injuries and diseases, including multiple sclerosis (MS), of the central nervous system. Nanoparticles have the potential to deliver drugs locally to sites of tissue damage, reducing the drug administered and limiting associated side effects, but efficient accumulation still remains a challenge. We developed peptide-functionalized polymeric nanoparticles to target blood clots and the extracellular matrix molecule nidogen, which are associated with areas of tissue damage. Using the induction of experimental autoimmune encephalomyelitis in rats to provide a model of MS associated with tissue damage and blood vessel lesions, all targeted nanoparticles were delivered systemically. In vivo data demonstrates enhanced accumulation of peptide functionalized nanoparticles at the injury site compared to scrambled and naive controls, particularly for nanoparticles functionalized to target fibrin clots. This suggests that further investigations with drug laden, peptide functionalized nanoparticles might be of particular interest in the development of treatment strategies for MS.

Topics: Animals; Carbocyanines; Drug Carriers; Encephalomyelitis, Autoimmune, Experimental; Female; Fibrin; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Lactones; Laminin; Membrane Glycoproteins; Multiple Sclerosis; Nanoparticles; Oligopeptides; Polyethylene Glycols; Rats; Rats, Inbred F344; Spinal Cord; Up-Regulation

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.

Topics: Acetone; Boron Compounds; Carbocyanines; Cells, Cultured; Chemical Precipitation; Flow Cytometry; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Lipids; Microalgae; Oxazines; Solvents; Staining and Labeling

A new micelle drug carrier that consists of a diblock polymer of propylene sulfide (PS) and N,N-dimethylacrylamide (poly(PS₇₄-b-DMA₃₁₀)) has been synthesized and characterized for site-specific release of hydrophobic drugs to sites of inflammation. Propylene sulfide was first polymerized using a thioacyl group transfer (TAGT) method with the RAFT chain transfer agent (CTA) 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (CEP), and the resultant poly(PS₇₄-CEP) macro-CTA was used to polymerize a second polymer block of DMA using reversible addition-fragmentation chain transfer (RAFT). The formation of the poly(PS₇₄-b-DMA₃₁₀) diblock polymer was confirmed by ¹H NMR spectra and gel permeation chromatography (GPC). Poly(PS₇₄-b-DMA₃₁₀) formed 100 nm micelles in aqueous media as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Micelles loaded with the model drugs Nile red and DiO were used to demonstrate the ROS-dependent drug release mechanism of these micelles following treatment with hydrogen peroxide (H₂O₂), 3-morpholinosydnonimine (SIN-1), and peroxynitrite. These oxidants were found to oxidize the micelle PPS core, making it more hydrophilic and triggering micelle disassembly and cargo release. Delivery of poly(PS₇₄-b-DMA₃₁₀) micelles dual-loaded with the Förster Resonance Energy Transfer (FRET) fluorophore pair DiI and DiO was used to prove that endogenous oxidants generated by lipopolysaccharide (LPS)-treated RAW 264.7 macrophages significantly increased release of nanocarrier contents relative to macrophages that were not activated. In vitro studies also demonstrated that the poly(PS₇₄-b-DMA₃₁₀) micelles were cytocompatible across a broad range of concentrations. These combined data suggest that the poly(PS₇₄-b-DMA₃₁₀) micelles synthesized using a combination of TAGT and RAFT have significant potential for site-specific drug delivery to tissues with high levels of oxidative stress.

Topics: Acrylic Resins; Animals; Carbocyanines; Cell Line; Cell Survival; Drug Carriers; Fluorescent Dyes; L-Lactate Dehydrogenase; Macrophages; Mice; Micelles; Oxazines; Reactive Oxygen Species; Sulfides

The aim of this research is to study the dynamics and efficiency of liposome accumulation in sensitive and resistant human breast cancer cells.. Methods of fluorescence microscopy, fluorescence microspectroscopy and MTT-test have been used.. The liposome-to-cell interaction and dye cellular uptake in sensitive, cisplatin-resistant and doxorubicin-resistant MCF-7 human breast cancer cells have been analyzed using time changes in both fluorescence resonance energy transfer signal from the donor probe DiO to the acceptor one DiI preloaded in liposomes and cell image brightness.. Obtained results show that resistant cells accumulate dye-loaded liposomes more effectively and reveal more effective dye molecule cellular uptake.

Topics: Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cisplatin; Doxorubicin; Drug Carriers; Drug Resistance, Neoplasm; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Lipid Bilayers; Liposomes; MCF-7 Cells; Microscopy, Fluorescence; Molecular Structure; Phosphatidylcholines; Spectrometry, Fluorescence; Tissue Distribution

The aim of the present work was to study the influence of surface hydrophilicity of biodegradable polymeric nanoparticles on cellular uptake by Caco-2 cells. Poly(D,L-lactide-co-glycolide acid) particles loaded with a fluorescent dye, 3,3'-dioctadecyloxacarbo-cyanine perchlorate (DiO), were prepared by the emulsion-evaporation process. Three batches of particles with narrow size distribution (100, 300 and 1000 nm) were produced using selective centrifugation. One set of particles was coated by adsorption of chitosan to increase the hydrophilicity of the particles. The interaction of particles with Caco-2 cells was determined by fluorescence spectroscopy and the number of particles associated with one single cell was then calculated. Interaction with cells was clearly dependant on particle size and surface hydrophilicity. Particles in the range of 100 nm presented higher interaction when compared to larger particles. Approximately 6000 uncoated particles and more than 30,000 chitosan-coated particles were quantified per cell. Confocal microscopy confirmed the spectroscopic measurements and revealed the location of the particles in the cell monolayer. Only small particles were observed intracellularly, whereas particles larger than 300 nm were associated with the apical membranes. The location of particles <300 nm appeared to be intracellular and some particles colocalized with the nucleus.

Topics: Biocompatible Materials; Caco-2 Cells; Carbocyanines; Cell Survival; Chitosan; Fluorescent Dyes; Humans; Intestinal Mucosa; Intracellular Space; Lactic Acid; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Nanoparticles; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Static Electricity; Surface Properties

During visual system development, multiple signalling pathways cooperate to specify axial polarity within the retina and optic tectum. This information is required for the topographic mapping of retinal ganglion cell axons on the tectum. Meis1 is a TALE-class homeodomain transcription factor known to specify anterior-posterior identity in the hindbrain, but its role in visual system patterning has not been investigated.. meis1 is expressed in both the presumptive retina and tectum. An analysis of retinal patterning reveals that Meis1 is required to correctly specify both dorsal-ventral and nasal-temporal identity in the zebrafish retina. Meis1-knockdown results in a loss of smad1 expression and an upregulation in follistatin expression, thereby causing lower levels of Bmp signalling and a partial ventralization of the retina. Additionally, Meis1-deficient embryos exhibit ectopic Fgf signalling in the developing retina and a corresponding loss of temporal identity. Meis1 also positively regulates ephrin gene expression in the tectum. Consistent with these patterning phenotypes, a knockdown of Meis1 ultimately results in retinotectal mapping defects.. In this work we describe a novel role for Meis1 in regulating Bmp signalling and in specifying temporal identity in the retina. By patterning both the retina and tectum, Meis1 plays an important role in establishing the retinotectal map and organizing the visual system.

Topics: Amino Acids; Animals; Animals, Genetically Modified; Apolipoprotein A-II; Body Patterning; Carbocyanines; Embryo, Nonmammalian; Gene Expression Profiling; Gene Expression Regulation, Developmental; Growth Differentiation Factor 6; Homeodomain Proteins; Mutation; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Oligodeoxyribonucleotides, Antisense; Retina; Retinal Ganglion Cells; RNA, Messenger; Signal Transduction; Smad5 Protein; Superior Colliculi; Zebrafish; Zebrafish Proteins

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.

Topics: Animals; Calibration; Capillaries; Carbocyanines; Data Interpretation, Statistical; Image Processing, Computer-Assisted; Male; Mice; Mice, Inbred C57BL; Microcirculation; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Microvessels; Nonlinear Dynamics; Phantoms, Imaging; Silicon Dioxide; Software

The present study investigated dynamic alteration of low-density lipoprotein receptor and its binding and uptake of low-density lipoprotein (LDL) after exposure to transforming growth factor-beta(2) (TGF-beta(2)) in human Tenon's capsule fibroblasts.. Tenon's capsule fibroblasts obtained from elective cataract surgery patients were cultured and stimulated with different concentrations (0.1-10 ng/mL) of TGF-beta(2) for 24, 48, and 72 h. The LDLr mRNA and protein levels were analyzed by relative quantification real-time RT-PCR and Western blot analysis, respectively. The binding and uptake of DiO (3,3'-dioctadecyloxacarbocyanine)-labeled LDL was assessed by confocal microscopy.. Real-time RT-PCR and Western blot analyses showed similar results revealing that after exposure to TGF-beta(2), the expression of protein and mRNA of LDLr occurred in a concentration-dependent and time-dependent manner with a peak at a concentration of 1.0 ng/mL at 72 h in Tenon's capsule fibroblasts. Confocal microscopy showed that DiO-LDL binding and uptake were time-dependent, reaching saturation at approximately 6 h.. This study shows that LDLrs were overexpressed in the activated Tenon's capsule fibroblasts in a concentration-dependent and time-dependent manner after exposure to TGF-beta(2). The results suggest that LDLr in the activated Tenon's capsule fibroblasts may become a novel focus as a target receptor for controlled drug delivery, particularly in anti-scarring therapy during excessive conjunctival wound healing.

Topics: Blotting, Western; Carbocyanines; Cells, Cultured; Dose-Response Relationship, Drug; Eye; Female; Fibroblasts; Gene Expression; Humans; Lipoproteins, LDL; Male; Microscopy, Confocal; Middle Aged; Protein Binding; Receptors, LDL; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor beta2

Drug-membrane interactions are well known but poorly understood. Here we describe dual measurements of membrane thickness change and membrane area change due to the binding of the amphipathic drug curcumin. The combined results allowed us to analyze the binding states of a drug to lipid bilayers, one on the water-membrane interface and another in the hydrocarbon region of the bilayer. The transition between the two states is strongly affected by the elastic energy of membrane thinning (or, equivalently, area stretching) caused by interfacial binding. The data are well described by a two-state model including this elastic energy. The binding of curcumin follows a common pattern of amphipathic peptides binding to membranes, suggesting that the binding states of curcumin are typical for amphipathic drugs.

Topics: Binding Sites; Carbocyanines; Curcumin; Dimethyl Sulfoxide; Lipid Bilayers; Membrane Fluidity; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Rhodamines; Unilamellar Liposomes

Information from both sides of the brain is integrated by axons that project across the midline of the central nervous system via numerous commissures present at all axial levels. Despite the accumulated experimental evidence, questions remain regarding the formation of commissures in the presence of strong repulsive signals in the ventral midline. Studies from invertebrates suggest that interaction at the midline between homologous axons of specific decussating neurons contributes to efficient midline crossing, but such evidence is lacking in vertebrate systems. We performed experiments to determine whether commissural axons of the caudal region of the hindbrain interact with their contralateral counterparts at the ventral midline and to evaluate the relevance of this reciprocal interaction. Double anterograde axon labeling with lipophilic tracers revealed close apposition between growth cones of contralateral pioneer decussating axons at the midline. Later, we detected fasciculation between contralateral axons that is maintained even after they have crossed the midline. Blocking axon projections unilaterally with a solid mechanical barrier decreased dramatically the midline crossing of the equivalent population from the contralateral side. Decussation was also blocked by a unilateral barrier permeable to diffusible molecules but not by an axon-permeable barrier. These results suggest that in the caudal region of the hindbrain, midline crossing is facilitated by interactions between decussating contralateral axon partners.

Topics: Amino Acids; Analysis of Variance; Animals; Axons; Body Patterning; Carbocyanines; Cell Communication; Cell Movement; Embryo, Mammalian; Functional Laterality; Gene Expression Regulation, Developmental; Mice; Nerve Tissue Proteins; Organ Culture Techniques; Rhombencephalon

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-beta-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.

Topics: Analysis of Variance; Animals; Animals, Newborn; Carbocyanines; Carrier Proteins; Cell Death; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Glucose; Hypoxia; Intracellular Signaling Peptides and Proteins; Ischemia; Ischemic Preconditioning; Membrane Potentials; Membrane Proteins; Microfilament Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Neurons; Patch-Clamp Techniques; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Synapses; Time Factors; Ubiquitin

Hypericin, a potent necrosis avid agent, features a peculiar affinity for necrotic tissue. Necrosis avid contrast agents have been investigated as markers for non-invasive imaging of different disorders. In view of the promising clinical applications, a more complete knowledge of the mechanism of action is important for the future development of new chemical structures with improved characteristics. To study whether a compound-specific or non-specific mechanism based on plasma lipoprotein transport is involved in the accumulation of hypericin in intratumoral necrosis, we performed a visual and quantitative fluoromicroscopic analysis of the colocalization of hypericin and DiOC18-labeled lipoproteins in subcutaneous murine radiation-induced fibrosarcoma tumors. Microscopic fluorescent overlay images of necrotic tumors demonstrated that hypericin already showed clear necrosis avid characteristics 4 h after injection, whereas a similar outstanding accumulation in necrosis was not demonstrated for the labeled lipoproteins. Moreover, a quantitative analysis of fluoromicroscopic images of tumor necrosis at 24 h after injection showed differences in normalized fluorescence intensities between hypericin and labeled lipoproteins of 50-100%, reflecting a shifted pattern in localization. We conclude that our results are indicative of a release of hypericin from the lipoprotein complex at some point along its way through the peri-necrotic tumor area and the necrotic tissue debris, which is in line with the hypothesis of a compound-specific mechanism.

Topics: Animals; Anthracenes; Carbocyanines; Female; Fibrosarcoma; Lipoproteins; Mice; Mice, Inbred C3H; Necrosis; Perylene; Sarcoma, Experimental; Xanthones

Upon cold and drought stress, sucrose and trehalose protect membrane structures from fusion and leakage. Similarly, these sugars protect membrane proteins from inactivation during dehydration. We studied the interactions between sugars and phospholipid membranes in giant unilamellar vesicles with the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine perchlorate incorporated. Using fluorescence correlation spectroscopy, it was found that sucrose decreased the lateral mobility of phospholipids in the fully rehydrated, liquid crystalline membrane more than other sugars did, including trehalose. To describe the nature of the difference in the interaction of phospholipids with sucrose and trehalose, atomistic molecular dynamics studies were performed. Simulations up to 100 ns showed that sucrose interacted with more phospholipid headgroups simultaneously than trehalose, resulting in a larger decrease of the lateral mobility. Using coarse-grained molecular dynamics, we show that this increase in interactions can lead to a relatively large decrease in lateral phospholipid mobility.

Topics: Carbocyanines; Fluorescent Dyes; Membrane Fluidity; Membranes, Artificial; Phosphatidylcholines; Spectrometry, Fluorescence; Sucrose; Trehalose

(51)Cr release assay (CRA) is still the standard method to study mast cell (MC)-mediated cytotoxicity in vitro. Non-radioactive methods e.g. MTT, Hoechst 22147 staining, have also been used. Though CRA has the benefit of being reproducible, it has several drawbacks e.g. spontaneous release and radioactivity. The basic strategy of this new flow cytometric assay involves labeling target cells with DIOC18, in addition to staining with propidium iodide to identify dead cells. 8-week-old human MCs were used as effectors. Human LAK-sensitive K-562; and LAK-resistant myeloid leukemia cell lines (DAMI, HL-60 and Meg-01) as well as 6 LAK-resistant myeloid leukemia patient samples were utilized. MCs/targets were co-incubated in certain ratios for short/long-term. Although there was some insignificant killing at 2 h/18 h, probably due to small sample size, significant killing in Meg-01 and HL-60 cells (27% and 39%; respectively) was observed at 48 h. This method clearly showed human MC-mediated cytotoxicity against human tumor cells. It was reproducible, reliable and cheaper without any radiation and spontaneous release. This is the first study to elucidate MC-mediated cytotoxicity by a flow cytometric assay.

Topics: Carbocyanines; Cell Separation; Cells, Cultured; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Flow Cytometry; HL-60 Cells; Humans; K562 Cells; Mast Cells

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.

Topics: Carbocyanines; Cell Cycle; Cell Line; Cell Survival; Flow Cytometry; Hepatocytes; Humans; Magnoliopsida; Plant Extracts

Size is the most studied parameter in the field of nanoparticle characterization but few studies have been performed on biodegradable particles with well-defined sizes. The aim of this work was to prepare fluorescent biodegradable polymeric particles having well-defined sizes and well-characterized surface properties. Poly(D,L-lactide-co-glycolide acid) particles were prepared by the emulsion evaporation process. Filtration and centrifugation were used to produce particle fractions in the narrow size range from polydispersed batches, and the efficiency of separation was compared. Selective centrifugation allowed for the preparation of five classes of particles having narrow size distribution (0.1, 0.3, 0.6, 1 and 2 microm). Particles were characterized in terms of size distribution, surface morphology, charge, residual surfactant and hydrophilicity. The results showed similar surface properties for all the batches. 3,3'-Dioctadecyloxacarbo-cyanine perchlorate has been successfully incorporated as a fluorescent dye and its ability to remain associated with the particles during cell culture experiments has been proven. Such particles may be used as an adequate tool for studying cellular uptake.

Topics: Caco-2 Cells; Carbocyanines; Centrifugation; Chemical Phenomena; Chemistry, Physical; Electrochemistry; Filtration; Fluorescent Dyes; Humans; Lactic Acid; Lasers; Light; Microscopy, Electron, Scanning; Nanoparticles; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Scattering, Radiation; Surface Properties; Surface-Active Agents; Water

We have characterized a method of labeling of axons in the post-mortem spinal cord using a silastic disc holding pins coated with DiI and DiO at the rostral and caudal ends of the cord. We optimized the DiI and DiO tracing techniques under different conditions of fixative concentration (1% versus 4% paraformaldehyde, PF), at room temperature (RT) versus 37 degrees C for up to 24 weeks. Crystal coated pins embedded in a silastic disc provided a novel method of dye application. Confocal microscopy of longitudinal sections showed DiI and DiO labeled both the axonal membrane and myelin sheath. DiI diffused significantly longer distances than DiO. Both dyes migrated greater distances at 37 degrees C compared with RT. No significant difference of dye labeling was found between 1% and 4% PF fixation. After prolonged incubation there was evidence that dye diffused through the aqueous medium and produced circumferential labeling of the cord. Placing a wax seal around the labeling site prevented this non-contiguous labeling. Labeling of myelin sheaths at extended distances into the cord suggested that dye could migrate between cells with prolonged incubation periods. Our data suggested that higher temperature facilitated dye diffusion along the axons, and demonstrated that with caution DiI and DiO could be used as specific tracers in the same spinal cords.

Topics: Animals; Axons; Carbocyanines; Coloring Agents; Diffusion; Female; Fixatives; Formaldehyde; Microscopy, Confocal; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Spinal Cord; Temperature; Tissue Fixation

Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

Topics: Carbocyanines; CD4-Positive T-Lymphocytes; Cell Aggregation; Cell Fusion; Coculture Techniques; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gene Products, env; Gene Products, rev; HIV Fusion Inhibitors; HIV-1; Humans; Jurkat Cells; rev Gene Products, Human Immunodeficiency Virus; Staining and Labeling

Low density lipoprotein (LDL) is a normal plasma component, which is of interest in a number of research areas such as hypercholesterolaemia, drug targeting in cancer chemotherapy and as a lipid supplement in tissue culture systems. Currently, however, it can only be obtained by extraction from fresh plasma samples, which yields only small quantities. Synthetic LDL (sLDL) has been prepared using readily available lipid components coupled with a synthetic amphiphatic peptide molecule containing the apoprotein B receptor sequence. sLDL was capable of supporting the growth of Chinese Hamster Ovary (CHO) and fibroblast cells in serum-free culture media in a cholesterol-dependent manner that was related to the presence of the receptor peptide molecule. sLDL could be fluorescently labelled with 3,3'-dioctadecyloxalocarbocyanine perchlorate (DiO), and once labelled was assimilated by CHO and fibroblast cells in a time- and temperature-dependent manner that was dependent upon the presence of the receptor peptide. In addition, assimilation was reduced by an excess of unlabelled native LDL. The results indicated that the interaction of sLDL with CHO and fibroblast cells occurred via a receptor dependent system, most likely the LDL cellular receptor. sLDL is therefore a useful, easily obtained substitute for native LDL with potential applications in the areas of drug targeting to cells and serum-free tissue culture systems.

Topics: Animals; Carbocyanines; Cell Culture Techniques; Cell Line; Cell Proliferation; CHO Cells; Cholesterol; Cricetinae; Culture Media, Serum-Free; Fibroblasts; Fluorescent Dyes; Lipoproteins, LDL; Microscopy, Confocal; Molecular Weight; Phospholipids; Receptors, Lipoprotein; Temperature; Time Factors; Triglycerides

Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chemical synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small molecules. Accordingly, we propose a novel biological principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.

Topics: Actins; Animals; Biological Transport; Carbocyanines; Cell Communication; Cell Line; Cell Membrane; Cell Surface Extensions; Endocytosis; Endosomes; Fluorescent Dyes; Green Fluorescent Proteins; Luminescent Proteins; Membrane Proteins; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microscopy, Video; Organelles; PC12 Cells; Protein Prenylation; Protein Transport; Pseudopodia; Rats; Recombinant Fusion Proteins; Synaptophysin

The vertebrate jaw is a mandibular-arch derivative, and is regarded as the synapomorphy that defines the gnathostomes. Previous studies (Kuratani et al., Phil. Trans. Roy. Soc. 356:15, 2001; Shigetani et al., Science 296:1319, 2002) have suggested that the oral apparatus of the lamprey is derived from both the mandibular and premandibular regions, and that the jaw has arisen as a secondary narrowing of the oral patterning mechanism into the mandibular-arch domain. The heterotopy theory of jaw evolution states that the lamprey upper lip is a premandibular element, leaving further questions unanswered as to the homology of the trabecula in the lamprey and gnathostomes, and to the morphological nature of the muscles in the upper lip. Using focal injection of vital dyes into the cheek process core of lamprey embryos, we found that the upper lip muscle and trabecula are both derived from mandibular mesoderm. Secondary movement of the muscle primordium is also evident when the expression of the early muscle marker gene, LjMA2, is visualized. A nerve-fiber labeling study revealed that the upper lip muscle-innervating neurons are located in the rostral part of the brain stem, where the trigeminal motor nuclei are not found in gnathostomes. We conclude that the lamprey upper lip is composed of premandibular ectomesenchyme and a lamprey-specific muscle component derived from the mandibular mesoderm innervated by lamprey-specific motoneurons. Furthermore, the lamprey trabecula is most likely equivalent to a mesodermally derived neurocranial element, similar to the parachordal element in gnathostomes, rather than to the neural-crest-derived prechordal element.

Topics: Animals; Biological Evolution; Carbocyanines; Fluorescent Dyes; Fresh Water; Gene Expression Regulation, Developmental; In Situ Hybridization; Japan; Lampreys; Mandible; Maxillofacial Development; Mesoderm; Morphogenesis

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.

Topics: Antigens, CD34; Carbocyanines; Cell Membrane; Chromium Radioisotopes; Coloring Agents; Cytotoxicity Tests, Immunologic; Dactinomycin; Flow Cytometry; Fluorescent Dyes; Humans; In Vitro Techniques; K562 Cells; Killer Cells, Natural

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO-labelled colon carcinoma cells (CC531s) in 100 microm thick liver sections. In vitro and in vivo experiments revealed that DiO-labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO-labelled CC531s cells could be traced all over the tissue depth and anti-metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine-phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO-labelled cells in tissue sections.

Topics: Animals; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Lasers; Liver Neoplasms; Male; Microscopy, Confocal; Rats; Staining and Labeling; Tumor Cells, Cultured

To examine distribution of sensory neurons of ventral and dorsal cervical cutaneous nerves in dorsal root ganglia (DRGs), DiO and DiI tracers were applied at the proximal section of nerves (transverse superficial cervical and anterior supraclavicular nerves were selected as ventral cervical cutaneous nerves; dorsal cutaneous branches of second, third and fourth cervical nerves were selected as dorsal cervical cutaneous nerves). Located distributions were observed in DRGs of C2, C3, and C4 (25/46 DRGs). Sensory neurons of the ventral cervical cutaneous nerves were distributed in dorso-lateral or dorso-medial portions; neurons of dorsal cervical cutaneous nerves were distributed in ventro-medial or ventro-lateral portions of DRGs. Moreover, sensory neurons of transverse superficial cervical and anterior supraclavicular nerves were mainly distributed from the caudal half of C2 to whole part of C4 DRGs. Results show that there is a tendency for located distribution in two group sensory neurons; also, sensory neurons of ventral cervical cutaneous nerves have a segmental distribution, which has been verified in the brachial and lumbar plexus.

Topics: Age Factors; Animals; Carbocyanines; Cervical Plexus; Female; Fluorescent Dyes; Ganglia, Spinal; Male; Neurons, Afferent; Rats; Rats, Wistar

The fusion of HIV and SIV with biological membranes was studied by photosensitized activation of a hydrophobic probe, [(125)I]iodonaphthylazide ([(125)I]INA), by a fluorescent lipid which is situated in the target membrane. Photosensitized labeling of viral envelope-resident proteins occurs only upon their insertion into target membranes. Photosensitized labeling as a result of HIV-1 Env-mediated cell fusion showed the same kinetics as aqueous dye transfer. We have for the first time measured kinetics of HIV and SIV virus-cell fusion. HIV-1(MN) virions were about 10x less fusion active than SIVmne virions. SIV inactivated by aldrithiol-2 retained fusion activity similar to that seen with untreated virus. The relatively slow time course of SIV-cell fusion (t(1/2) = 19 min) indicates that the fusion events are stochastic. This feature provides a basis for understanding the mode of action of HIV/SIV entry inhibitors that target transition states.

Topics: 2,2'-Dipyridyl; Animals; Azides; Carbocyanines; Cell Line; Disulfides; Fluorescent Dyes; Glycoproteins; HIV-1; HLA-DR Antigens; Humans; Iodine Radioisotopes; Kinetics; Membrane Fusion; Mice; Photochemistry; Photosensitizing Agents; Simian Immunodeficiency Virus; Sulfhydryl Reagents; Viral Envelope Proteins

To determine the effect of particle size and ligand surface density on the cellular association of poly lactide-co-glycolide nanoparticles covalently coated with bacterial invasin.. Poly lactide-co-glycolide nanoparticles containing a flourescent probe were prepared at four diameters 155 nm, 200 nm, 375 nm and 600 nm using standard techniques. Bacterial invasin was covalently coupled to the particles surface at varying surface concentrations using a water soluble carbodiimide. The modified particle's cellular association with HEp2 2B cells in tissue culture was determined using flow cytometry.. Cellular association of modified nanoparticles was time dependent, abolished at low temperature, competitively inhibited by free invasin or the RGD peptide ligand and saturable. Increased cell association was produced by increasing the particle's surface invasin concentration however, this effect was size dependent. Small particles (155 nm and 200 nm) exhibiting a maximal association with increasing invasin concentration whilst the larger particles (375 nm and 600 nm) provide a minimum at low invasin concentrations.. Modified particle cell association provided results commensurate with a receptor dependent uptake mechanism related to the presence of invasin. The size and surface concentration dependency however illustrate that application of these ligands to particulate drug delivery or targeting systems will be controlled by their natural cellular association properties.

Topics: Adhesins, Bacterial; Bacterial Proteins; Biocompatible Materials; Carbocyanines; Carrier Proteins; Cell Line; Drug Carriers; Flow Cytometry; Fluorescent Dyes; Humans; Kinetics; Lactic Acid; Maltose-Binding Proteins; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Recombinant Fusion Proteins

This laboratory has demonstrated that lipid-coated microbubbles (LCMs) effectively aggregate and deliver chemotherapeutic drugs into rat brain tumor cells and antigliosis agents into maturing rat brain injury sites. In this study, we report the affinity of tail vein-injected LCMs to the injured rat spinal cord by a compressive lesion to the upper thoracic region.. The accumulation of LCMs in the injured spinal cord was analyzed by labeling it with a lipid-soluble fluorescent dye, 3,3'-dioctadecyloxacarbocyanine perchlorate. Indices of glial fibrillary acidic protein were measured concomitantly with 3,3'-dioctadecyloxacarbocyanine perchlorate-labeled LCMs using confocal microscopy.. There was no aggregation of LCMs accumulated 1 and 6 hours after injury; however, when given 2, 4, and 7 days after injury, LCMs showed a clear affinity for the injured region. LCM aggregation shifted from the central necrotic area of the injury on postinjury Day 2 and postinjury Day 4 to the white matter among glial fibrillary acidic protein-positive astrocytes by postinjury Day 7.. Affinity of LCMs for spinal cord injury sites may be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by glial fibrillary acidic protein-positive astrocytes in adjacent white matter. These findings suggest a potential for using LCMs as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites.

Topics: Animals; Astrocytes; Carbocyanines; Fluorescent Antibody Technique; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Injections, Intravenous; Lipids; Microscopy, Fluorescence; Microspheres; Necrosis; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Surface Properties; Tail; Thorax; Time Factors; Wounds, Nonpenetrating

Endometriosis is an adhesion disorder characterized by the presence of endometrial tissue in ectopic sites outside the uterus. The disease is associated with dysmenorrhea, pelvic pain and infertility. Although endometriosis is the most common gynecologic disorder, relatively little is known regarding its etiology, pathogenesis and the course of the disease. This situation is primarily due to the absence of experimental systems to examine the mechanism of endometrial cell adhesion, role of inflammatory cells and the interactions of epithelial, and stromal cells with the peritoneum and ovarian tissue leading to the development of this disorder. Dissociated human endometrial cells were suspended in peritoneal fluids of individuals with and without endometriosis and were injected into the peritoneal cavity of athymic mice. This led to development of ectopic adhesions of endometrial cells at the peritoneal and ovarian surfaces. Endometrial cells which were marked with fluorescent lipophylic dyes, prior to intraperitoneal injection, could be visualized without surgery at such sites. The studies demonstrate a model for endometriosis in athymic mice.

Topics: Adult; Animals; Ascitic Fluid; Carbocyanines; Disease Models, Animal; Endometriosis; Endometrium; Female; Fluorescent Dyes; Humans; Injections, Intraperitoneal; Leukocyte Transfusion; Mice; Mice, Nude; Microscopy, Fluorescence; Middle Aged

Developing axons reach their final targets as a result of a series of axonal projections to successive intermediate targets. Long-range chemoattraction by intermediate targets plays a key role in this process. Growing axons, however, do not stall at the intermediate targets, where the chemoattractant concentration is expected to be maximal. Commissural axons in the metencephalon, initially attracted by a chemoattractant released from the floor plate, were shown to lose responsiveness to the chemoattractant when they crossed the floor plate in vitro. Such changes in axon responsiveness to chemoattractants may enable developing axons to continue to navigate toward their final destinations.

Topics: Animals; Axons; Carbocyanines; Cell Adhesion Molecules, Neuronal; Chemotaxis; Contactin 2; Culture Techniques; Fluorescent Dyes; Membrane Glycoproteins; Nerve Growth Factors; Netrin-1; Pons; Rats; Rats, Wistar; Rhombencephalon; Tumor Suppressor Proteins

This laboratory has previously described the aggregation of intravenously administered lipid-coated microbubbles (LCM) around tumors and areas of injury. 7Beta-hydroxycholesterol has been used to inhibit astrocytic proliferation in nervous system injury models. The compound has been given by direct infusion, by epidural catheter, or in liposomes (delivered stereotactically to the injury site). In this article, we report the use of LCM to deliver 7beta-hydroxycholesterol to a radiofrequency injury site in the rat cerebrum.. First, the ability of LCM to target the thermal lesion in the rat brain was characterized using a lipid-soluble fluorescent dye 3,3-dioctadecyloxacarbocyanine perchlorate. Then, the effectiveness of this delivery system in suppression of glial proliferation was measured by glial fibrillary acidic protein immunoreactivity.. Glial fibrillary acidic protein immunoreactivity was significantly reduced when 7beta-hydroxycholesterol was administered via LCM but not alone, suggesting that astrocytic proliferation would correspondingly be diminished.. LCM were assessed as a delivery vehicle for 7beta-hydroxycholesterol in a rat brain radiofrequency lesion and found to be efficient in reducing astrogliosis, as measured by glial fibrillary acidic protein immunoreactivity.

Topics: Animals; Astrocytes; Brain; Brain Injuries; Carbocyanines; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Gliosis; Hydroxycholesterols; Immunohistochemistry; Lipids; Microspheres; Radio Waves; Rats; Rats, Sprague-Dawley; Surface Properties

The goal of this study was to evaluate whether there is any relationship between survival of patients with brain tumor and tumor proliferation or tumor invasion in vitro.. Samples of freshly resected brain tumors from 14 patients with glioblastoma multiforme (GBM) were directly grown as three-dimensional multicellular spheroids. The tumor spheroids were cocultured with fetal rat brain cell aggregates (BCAs), used to represent an organotypical normal brain tissue model. Before the coculture, the tumor spheroids and the BCAs were stained with two different carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecycloxacarbocyanine perchlorate (DiO), respectively. During the coculture, confocal laser scanning microscopy allowed a sequential analysis of tumor cell invasion by visualizing dynamic aspects of the invasive process. Single cocultures were examined at three different time points (24, 48, and 96 hours). During the observation period there was a change in the structural morphology of the cocultures, with a progressive decrease in BCA volume. Furthermore, the scanning confocal micrographs revealed a bidirectional movement of tumor cells and normal cells into brain and tumor tissue, respectively. It is also shown that there is a considerable variation in the rate of BCA destruction in cocultures of glioma spheroids generated directly from biopsy specimens. This variation is seen both between spheroids generated from the same biopsy as well as between spheroids that are grown from different biopsy specimens. Cell proliferation measured by Ki-67 immunohistochemical analysis of biopsy samples obtained in the same patients revealed a correlation between tumor cell proliferation and tissue destruction of the BCAs, as determined by a reduction in BCA volume (p = 0.0338). No correlation was found when survival was related to the same parameters (p > 0.05).. The present work provides a model for quick and efficient assessment of dynamic interactions between tumor and normal brain tissue shortly after surgery.

Topics: Adult; Aged; Animals; Biopsy; Brain; Brain Neoplasms; Carbocyanines; Cell Aggregation; Cell Division; Cells, Cultured; Female; Fluorescent Dyes; Glioblastoma; Humans; In Vitro Techniques; Ki-67 Antigen; Male; Microscopy, Confocal; Middle Aged; Neoplasm Invasiveness; Rats; Spheroids, Cellular; Survival Rate; Time Factors; Tumor Cells, Cultured

Fluorescent lipid analogue 3,3'-dioctadecyloxacarbocyanine incorporated into biological membranes was used to induce photoactivation of a hydrophobic probe 5-[125I]iodonaphthyl-1-azide (125INA) by energy transfer and to thereby confine subsequent radiolabelling of proteins to the lipid bilayer. This approach was applied in bovine chromaffin cells to discover cytosolic proteins that reversibly penetrate into membrane domains. alpha-Glutathione S-transferase (alpha-GST) was identified as the only labelled protein in bovine chromaffin-cell cytosol, indicating that it inserts reversibly into the membrane lipid bilayer. The selectivity of the labelling towards the lipid bilayer is demonstrated by showing that influenza virus haemagglutinin becomes labelled by 125INA only after the insertion of this protein into the target membrane. The molar 125INA:protein ratio was used as a quantitative criterion for evaluation of the penetration of proteins into the membrane lipid bilayer. This ratio was calculated for four integral membrane proteins and four soluble proteins that interact with biological membranes. The values for four integral membrane proteins (erythrocyte anion transporter, multidrug transporter gp-170, dopamine transporter and fusion-competent influenza virus haemagglutinin) were 1, 8, 2 and 2, respectively, whereas for soluble proteins (annexin VII, protein kinase C, BSA and influenza virus haemagglutinin) the values were 0.002, 0, 0.002 and 0.02, respectively. The molar ratio for alpha-GST was found to be 1, compatible with the values obtained for integral membrane proteins.

Topics: Adrenal Medulla; Animals; Azides; Carbocyanines; Cattle; Cell Membrane; Cells, Cultured; Chromaffin Cells; Chromaffin Granules; Cytosol; Energy Transfer; Fluorescent Dyes; Glutathione Transferase; Iodine Radioisotopes; Lipid Bilayers

The stain toluidine blue-O (tol blue), applied to sections of neural tissue, is shown to be compatible with the vivid fluorescent lipophilic neural tracers 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (Di-ASP), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). As with other Nissl stains, toluidine blue-O fluoresces in the red end of the spectrum but such fluorescence quenches upon binding with tissue. Moreover, progressive staining occurs at concentrations low enough to minimise any background fluorescence attributable to non-specific residence of the stain. The bright yellow Di-ASP and vivid green DiO signals are spectrally removed from the red fluorescence of toluidine blue-O. With toluidine blue-O counterstaining, Di-ASP generally offers contrast superior to that with DiI, however, the latter is improved by viewing in a polarised green bright field. Visible Di-ASP emission, although broad, peaks at a more film-sensitive region of the spectrum than that for DiI, thus reducing the photographic exposure required.

Topics: Animals; Axons; Carbocyanines; Female; Fluorescent Dyes; Lipids; Macropodidae; Optic Nerve; Pregnancy; Pyridinium Compounds; Staining and Labeling; Tolonium Chloride

The interaction of VSV glycoprotein (VSV G) with biological membranes was studied by photosensitized labeling. The method is based on photosensitized activation by the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine (DiO) of a hydrophobic probe, [125I]iodonaphthyl azide (125INA), that rapidly partitions into the membrane bilayer of virus and cells. 125INA labeling of proteins and lipids can be confined to the site of chromophore localization by photosensitized labeling. Photoactivation using visible light of target membrane labeled with DiO and 125INA, to which unlabeled virions are bound, results in exclusive labeling of envelope glycoproteins inserted into the target membrane [Pak et al. (1994) J. Biol. Chem. 269, 14614]. In this study, we labeled lipid symmetric erythrocyte ghosts with 125INA and DiO. Photosensitized activation of VSV prebound to labeled ghosts with visible light resulted in VSV G labeling under fusogenic conditions. Photoactivation of 125INA by UV light, which is nonspecific, produced labeled VSV G at both acidic and neutral pH. Photosensitized labeling of VSV G by DiO-125INA-ghosts was also observed at pH 5.5, 4 degrees C, in the absence of mixing between viral and cellular lipids, suggesting insertion of the ectodomain of VSV G. Soluble VSV G lacking the transmembrane domain inserted into DiO-125INA-ghosts under the same conditions as intact VSV G. DiO inserted into intact VSV appeared to be a suitable fluorophore for continuous kinetic measurements of membrane fusion by fluorescence dequenching. Our photosensitized labeling results establish biochemical correlates for the three states of VSV G, which we had proposed based on kinetic data [Clague et al., Biochemistry 29, 1303]. In addition, we found that VSV G insertion into the target membrane is reversible, suggesting a "velcro"-like attachment of the fusogenic domain with the target membrane.

Topics: Affinity Labels; Azides; Carbocyanines; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Fluorescent Dyes; Glycoproteins; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Iodine Radioisotopes; Membrane Glycoproteins; Photosensitizing Agents; Protein Conformation; Ultraviolet Rays; Viral Envelope Proteins; Viral Fusion Proteins

A group of efferent neurons whose bodies are located contralaterally and extend projections across the ventral midline of the hindbrain is considered as a rhombomere 4-specific characteristic. These neurons contribute to the vestibulo-acoustic nerve. At the level of rhombomere 2, a similar kind of efferents have only been described as a result of several experimental manipulations and have been interpreted as being due to rhombomere 2 acquiring rhombomere 4 identity. Here is shown that contralateral efferents can also be detected in rhombomere 2 of normal mouse and chicken hindbrains. These findings indicate that neural processes crossing the midline should not be considered as a rhombomere 4-specific characteristic. They also imply that the formation of the contralateral efferents at different rostro-caudal levels might be under different genetic controls, because Hoxb-1, which is not expressed in rhombomere 2, seems to be essential for their proper formation in rhombomere 4.

Topics: Animals; Carbocyanines; Chick Embryo; Cranial Nerves; Fluorescent Dyes; Mice; Neurons, Efferent; Rhombencephalon; Vestibulocochlear Nerve

We investigated the potential role of rostral-caudal and dorsal-ventral subdivisions of the early rostral brain by relating these subdivisions to the early patterning of neuron cell bodies and their axon projections. The earliest neurons were mapped using the lipophilic axon tracers diI and diO on embryos fixed on embryonic days 9.5-10.5 (E9.5-E10.5); neuromeric boundaries were marked by diO. The tracts were small in number, were organized orthogonally (2 dorsal-ventral and 4 rostral-caudal), and originated from groups of cell bodies which we term "sources." Two parallel longitudinal axon systems, one dorsal (the tract of the postoptic commissure and the mesencephalic tract of the trigeminal nerve) and one ventral (the mammillotegmental tract and the medial longitudinal fasciculus), projected caudally from the prosencephalon into the rhombencephalon. We argue that the dorsal longitudinal pathway marked the boundary between the alar and basal plates along the entire neuraxis. The dorsal-ventral axons coursed circumferentially and either crossed the midline (forming the posterior and ventral tegmental commissures) or turned caudally without crossing the midline. The dorsal-ventral axons were not generally restricted to the interneuromeric boundaries, as others have suggested. Earlier, all neighboring neurons projected their axons together; later, nearby neurons projected into different pathways. Some tracts originated in single neuromeres, while other tracts had origins in two or more neuromeres. The dorsal longitudinal axons altered course at several of the borders, but the ventral longitudinal axons did not. In summary, the early subdivisions appeared to influence some, but not all, aspects of tract formation.

Topics: Animals; Carbocyanines; Immunohistochemistry; Mesencephalon; Mice; Molecular Probes; Neural Pathways; Neurons; Oculomotor Nerve; Prosencephalon; Trigeminal Nerve; Trochlear Nerve

The DNA content of nuclei during the 2-cell stage as well as in presumptive tetraploid embryos was investigated. In vivo produced pig zygotes were cultured to the 2-cell stage and either monitored for cleavage to the 4-cell stage or mounted at various times post-cleavage and DNA content determined. The length of the 2-cell stage was 14.8 +/- 3.0 hr. There was a significant increase in the length of the 2-cell stage due to the time in vitro as a zygote (P < 0.001: R2 = 0.866). The DNA content increased (P < 0.05) each 2 hr postcleavage until 10 hr postcleavage. This suggested that there is a short G1 and G2 phase and a relatively long phase of DNA synthesis. Next, 2-cell stage embryos were pulsed with electricity to induce cell-to-cell fusion. Whereas only about half fused within 30 min (55%), most (96%) developed to the blastocyst stage. The DNA content of the nuclei of the embryos was consistent with them being tetraploid. A final experiment was designed to evaluate the ability of the tetraploid embryo to form a chimera with isolated inner cell mass (ICM) cells. Inner cell masses were isolated from d 6 embryos, cut into thirds, labeled with DiO (a membrane die) and injected into the perivitelline space of 4-cell-stage tetraploid embryos. Twelve of 17 formed blastocysts. In most (8/12), the ICM of the resulting blastocyst was labeled, whereas in one the only fluorescence was in the trophectoderm, and in two fluorescence was evenly distributed between the ICM and trophectoderm. These results suggest that it may be possible to create a fetus derived from ICM cells, or potentially stem cells, that has a tetraploid trophoblast.

Topics: Animals; Blastocyst; Carbocyanines; Cell Culture Techniques; Cell Division; Cell Fusion; Chimera; DNA; Embryo, Mammalian; Insemination, Artificial; Oocytes; Ploidies; Swine; Zygote

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi. Repetitive compression and expansion of the monolayers containing DPPC:DOPC:NBD-PC 49:50:1 at an initial rate of 3.2 A2 molecule-1 s-1 produced monolayers with visual properties consistent with there being a preferential exclusion of the unsaturated lipid from the monolayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 4-Chloro-7-nitrobenzofurazan; Biophysical Phenomena; Biophysics; Carbocyanines; Fluorescent Dyes; Membranes, Artificial; Microscopy, Fluorescence; Phosphatidylcholines; Pulmonary Surfactants

Red blood cells labeled with the carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), were evaluated for use in making microvascular measurements in rat small intestine and spinotrapezius muscle. We determined the minimum concentration of each dye which produced near maximal fluorescent intensity and labeled cell fraction. These dyes, which have excitation and emission spectra similar to fluorescein and rhodamine derivatives, have a number of advantages over the isothiocyanates: (1) the labeling procedure is quicker, easier, and less expensive; (2) the labeled cell fraction and the fluorescent intensity of DiI and DiO cells are stable for long periods of time in the rat circulation; and (3) DiI-labeled cells are brighter and transmit light through overlying erythrocytes better than rhodamine X isothiocyanate. However, in vitro and in vivo evaluations illustrate the potential limiting effects of vessel diameter and cell velocity on the accuracy of microvascular measurements made using this technique. In the small intestine and spinotrapezius muscle preparations, measurements of labeled cell flux were readily reproducible and could be partly automated with image analysis only in capillaries and small venules. Counting labeled cells in larger vessels by human observation or with automation was not reproducible, presumably due to absorption and dispersion of the fluorescent signal by overlying erythrocytes and smearing of the cell image at high cell velocities.

Topics: Animals; Carbocyanines; Erythrocytes; Fluorescent Dyes; Image Processing, Computer-Assisted; Intestine, Small; Microcirculation; Muscles; Rats