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PubMed2005

Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures.

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline to the culture medium confirmed that both N-methyl-D-aspartate and non-N-methyl-D-aspartate ionotropic glutamate receptors were involved in the oxygen-glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the DL-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of DL-threo-beta-benzyloxyaspartate in concentrations of 25 microM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen-glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10 microM) dose of DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation (but not during the next 48 h after oxygen-glucose deprivation) significantly reduced the oxygen-glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen-glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen-glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.

Amino Acid Transport System X-AG↗

PubMed2004

Viability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure.

Troglitazone, bosentan and glibenclamide inhibit the bile salt export pump (Bsep) which transports taurocholate into bile. Sandwich-cultured rat hepatocytes maintain functional sodium taurocholate co-transporting polypeptide and Bsep transport proteins, and may be useful to study inhibition of transport by xenobiotics at concentrations below the lowest observable adverse effect level (LOAEL). The purpose of this study was to compare viability assessments determined with the neutral red, lactate dehydrogenase (LDH), alamar blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide assays in sandwich-cultured rat hepatocytes following exposure to xenobiotics known to inhibit Bsep, and to define the LOAEL for these xenobiotics in this system. The neutral red assay was not amenable to use in this model due to crystal formation on the collagen. Troglitazone decreased viability in every assay examined, with a LOAEL approximately 100 microM. Bosentan also decreased viability as measured by the LDH, MTT and propidium iodide assays, with a LOAEL approximately 200 microM; however, a significant decrease in viability was not observed with the alamar blue assay. Glibenclamide did not decrease viability with any assay at the xenobiotic concentrations examined in this study. Based on the results of this study, the LDH or propidium iodide assays would be the methods of choice to assess viability in sandwich-cultured rat hepatocytes after xenobiotic exposure.

Animals↗

PubMed1998

Effects of PEG-lipids on permeability of phosphatidylcholine/cholesterol liposomes in buffer and in human serum.

The permeability of liposomal membranes was studied as a function of the amount of incorporated PEG-lipid. The fluorescent dyes ethidium, propidium and 5(6)-carboxy fluorescein were used as markers for measurements of spontaneous leakage. The results show that addition of up to 8 mol% of PEG(2000)-DSPE into liposomal membranes of DSPC/Cho and EPC/Cho reduces the permeability of carboxyfluorescein in buffer solution. In contrast, the leakage of the more amphiphilic dye ethidium was not to any measurable extent affected by PEG-lipid inclusion. Another important difference was that ethidum leakage showed a clear dependence on temperature whereas leakage of carboxyfluorescein from pegylated liposomes did not. We conclude that the mechanisms by which the two dyes permeate the liposomal bilayer are qualitatively different. Both ethidium and carboxyfluorescein did interact with human serum components in a way that made measurements in serum unreliable. The more hydrophilic ethidium analogue propidium was shown not to interact with human serum components to any detectable extent. This made propidium suitable for permeability determinations in human serum. It was found that liposomes composed of pure EPC or EPC with 5 mol% DSPE-PEG, displayed a dramatic increase in permeability when subjected to a medium composed of 20% human serum in buffer. Addition of 40 mol% cholesterol to the EPC bilayers reduced the observed release rate in human serum substantially, whereas no stabilizing effect was observed upon PEG-lipid inclusion.

Buffers↗

PubMed1984

A rapid method for preparation of urinary bladder epithelium for flow cytometric analysis.

A rapid and reliable method is described for preparation of urinary bladder epithelium for flow cytometric analysis. This method has proven useful for measurement of cell cycle kinetics in bladder epithelium from normal fetal, neonatal and adult mice as well as animals that have been exposed to urinary bladder toxicants. A modified Vindelov 's propidium iodide stain was instilled directly into the urinary bladder through the urethra, allowing the propidium iodide to lyse the epithelial cells and stain the DNA in the liberated nuclei. The stained epithelial cell nuclei were then removed from the lumen and analyzed by flow cytometry without centrifugation or filtration techniques. This method allows examination of epithelial cell nuclei without contamination from other tissue elements due to the fact that the propidium iodide did not act beyond the epithelial basement membrane under our experimental conditions.

Animals↗

PubMed2002

Inhibition of acetylcholinesterase from Electrophorus electricus (L.) by tricyclic antidepressants.

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.

Acetylcholinesterase↗

PubMed2003

Identification of neuronal cell death in a model of degeneration in the hippocampus.

Neuronal cell death and microglial changes are both hallmarks of neurodegenerative disorders. Therefore, analysis of degenerating neurons related to microglial changes are addressed in many studies of neurosciences. Here we compared different lesion models and two markers for neurodegeneration (Fluoro-Jade and propidium iodide) in an in vivo as well as an in vitro approach. Fluoro-Jade is a specific and selective marker to identify neurons undergoing degeneration. We also tested this marker to analyze neurodegeneration in organotypic hippocampal slice cultures. We could show that activation of microglia is followed by neuronal cell death. Most degeneration markers, such as propidium iodide, only stain the neuronal cell body excluding the axonal and dendritic processes. Fluoro-Jade is able to stain the distal portion as well as the proximal portion of the dissected axon including the axotomized neuron, as so called anterograde and retrograde degeneration after axotomy. To analyze the specificity of Fluoro-Jade, we used primary microglial and BV-2 cells, a well-described murine microglial cell line. Treatment of microglial and BV-2 cells with an excess of L-glutamate induces cell death which could be detected by propidium iodide staining, but not by Fluoro-Jade, demonstrating its specificity to monitor neuronal cell death.

Animals↗

PubMed1990

Drug binding by branched DNA molecules: analysis by chemical footprinting of intercalation into an immobile junction.

Branched DNA structures interact with drugs differently from unbranched control duplexes of similar sequence. A specific interaction between the reagent (methidiumpropyl-EDTA).Fe(II) [MPE.Fe(II)] and a branched DNA molecule formed from 16-mer oligonucleotide strands has been reported [Guo, Q., Seeman, N. C., & Kallenbach, N. R. (1989) Biochemistry 28, 2355-2359]. The structure of the branched molecule is thought to be made up of two double-helical stacking domains with an overall twofold symmetry across the branch site. The MPE-Fe(II) interaction occurs predominantly at or adjacent to the branch site and is eliminated by a second intercalator, propidium iodide. Further studies on the nature and properties of this site are presented here. Comparison of the patterns of scission of linear duplex and branched tetramer by EDTA.Fe(II), MPE.Fe(II), and Cu(I)-(o-phenanthroline)2 [(OP)2Cu(I)] provides a higher resolution picture of the site of enhanced binding. In particular, the sensitive footprinting afforded by (OP)2Cu(I) allows us to localize the major site of preferential interaction with propidium precisely to the branch point itself, with a roughly twofold symmetric pattern of cuts resulting. In detail, the differential pattern with respect to each duplex control is distinct for each arm of the junction. Excess propidium results in apparent reversal of the crossover isomer of the junction, indicating a possible additional avenue for the action of drugs in biological systems--effects on the products of recombination.

Base Sequence↗

PubMed1998

Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligands.

The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripheral site at the mouth of the gorge some 10-20 A from the acylation site. Many ligands that bind exclusively to the peripheral site inhibit substrate hydrolysis at the acylation site, but the mechanistic interpretation of this inhibition has been unclear. Previous interpretations have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. These analyses indicate that inhibitors bound to the peripheral site decrease acylation and deacylation rate constants and/or decrease substrate affinity at the acylation site by factors of up to 100. Conformational interactions have been proposed to account for such large inhibitory effects transmitted over the distance between the two sites, but site-specific mutagenesis has failed to reveal residues that mediate the proposed conformational linkage. Since examination of individual rate constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an alternative nonequilibrium analysis of the steady-state inhibition patterns. This analysis incorporates a steric blockade hypothesis which assumes that the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Simulations based on this nonequilibrium steric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if binding of these ligands slows substrate association and dissociation rate constants by factors of 5-70. Direct measurements with the acylation site ligands huperzine A and m-(N,N, N-trimethylammonio)trifluoroacetophenone showed that bound propidium decreased the association rate constants 49- and 380-fold and the dissociation rate constants 10- and 60-fold, respectively, relative to the rate constants for these acylation site ligands with free AChE, in reasonable agreement with the nonequilibrium steric blockade model. We conclude that this model can account for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.

Acetophenones↗

PubMed2003

Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites.

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.

Acetylcholinesterase↗

PubMed2005

Corticosterone increases damage and cytosolic calcium accumulation associated with ethanol withdrawal in rat hippocampal slice cultures.

BACKGROUND: Evidence suggests that stress hormones (i.e., glucocorticoids) may be increased during acute or chronic consumption of ethanol and during withdrawal from ethanol consumption, effects that may contribute to the development of cognitive impairment. The goal of the current studies was to examine the hypothesis that increased glucocorticoid levels in conjunction with ethanol exposure and withdrawal may cause hippocampal damage. METHODS: Organotypic hippocampal slice cultures were exposed to 50 mM ethanol for 10 days and withdrawn for 1 day. After withdrawal, cytotoxicity and cytosolic Ca2+ accumulation were measured using the nucleic acid stain propidium iodide and Calcium Orange, AM, respectively. Cultures were also treated with nontoxic concentrations of corticosterone (0.001-1 microM) during ethanol exposure and withdrawal or only during withdrawal. Additional cultures were coexposed to corticosterone and RU486 (0.1-10.0 microM), spironolactone (0.1-10.0 microM), or MK-801 (20 microM) during ethanol exposure and/or withdrawal. RESULTS: Ethanol withdrawal did not increase propidium iodide fluorescence and cytosolic Ca2+ levels. However, significant increases in propidium iodide fluorescence and in cytosolic Ca2+ accumulation were observed in cultures when corticosterone (> or = 100 nM) was exposed during ethanol treatment and/or withdrawal. These effects of corticosterone on ethanol withdrawal were attenuated by RU486 and MK-801 but not by spironolactone coexposure. CONCLUSIONS: This report demonstrated that corticosterone exposure during ethanol treatment and/or withdrawal resulted in significant hippocampal damage, possibly via activation of glucocorticoid receptors and enhancement of the glutamatergic cascade. The findings from these studies suggest that glucocorticoids contribute to the neuropathological consequences of alcohol dependence in humans.

Animals↗

PubMed1985

Selective extraction of small and large molecules from the cerebrospinal fluid by Purkinje neurons.

Cerebellar Purkinje neurons accumulated propidium iodide, granular blue, and horseradish peroxidase conjugated to wheat germ agglutinin but not unconjugated horseradish peroxidase, bisbenzimide, or Evans blue when these compounds were infused into the lateral cerebral ventricles of awake, unrestrained rats. Accumulation of propidium iodide by Purkinje neurons of the vermis was associated with a reproducible behavioral abnormality characterized by truncal tremor, ataxia, and nystagmus. Both the accumulation of propidium iodide in Purkinje cells and the behavioral abnormality were prevented by prior intracerebroventricular administration of ouabain or colchicine, drugs that block neuronal transport processes. The ability of cerebellar Purkinje neurons to extract small and large molecules from the cerebrospinal fluid has important implications for their physiology and pathology.

Animals↗

PubMed1999

LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.

LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy.

Anidulafungin↗

PubMed2000

Huprine X is a novel high-affinity inhibitor of acetylcholinesterase that is of interest for treatment of Alzheimer's disease.

Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.

Acetylcholinesterase↗

PubMed2002

Mitochondrial cytochrome c release in radiation-induced apoptosis of human peripheral T cells.

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, using a microscopy-video system, mitochondrial membrane potential assay, annexin V, propidium iodide, and cytochrome c ELISA kit. After 5 Gy irradiation with 10 MV X-ray from a linear accelerator, the percentages of apoptotic T cells were estimated as approximately 5, 10, 20, 35, and 70%, at 0, 3, 6, 10, and 20 h after irradiation, respectively, as observed with the microscopy-video system. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, approximately half of the T cells showed dysfunction of mitochondrial membrane potential at 10 h after 5 Gy irradiation. With regard to annexin V and propidium iodide, approximately 40 and 5% of the human peripheral T cells showed positivity against annexin V and propidium iodide at that time, respectively. Mitochondrial cytochrome c release from the mitochondria to the cytosol was confirmed to start at 10 h and to reach a maximum at 20 h after 5 Gy of irradiation. These results demonstrated that mitochondrial cytochrome c release occurred following dysfunction of mitochondrial membrane potential in radiation-induced T cell apoptosis.

Annexin A5↗

PubMed1991

Quantitative assessment of chromatin stability alteration in human spermatozoa induced by freezing and thawing. A flow cytometric study.

The authors examined the effect of cryopreservation on chromatin stability in human spermatozoa from 21 ejaculates. Each ejaculate was divided into four aliquots: (1) fresh aliquot, (2) frozen and thawed aliquot, (3) fresh aliquot subjected to an in vitro decondensation method, and (4) frozen and thawed aliquot subjected to the same in vitro decondensation method; all were then fixed with an ethanol fixative agent. Chromatin stainability was quantified by flow cytometric measurement of fluorochrome uptake by DNA. Study of 21 fresh aliquots showed that 37.9% of the DNA was accessible to propidium iodide. The comparative stainability between the 21 fresh and 21 frozen-thawed, undecondensed aliquots demonstrated a low but significant increase in accessibility of DNA by propidium iodide for the thawed samples: 38.7 +/- 1.7% (mean +/- SD) versus 37.9 +/- 1.3%. The biochemical action of the nuclear decondensation solution increased the accessibility of propidium iodide, but in different ways: 57.2 +/- 12.9% versus 54.7 +/- 13.7%, respectively, for fresh and frozen-thawed aliquots. Analysis of the flow cytometric histograms revealed an intermediate population of spermatozoa adjacent to the main germinal peak. This population increased significantly: 9.6 +/- 1.9% for the fresh versus 12.3 +/- 4.9% for the frozen-thawed undecondensed aliquots and 8.6 +/- 3.5% versus 12.3 +/- 4.9%, respectively, for fresh and frozen-thawed, decondensed aliquots. Because the chromatin stability of thawed spermatozoa may be a critical factor in assisted procreation, the authors discuss the effect of thermal denaturation on the nucleoprotein structures and the origin of the intermediate population of spermatozoa.

Chromatin↗

PubMed1985

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody.

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.

Adenocarcinoma↗

PubMed1986

Changes in accessibility of DNA to various fluorochromes during spermatogenesis.

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.

Acridine Orange↗

PubMed2000

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape.

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.

Administration, Topical↗