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PubMed2001

Flow cytometry, a useful tool for detecting the lethal effect of pentamidine on Leishmania viannia complex promastigote forms.

Flow cytometry (FC), combined with propidium iodide as supravital colorant, was used to study Leishmania(subgenus Viannia)panamensis [L (V) panamensis] complex strain susceptibility to meglumine antimoniate, sodium stibogluconate and pentamidine. Despite all drugs examined being leishmanicidal to axenic forms in in vitro trials (in the presence of macrophages), axenic amastigotes directly exposed to these drugs were highly resistant under our experimental conditions. A direct lethal effect on promastigotes, detectable by FC, was only obtained with pentamidine after in vitro treatment of both promastigotes and axenic amastigotes with the drugs. Pentamidine's rapid lethal effect, as detected by FC, could be further confirmed in short- and long-term parasite cultures after exposure to a drug. FC's suitability for measuring L (V) panamensis complex's promastigote susceptibility to pentamidine, shortly after in vitro drug exposure, might be useful in monitoring clinical trials with this drug and facilitating rapid pentamidine-resistant natural isolate identification.

Animals↗
PubMed1996

Alterations in nuclear ploidy and cell phase distribution of rat liver cells in experimental alcoholic liver disease: relationship to antioxidant enzyme gene expression.

The ability of a cell to withstand oxidative stress has been hypothesized to be related to its ploidy status. We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship between severity of liver injury, antioxidant mRNA levels, and DNA ploidy of liver cells. Rats were fed ethanol with different dietary fats (saturated fat, corn oil, and fish oil); pair-fed control animals received isocaloric amounts of dextrose. All animals were euthanized at 1 month and had evaluation of pathologic changes in the liver, DNA content by flow cytometry, and mRNA levels for catalase and glutathione peroxidase. The fish oil-ethanol group exhibited the most severe pathology, the corn oil-ethanol group had intermediate pathologic changes, and no pathologic changes were seen in the saturated fat-ethanol and dextrose-fed controls. Flow cytometric analysis of propidium iodide-stained nuclei revealed that saturated fat-dextrose and corn oil-dextrose groups had about 65% of cells with (diploid) G1 DNA content and about 30% of cells with tetraploid (4C) nuclei. The fish oil-dextrose had a significantly higher (p < 0.001) number of 4C cells (67.4 +/- 2.1%) compared to the other two dextrose-fed groups. In the animals showing pathologic liver injury, there was a higher percentage of cells with hypertetraploid nuclei. The highest percentage of these hypertetraploid cells was seen in the fish oil-ethanol group. Catalase and glutathione peroxidase mRNA levels correlated significantly with polyploidy. A significant correlation was seen between the number of cells in the greater than G2 + M phase and glutathione peroxidase mRNA levels (r = 0.91, p < 0.01) and catalase mRNA. The different slopes of correlation analysis between catalase mRNA and dietary fats show that the degree of saturation of fatty acids may influence catalase mRNA expression in cells with different ploidy states. We propose that polyploidization of liver cell nuclei may serve as a defense mechanism against ethanol-induced hepatotoxicity. This defense mechanism may also, in part, account for the antiregenerative effect of ethanol on hepatocytes.

Animals↗
PubMed2001

Progressive disruption of the plasma membrane during renal proximal tubule cellular injury.

The goal of this study was to examine the progression of plasma membrane disruption during cell injury using rabbit renal proximal tubules (RPT). The results demonstrated that the plasma membrane became permeable to larger and larger molecules as anoxia proceeded. At least three distinctive phases of membrane disruption were differentiated during anoxia. In phases 1, 2, and 3, plasma membranes became permeable to propidium iodide (PI, molecular weight = 668), 3 kDa dextrans, and 70 kDa dextrans or lactate dehydrogenase (LDH, molecular weight = 140 kDa), respectively. Phase 1 was reversible by reoxygenation but not prevented by the glycine. Phase 2 was inhibited by glycine. Phase 3 was inhibited by several membrane-permeable homobifunctional crosslinkers, dimethyl-pimelimidate (DMP), ethylene-glycolbis(succinimidylsuccinate), and dithiobis(succinimidylpropionate), but not by the membrane-impermeable crosslinker dithiobis(sulfosuccinimidylpropionate). In addition, DMP decreased RPT LDH release produced by mitochondrial inhibition (antimycin A), an oxidant (t-butylhydroperoxide) and a nephrotoxicant that is metabolized to an electrophile (tetrafluoroethyl-l-cysteine). These results identify (1) different phases of plasma membrane damage with increasing permeability during cell injury, (2) the reversibility of phase 1, (3) the relative site of action of the cytoprotectant glycine (prevents phase 2), and (4) the protective effects of chemical crosslinkers in RPT cell death produced by different toxicants.

Animals↗
PubMed2001

Marijuana smoke and Delta(9)-tetrahydrocannabinol promote necrotic cell death but inhibit Fas-mediated apoptosis.

Marijuana smoke shares many components in common with tobacco smoke except for the presence of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychotropic compound found only in Cannibis sativa. Delta(9)-THC has been shown to potentiate smoke-induced oxidative stress and necrotic cell death. In the present study, our objective was to determine the effects of Delta(9)-THC on the balance between Fas-induced apoptosis and necrosis in A549 lung tumor cells. We found that Fas-induced activation of caspase-3 was inhibited by whole smoke from both tobacco and marijuana cigarettes. Gas-phase smoke, which generates high levels of intracellular reactive oxygen species, had no effect on caspase-3 activity. However, particulate-phase smoke (tar) was a potent inhibitor of Fas-induced caspase-3 activity, with marijuana tar being more potent than either tobacco or placebo marijuana tar (lacking Delta(9)-THC). Delta(9)-THC also inhibited Fas-induced caspase-3 activity in A549 cells. In contrast, no inhibition was observed when Delta(9)-THC was incubated with activated caspase-3 enzyme, suggesting that Delta(9)-THC acts on the cell pathway(s) leading to caspase-3 activation and not directly on enzyme function. Flow cytometry was used to measure the percentage of cells undergoing apoptosis (staining for annexin V) versus necrosis (staining for propidium iodide) and confirmed that both marijuana tar extract and synthetic Delta(9)-THC inhibit Fas-induced apoptosis while promoting necrosis. These observations suggest that the Delta(9)-THC contained in marijuana smoke disrupts elements of the apoptotic pathway, thereby shifting the balance between apoptotic and necrotic cell death. This shift may affect both the carcinogenic and immunologic consequences of marijuana smoke exposure.

Annexin A5↗
PubMed1990

Effects of lactacidosis on volume and viability of glial cells.

The effect of lactacidosis was analyzed in vitro by employment of C 6 glioma cells and astrocytes from primary culture. The cells were suspended in an incubation chamber under continuous control of pH, temperature and pO2. Cell swelling and viability were quantified by flow cytometry using propidium iodide for staining of dead cells. After a control period, the pH of the suspension medium was titrated to levels between pH 6.8 down to 4.2 by addition of isotonic lactic acid. Acidification below pH 6.8 led to an immediate swelling of C 6 glioma cells as well as of astrocytes. The degree of cell swelling was related to the decrease in pH and the duration of exposure. For instance, lactacidosis of 60 min at pH 6.2 resulted in an increase of glial volume to 124.5 +/- 4.6%, while pH 4.2 in an increase to 190.9 +/- 8.4%. Cell viability remained unchanged down to pH 6.2. At pH 5.6 and below viability decreased in relation to the severity of acidosis. When sulfuric acid was used, the extent of cell swelling at pH 5.6 was only 50% of what was found by addition of lactic acid, whereas cell viability was not differently affected. The results demonstrate a specific efficacy of lactic acid to induce glial swelling, which might be due to a cellular accumulation of the compound.

Acidosis, Lactic↗
PubMed1994

Sequential changes in intercellular junctions between hepatocytes during the course of acute liver injury and restoration after thioacetamide treatment.

Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal. These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions.

Animals↗
PubMed1989

Olivary branching projections to the flocculus, nodulus and uvula in the rabbit. II. Retrograde double labeling study with fluorescent dyes.

The previous electrophysiological study reported that some of the Purkinje cells in the vestibulocerebellum were innervated by branching climbing fibers. To identify the cells of origin of these branching climbing fibers, a pair of retrograde fluorescent tracers, propidium iodide in combination with 4', 6-diamidino-2-phenylindole or diamidino yellow, were injected into two sites in the flocculus, nodulus and uvula. Proportions of double labeled neurons out of all labeled neurons in each subdivision of the contralateral inferior olive were studied. In cases of injections into the flocculus and nodulus, 13% (maximum 27%) of all the labeled neurons in the dorsal cap and 6% (maximum 12%) in the ventrolateral outgrowth were double labeled. In cases of injections into the nodulus and uvula, 20% neurons (maximum 34%) of the labeled neurons in the dorsal cap and 8% of those in the nucleus beta were double labeled. The present retrograde labeling study revealed that some dorsal cap neurons project to the uvula, which has not been reported previously in the rabbit. Injections into the flocculus and uvula caused only slight double labeling (0.4%) in the inferior olive.

Amidines↗
PubMed1986

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping.

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.

Animals↗
PubMed1988

Gradient fractionation of cycling and resting cells monitored by BrdUrd incorporation.

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium Iodide (PI), showed the S compartment highly concentrated in the 1.073/77 g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.

Animals↗
PubMed1980

A rapid cytofluorometric method for quantitative DNA determination on fixed smears.

A study has been made of the possibility of using propidium iodide (PI) for the cytofluorometric determination of DNA in fixed smears. A preliminary spectrofluorometric study made it possible to define the optimal conditions for the excitation wavelength and the intensity of the emitted fluorescence. The stoichiometry and specificity of the DNA-PI bond were studied in different materials and for different fixation procedures. In order to check whether RNA might interfere, it was extracted enzymatically from test preparations selectively. The data from such treated sections were not significantly different from those obtained by means of the fluorescent Feulgen reaction (Pararosaniline-SO2) on the same material. On the other hand, some of the advantages of the PI method are important: (a) the time required for making ready and staining the preparations is very short, and in any case is considerably shorter than the Feulgen method; (b) the high quantum yield of the DNA-PI complex induces very high fluorescence intensities, which can, therefore, be easily measured, even with low sensitivity instruments; (c) the spectral conditions are particularly favourable for excluding the inner filter effect from the measurement; (d) the photo-decomposition is considerably lower compared to that found in preparations stained by the conventional Feulgen method. The wide possibility of excitation (from the u.v. to the green), together with the limited extent of the emission band (which is mainly in the red) are also conditions that are particularly favourable for obtaining multi-parametric determinations simultaneously from the same cell.

DNA↗
PubMed1997

"Micronucleoli" in the Xenopus germinal vesicle.

The germinal vesicle of the Xenopus oocyte contains 1500 or more extrachromosomal nucleoli that are assembled on amplified copies of the rRNA genes. Many of these nucleoli have diameters of 10-15 micron, but some are much smaller, ranging down to 1 micron or less. Morphologically the smaller nucleoli or "micronucleoli" resemble the similarly sized B snurposomes, but they can be recognized with appropriate antibody probes (e.g., anti-nucleolin and anti-fibrillarin). We describe here a sensitive fluorescent staining technique that uses avidin and propidium iodide to visualize the rDNA in the amplified nucleoli. Many large nucleoli stain about as brightly as haploid yeast nuclei on the same slides. They presumably contain about 12 Mb of DNA, equivalent to 900 rDNA repeats. The smallest micronucleoli display only a tiny dot of stain, which must correspond to relatively few rDNA repeats.

Animals↗
PubMed1992

Viability measurements of hybridoma cells in suspension cultures.

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.

Adenosine Triphosphate↗
PubMed1998

Spontaneous and follicular fluid-induced acrosome reaction in sperm samples from in vitro fertilizing and nonfertilizing normozoospermic patients.

PURPOSE: One of the most challenging and intriguing groups of infertile patients is that of normozoospermic men who have repeatedly not achieved fertilization in vitro. These cases probably present a wide range of gamete disorders manifested at different stages of the fertilization process. The occurrence of spontaneous and follicular fluid (FF)-induced acrosome reactions (ARs) in in vitro fertilizing and nonfertilizing specimens from normozoospermic IVF patients was assessed, and the effect of Percoll and cryopreservation on the incidence of ARs was evaluated. METHODS: Semen samples from 62 normozoospermic (15 in vitro nonfertilizing and 47 fertilizing) patients were analyzed. Spermatozoa were double-stained with FITC-conjugated Pissum sativum lectin, to assess their acrosomal status, and propidium iodide, to evaluate their vitality, using flow cytometry analysis. RESULTS: A lower average of AR incidence was observed in the nonfertilizing than in the fertilizing group with all treatments. Both groups exhibited an increase in the proportion of ARs following incubation with FF. This rise was most prominent when Percoll-separated fractions were used (15.8 and 25.6% AR in the nonfertilizing and fertilizing groups, respectively). Thawed cryopreserved and fresh fertilizing samples exhibited similar AR rates. CONCLUSIONS: That normozoospermic recurrently nonfertilizing compared to fertilizing semen samples have a lower capacity to undergo ARs is suggested.

Acrosome↗
PubMed2003

Relationship between temperature and apoptosis of human peripheral blood mononuclear cells.

To examine the effect of various incubation temperatures on the apoptotic death of human peripheral blood mononuclear cells (PBMC), we incubated cells at 37 degrees C, 22 degrees C, and 4 degrees C for 1 and 24 hours. In addition, cells incubated at 4 degrees C for 3, 6, and 9 hours were rewarmed to 37 degrees C until a total incubation time of 24 hours was reached. The percentage of apoptotic cells was detected by a flow cytometric assay using propidium iodide staining. Incubation of PBMC at the above-mentioned temperatures for 1 hour did not affect the percentage of apoptotic cells. However, incubation at 4 degrees C for 24 hours resulted in the lowest percentage of apoptotic cells compared to those incubated at 22 degrees C and 37 degrees C. Rewarming of the cells to 37 degrees C increased the percentage of apoptotic cells to a level similar to that of the controls (incubated at 37 degrees C). Because PBMC are closely involved in the normal function of the immune system, the results of the study should be considered in cases in which these cells are exposed to various thermal conditions.

Apoptosis↗
PubMed2000

Effect of auranofin, an antirheumatic drug, on neutrophil apoptosis.

OBJECTIVE: The effects of auranofin (AF) on apoptosis and on the biological functions of neutrophils were investigated. METHODS: Neutrophils were incubated with various concentrations of AF for different periods. Cell viability was determined by the MTS assay and apoptosis was evaluated by flow cytometric analysis of propidium iodide (PI)- staining of the nuclei and annexin-V staining of phosphatidylserine in the cell membrane. The effect of AF on the expression of adhesion molecules (CD62L and CD11b/CD18) and on the generation of O2- by neutrophils was also determined. RESULTS: At a low concentration (1 microM), AF significantly prolonged neutrophil survival by delaying spontaneous apoptosis. Neutrophils incubated with AF for 12 and 24 hours maintained the capacity to express adhesion molecules and generate O2-. In contrast, a higher AF concentration (5 microM) shortened neutrophil survival by the induction of cell necrosis. CONCLUSION: Although the biological significance of inhibitory effect of AF on neutrophil apoptosis remains unclear, it seems to be unlikely that AF exerts the anti-inflammatory effect in vivo by directly suppressing neutrophil functions. Since AF has a wide range of effects on leukocytes, its therapeutic benefit in rheumatoid arthritis may be mediated in a complex manner.

Annexin A5↗
PubMed2006

Cytotoxic effects of BADGE (bisphenol A diglycidyl ether) and BFDGE (bisphenol F diglycidyl ether) on Caco-2 cells in vitro.

Bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) are used as starting substances for the manufacturing of epoxy resins used in internal can coatings. They are obtained by a condensation reaction between epichlorohydrin with bisphenol A and bisphenol F, respectively. These potential endocrine disrupting chemicals are able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal cells. The aim of this study was to characterize BADGE and BFDGE toxicity in Caco-2 cells, in particular, at the cellular and molecular level. Using several approaches, we characterized BADGE- and BFDGE-induced cell toxicity in Caco-2 cells. The treatment was done using different concentrations up to cytotoxic doses and different times of exposure to the agents. We evaluated the effect of these compounds on cell morphology, cell detachment, cell proliferation, F-actin disruption and plasma membrane integrity. Both compounds are able to induce morphological changes, cell detachment from the substratum and to inhibit cell proliferation, being these effects time and dose-dependent. Moreover, BADGE and BFDGE induce F-actin depolymerization, this effect is very potent at 24 h of incubation with the agents and a complete F-actin disruption can be observed at 200 microM BADGE or BFDGE. In addition, cell integrity is not damaged, since neither propidium iodide uptake nor LDH release takes place in Caco-2 cells exposed to high doses of these agents for 24 h.

Actins↗
PubMed2001

Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration.

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to approximately 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigmae is smaller than that of the cytosol sigmai. The stretching force vanishes when sigmae is equal to or larger than sigmai. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).

Animals↗
PubMed2002

NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer.

500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.

Antibiotics, Antineoplastic↗