Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Propidium”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed1981

Four fluorochromes for the demonstration and microfluorometric estimation of RNA.

Microfluorometric estimates of total RNA were made in selected test material stained with berberine sulfate, acridine orange, and Hoechst 33258. These measurements were compared with those obtained with propidium iodide, which is known to interact only with double-stranded nucleic acids. It was observed that all of the fluorochromes, including propidium iodide, yielded very similar patterns of fluorescence in the various types of material tested. In isolated thymocyte and hepatocyte nuclei stained with either propidium iodide or Hoechst 33258 at pH 2, it was evident that RNA could be estimated only indirectly by measuring the amount of fluorescence before and after extraction with RNase. It was apparent that the total fluorescence of small thymocyte nuclei was affected much less by RNase extraction than that of 2c hepatocyte nuclei. Attempts to obtain direct estimates of RNA by exposing the preparations to DNase were not successful: the fluorescence of thymocyte nuclei dropped almost to zero, and hepatocyte nuclei could no longer be assigned to distinct ploidy classes. In addition, since the highly condensed chromatin of thymocyte nuclei was stained much more prominently than the looser chromatin of hepatocyte nuclei with Hoechst 33258, it was apparent that this fluorochrome - when used at pH 2 - has potential usefulness as a "probe" of organizational differences in chromatin.

Acridine Orange↗
PubMed2001

C-phycocyanin protects cerebellar granule cells from low potassium/serum deprivation-induced apoptosis.

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.

Animals↗
PubMed2004

Formation of transient non-protein calcium pores by lysophospholipids in S49 Lymphoma cells.

Palmitoyl-lysophosphatidylcholine promotes a transient calcium influx in lymphoma cells. Previously, it was observed that this influx was accompanied by a temporary increase in propidium iodide permeability that appeared linked to calcium entry. Those studies demonstrated that cobalt or nickel could block the response to lysophosphatidylcholine and raised the question of whether the calcium conductance involved specific channels. This communication describes a series of experiments to address that issue. The time dependence and structural specificity of the responses to lysophosphatidylcholine reinforced the hypothesis of a specific channel or transporter. Nevertheless, observations using patch clamp or calcium channel blockers suggested that this "channel" does not involve proteins. Alternative protein-mediated mechanisms such as indirect involvement of the sodium-calcium exchanger and the sodium-potassium ATPase were also excluded. Experiments with extracellular and intracellular calcium chelators suggested a common route of entry for calcium and propidium iodide. More directly, the ability of lysophosphatidylcholine to produce cobalt-sensitive permeability to propidium iodide was reproduced in protein-free artificial membranes. Finally, the transient nature of the calcium time course was rationalized quantitatively by the kinetics of lysophosphatidylcholine metabolism. These results suggest that physiological concentrations of lysophosphatidylcholine can directly produce membrane pores that mimic some of the properties of specific protein channels.

Animals↗
PubMed1985

Organization of the striatum: collateralization of its efferent axons.

The anatomical structure of the basal ganglia indicates that the input from the cerebral cortex is funnelled through the striatum to the globus pallidus and substantia nigra. This structure implies integration of the information as it is transferred through the basal ganglia. In order to investigate this integration, we studied the collateralization of striatal efferents to the globus pallidus and the substantia nigra-ventral tegmental area. Retrogradely transported fluorescent tracers were injected into the target areas of striatal efferents. Nuclear yellow or propidium iodide was injected into the substantia nigra-ventral tegmental area (SN-VTA) and 4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) into the globus pallidus (GP) of adult albino rats. SITS was chosen for the pallidal injections because it is not taken up by fibers-of-passage. The pressure injections resulted in large injection sites which covered the majority of each efferent target area, and as a result retrogradely labeled cell bodies were found throughout the entire extent of the striatum. Cell bodies double-labeled with both dyes were found intermingled with single-labeled cell bodies. In rats injected with propidium iodide in the SN-VTA and SITS in the GP, 70% of all neurons (as revealed by Nissl staining) were labeled. Of these labeled cells, 40% were double labeled, 20% contained only SITS and 40% contained only propidium iodide. Thus a substantial number of the striatal neurons that project to the SN-VTA also possess collateral axons to the GP. Some striatal neurons appear to project to only the SN-VTA or only to the GP. The cells projecting to only one of these striatal target regions tend to cluster together in patches. The organizational pattern of these patches does not seem to coincide in any simple way with the mosaic pattern of striatal opiate receptors, nor with the previously described mosaic pattern of striatal afferents and various neurotransmitter substances.

Animals↗
PubMed1979

Simultaneous use of retrograde fluorescent tracers and fluorescence histochemistry for convenient and precise mapping of monoaminergic projections and collateral arrangements in the CNS.

A procedure is described for the use of fluorescent retrograde tracers in conjunction with monamine fluorescence histochemistry for single or double labeling studies of the projections of identified catecholamine or indoleamine neurons in the CNS. A number of fluorescent tracers, including Evans Blue, bisbenzimide, DAPI, 'True Blue', 'Granular Blue' and propidium iodide, have been tested and characterized microspectrofluorometrically. Of these, 'True Blue', propidium iodide and Evans Blue were found to have the most suitable properties to be studied concomitant with the monoamine fluorphores. A combination of two of these tracers--'True Blue' fluorescing blue and propidium iodide or Evans Blue fluorescing orange to red--and the catecholamines and indoleamines fluorescing yellow-green and yellow to brownish yellow, respectively, makes possible simultaneous use of four different fluorescent markers in one and the same section. The present procedure has great practical advantages over available HRP techniques and should in modified form also be applicable to other types of transmitter-specific neuronal tracing based on immunocytochemistry or enzyme histochemistry.

Animals↗
PubMed2004

Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis.

LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates. Previously, two membrane-embedded acidic residues, Asp142 and Glu327, have been reported to be important for multidrug transport activity of LmrP. Here we show that neither Glu327 nor Asp142 is essential for ethidium binding but that Glu327 is a critical residue for the high affinity binding of Hoechst 33342. Substitution of these two residues, however, negatively influences the transport activity. The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively. Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Deltapsi) and transmembrane proton gradient (DeltapH), while extrusion of propidium predominantly depends on the DeltapH only. The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate DeltapH-dependent extrusion of propidium. These data indicate that Asp142 and Glu327 are involved in proton translocation.

Bacterial Proteins↗
PubMed1998

Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity.

Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen. PicoGreen has been shown to detect as little as 0.5 ng pure DNA or 10(2) cells (interassay SD < 10%, intraassay SD < 5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen, cells were digested with papain for 20 h at 60 degrees C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.

Animals↗
PubMed2000

Alzheimer's disease presenilin-1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis.

Mutations in the presenilin-1 (PS1) and presenilin-2 (PS2) genes account for the majority of early-onset familial Alzheimer's disease cases. Recent studies suggest that presenilin gene mutations predispose cells to apoptosis by mechanisms involving altered calcium homeostasis and oxidative damage. In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed to an overnight (17 h) serum deprivation, followed by a 30 min treatment with either 20 mM glucose, 10 nM insulin-like growth factor-1 or 20 mM glucose + 10 nM insulin-like growth factor-1. Cells were then cultured for a further 3, 6 or 24 h and stained for apoptotic condensed nuclei using propidium iodide. Confirmation that cells were undergoing an active apoptotic process was achieved by labelling of DNA strand breaks using the terminal dUTP nick end labelling (TUNEL) technique. We also determined cell viability using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Propidium iodide staining revealed that all cell lines and controls showed an increased number of apoptotic cells appearing with condensed nuclei at 24 h compared with 6 h and 3 h. High glucose-induced hyperosmotic stress resulted in significantly more apoptotic cells in the PS1 deltaE9 and PS1 L250S mutation cell lines at 24 h, compared with the wild-type PS1 lines (P < 0.001, ANOVA for both comparisons). Mean values (+/-S.D.) for the percentage number of apoptotic cells at 24 h following high glucose treatment were 16.1 +/- 3.5%, 26.7 +/- 5.5% and 31.0 +/- 5.7% for the wild-type PS1, PS1 deltaE9 and PS1 L250S lines, respectively. The pro-apoptotic effects of high glucose treatment were reversed by 10 nM insulin-like growth factor-1, although to a lesser extent in the mutation cell lines (5.8 +/- 2.4%, 15.2 +/- 7.3% and 13.2 +/- 2.0% for the wild-type PS1, PS1 deltaE9 (P < 0.01 for comparison with wild-type PS1) and PS1 L250S (P < 0.01 for comparison with wild-type PS1) transfected lines, respectively. TUNEL labelling of cells at 24 h following treatment gave essentially the same results pattern as obtained using propidium iodide. The percentage number of apoptotic cells with DNA strand breaks (means +/- S.D.) following high glucose treatment was 15.4 +/- 2.6% for the wild-type PS1, 26.8 +/- 3.2% for the PS1 deltaE9 (P < 0.001 for comparison with wild-type PS1) and 29.7 +/- 6.1% for the PS1 L250S transfected lines (P < 0.001 for comparison with wild-type PS1). The PS1 deltaE9 and PS1 L250S transfected lines also showed a higher number of apoptotic cells with DNA strand breaks at 24 h following high glucose plus insulin-like growth factor-1 treatment (11.4 +/- 2.0% and 14.3 +/- 2.8%, respectively), compared with values for the wild-type PS1 lines (8.5 +/- 2.4%). These differences were significant (P < 0.01) for the comparison of wild-type PS1 and PS1 L250S, but not PS1 deltaE9 lines. The mutation-related increases in number of apoptotic cells at 24 h following high glucose treatment were not accompanied by significant differences in cell viability at this time-point. Our results indicate that PS1 mutations predispose to hyperosmotic stress-induced apoptosis and that the anti-apoptotic effects of insulin-like growth factor-1 are compromised by these mutations. Perturbations of insulin-like growth factor-1 signalling may be involved in PS1 mutation-related apoptotic neuronal cell death in Alzheimer's disease.

Alzheimer Disease↗
PubMed1998

Allosteric control of acetylcholinesterase activity by monoclonal antibodies.

Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.

Acetylcholinesterase↗
PubMed2001

Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia.

Using confocal microscopy, we investigated mechanisms underlying loss of plasma membrane integrity during necrotic death of cultured hepatic sinusoidal endothelial cells exposed to 2.5 mM potassium cyanide (chemical hypoxia). After 2-3 h, the anionic fluorophore calcein abruptly began to enter the cytosol, and nuclei labeled with cationic propidium after another 2-5 min. As calcein permeated, growth of blebs on the plasma membrane accelerated. Lucifer yellow, another anionic fluorophore, entered identically to calcein, whereas high molecular weight dextrans (40-2000 kDa) entered like propidium. Glycine slowed, but did not prevent calcein entry, whereas permeation of propidium and high molecular weight dextrans was blocked completely by glycine. These findings suggest that opening of a glycine-sensitive organic anion channel, or death channel, precipitates a metastable state characterized by rapid cell swelling and bleb growth. This metastable state culminates in non-specific breakdown of the plasma membrane permeability barrier and irreversible cell death.

Cell Death↗
PubMed2001

Comparison of several techniques for the detection of apoptotic astrocytes in vitro.

Implication of apoptosis in numerous physiological and pathological processes has resulted in the development of numerous methods to detect apoptosis, but none of them is adapted to all cell types. In this study, we induced apoptosis on murine immortalized astrocytes with urine from multiple sclerosis (MS) patients. Among techniques allowing the detection of apoptotic cells, only a few are adapted to adherent cells such as astrocytes. We compared several techniques (propidium iodide labelling and flow cytometry analysis, TUNEL and annexin V labelling in immunofluorescence, DNA ladder, ELISA tests to detect nucleosomes) in order to choose the method best adapted to our adherent cellular model and to discuss their practicability for the detection of apoptosis on adherent cells. For technical course, propidium iodide labelling followed by flow cytometry analysis as a quantitative technique, and TUNEL in IF (easier and quicker than propidium iodide) as a semiquantitative test were both retained as best adapted to our case. Moreover, in our model, we have observed that phosphatydilserine externalization and DNA fragmentation were concomittant after induction of apoptosis. Techniques studied in this article would allow an enlarged study of the apoptotic mechanism in several pathologies by culture of adherent cells sensitive to apoptosis in vitro.

Animals↗
PubMed2000

Assessment of donor T-cell function in cellular blood components by the CD69 induction assay: effects of storage, gamma radiation, and photochemical treatment.

BACKGROUND: Functional donor T-lymphocytes in blood components may cause a variety of transfusion complications. A flow cytometric assay based on the measurement of induced CD69 expression may be an alternative to cell proliferation methods in determining the functional status of these cells in blood components. STUDY DESIGN AND METHODS: Seven units of whole blood, RBCs, and platelet concentrates (PCs) were stored under blood bank conditions. Half of 3 PCs each were gamma-radiated or treated with UVA+psoralen; the other half served as controls. Samples were analyzed for phorbolester-induced expression of CD69 as an indicator of cell responsiveness and for exclusion of propidium iodide as a measure of cell membrane integrity and viability. RESULTS: CD69 inducibility and propidium iodide exclusion decreased exponentially (half-life, 3. 3 and 8.1 days, respectively) during cold blood storage. Irradiation and UVA+psoralen treatment of PCs immediately reduced CD69 inducibility to 21 percent (controls, 82%; p = 0.004) and 12 percent (controls, 95%; p = 0.0008), respectively. The proportion of cells capable of propidium iodide exclusion was similar in treated samples and controls, but it declined faster in the treated samples during subsequent storage. CONCLUSION: Flow cytometric measurement of CD69 induction can be adapted to provide quantitative assessment of T-cell function in blood components. Results obtained by the CD69 assay are in general agreement with those previously reported by use of proliferation methods; the assay may be useful for special applications in transfusion medicine.

Antigens, CD↗
PubMed1977

Sequence specificity of mutagen-nucleic acid complexes in solution: intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes.

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex.

Acridines↗
PubMed1994

Rapid (one-shot) staining method for two-color multiparametric DNA flow cytometric analysis of carcinomas using staining for cytokeratin and leukocyte common antigen.

The authors present an improved method for rapid two-color staining with direct conjugated antibodies to cytokeratin and CD45 antigen (leukocyte common antigen) for whole-cell, ethanol-fixed preparations of human carcinomas. This method was quality controlled with the T24 human bladder tumor cell line and compared in parallel analysis of 24 fresh human carcinomas with the original two-color method of multiparametric analysis that had been published in 1989. This rapid method was designed to achieve comparable staining intensities of both green (phenotype directed monoclonal antibody label) and red (propidium iodide labeled DNA) fluorescence, identical DNA indexes, comparable coefficients of variation, and subjective visual quality of DNA histograms. This is accomplished in a single (one-shot), abbreviated incubation with monoclonal antibody diluted in propidium iodide-RNase, thereby eliminating two incubations and three wash steps required with the original method. The single rinse is done in the propidium iodide-RNase staining solution with resuspension in fresh staining solution before analysis. With the rapid method, the preparation time is reduced by 130 minutes, resulting in a 60% time savings in batch staining mode compared with the original method. The time reduction and fewer wash steps, which should avoid excessive cell loss and cytoplasmic stripping, may advance the adoption of this two-color method in clinical practice.

Color↗
PubMedDate not supplied

Bile acid-induced depolarization of mitochondrial membrane potential preceding cell injury in cultured gastric mucosal cells.

Changes in energy metabolism elicited by sodium taurocholate and their relation to cell viability were determined in gastric mucosal cells. Cultured mucosal cells were labelled with rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, or by propidium iodide, a fluorochrome which labels the nuclei of non-viable cells. The cells were observed under a fluorescence microscope with a laser scanning confocal imaging system. After the addition of sodium taurocholate at concentrations > 5 mol/L, mucosal cells showed a rapid and significant decrease in rhodamine-123 fluorescence. A decrease to 40% of the pretreated values at 30 min was seen with a concentration of sodium taurocholate of 7.5mmol/L. A marked increase in the percentage of propidium iodide-positive cells was noted when the concentration of sodium taurocholate exceeded 5mmol/L. However, the extent of the decrease in rhodamine-123 fluorescence was always greater than the increase in the percentage of propidium iodide-positive cells, suggesting that most of these gastric mucosal cells remained viable. It is therefore suggested that the decrease in rhodamine-123 fluorescence is largely due to the disturbed oxidative phosphorylation of mitochondria. Pretreatment of gastric mucosal cells with low concentrations of ethanol resulted in a significant cytoprotective effect against sodium taurocholate injury with significant prevention of a decrease in rhodamine-123 fluorescence. It is concluded that sodium taurocholate induces a depolarization of the mitochondrial membrane potential preceding cell injury and that the cytoprotective effect of ethanol relates to its attenuation of the uncoupling effect.

Animals↗
PubMed2000

Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp.

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10(5) to 10(6)/ml) at 22 degrees C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.

Acanthamoeba↗
PubMed1994

Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation.

Peripheral-blood human T lymphocytes were treated with Staphylococcus aureus alpha-toxin. Membrane permeabilization was assessed by measuring efflux of K+ and Rb+ and influx of Na+, Ca2+, and propidium iodide. Cellular ATP and [3H]thymidine incorporation following lectin stimulation were measured as parameters for cell viability. Internucleosomal cleavage characteristic of programmed cell death was assessed by agarose gel electrophoresis and by quantifying low-molecular-weight, [3H]thymidine-labeled DNA fragments. Nanomolar concentrations of alpha-toxin evoked protracted, irreversible ATP depletion in both activated and resting T lymphocytes. Toxin-damaged cells also lost their ability to incorporate [3H]thymidine upon subsequent stimulation with phytohemagglutinin. These cells carried toxin hexamers, and their plasma membranes became permeable for monovalent ions but not for Ca2+ and propidium iodide. The permeabilization event was followed by internucleosomal DNA degradation characteristic of programmed cell death. Membranes of cells treated with high toxin doses (> 300 nM) became permeable to both Ca2+ and propidium iodide. In this case, ATP depletion occurred within minutes and no DNA degradation was observed. When cells were suspended in Na(+)-free buffer, alpha-toxin applied at low doses still bound and formed hexamers. However, these cells displayed neither DNA degradation nor loss of viability. The data indicate that formation of very small but not of large alpha-toxin pores may trigger programmed cell death in lymphocytes and that uncontrolled flux of Na+ ions may be an important event precipitating the suicide cascade.

Adenosine Triphosphate↗
PubMed2001

Annexin V staining during reperfusion detects cardiomyocytes with unique properties.

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and further elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in approximately 3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.

Animals↗