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PubMed1992

Cryptosporidium parvum sporozoite staining by propidium iodide.

Modified Ziehl-Neelsen (ZN) acid-fast stain is the usual method for detection of Cryptosporidium oocysts in feces. Propidium iodide permitted us to stain free or intra-oocyst sporozoites. With the ZN method only 3-5% of the oocysts purified from three human and one experimentally infected lamb dichromate-preserved feces were stained by carbol fuchsin. These fuchsin-stained oocysts were free of intact sporozoites as identified by propidium iodide staining. Treatment with 10% formalin or 0.5% sodium hypochlorite increased the percentage of acid-fast stained oocysts and thus the sensitivity of acid-fast staining. Treatment with sodium hypochlorite induced intra-oocyst sporozoite alterations as demonstrated by flow cytometric analysis of the oocysts' DNA content. Propidium iodide staining of fixed oocysts is a simple and rapid method to visualize sporozoites and to assess oocyst preservation after different treatments.

Animals↗
PubMed1990

Hydration of dA.dT polymers: role of water in the thermodynamics of ethidium and propidium intercalation.

We report differences in the interaction of two structurally similar phenanthroline intercalators, ethidium and propidium, with poly(dA).poly(dT) and poly[d(A-T)] as a function of ionic strength based on titration microcalorimetry, fluorescence titration, and hydrostatic pressure measurements. Both ethidium and propidium bind more strongly to poly[d(A-T)].poly[d(A-T)] than to poly(dA).poly(dT). Ethidium intercalation into the latter polymer displays titrations with positive cooperativity; this is not found with propidium. The enthalpy of intercalation (delta H degrees) is exothermic for both dyes with poly[d(A-T)].poly[d(A-T)]; however, the value of this parameter is nearly zero in the case of poly(dA).poly(dT). The molar volume change (delta V degrees) accompanying dye intercalation is negative under all conditions for poly[d(A-T)].poly[d(A-T)] whereas it is positive for poly(dA).poly(dT). The changes observed in delta V degrees correlate well with the entropy changes derived from the titration and calorimetric data for this reaction. The results, interpreted in terms of the relative hydration of these two polymers, are consistent with a higher extent of hydration of poly(dA).poly(dT) relative to poly[d(A-T)].poly[d(A-T)].

Binding Sites↗
PubMed1999

Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands. Distinct effects of propidium and fasciculin.

Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.

Acetylcholinesterase↗
PubMed2002

Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide.

The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4',6-diamino-2-phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C-PI or C-DAPI) was compared with that of the standard, petunia (P-PI or P-DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R-PI or R-DAPI) is expected to be proportional to the genome size. Between-tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between-tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between-tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed.

Cell Nucleus↗
PubMed1980

Flow microfluorometric analysis of cellular DNA: Critical comparison of mithramycin and propidium iodide.

Mithramycin and propidium iodide were used to stain HeLa cells, human lymphoma cells, and phytohemagglutinin-stimulated lymphocytes for flow microfluorometric analysis of cellular DNA. The stains provided similar estimates for the proliferative fraction of the populations. However, significant differences in the relative fluorescent intensity were demonstrated in the three cell populations. Fluorescent intensity of HeLa and lymphoma cells stained with mithramycin was higher than matched propidium iodide-stained cells. Normal lymphocytes showed greater fluorescent intensity when stained with propidium iodide. Differences in the staining behavior of these two dyes may prove to be highly informative probes of chromatin structural differences.

Animals↗
PubMed1985

Esterase activity, exclusion of propidium iodide, and proliferation in tumor cells exposed to anticancer agents: phenomena relevant to chemosensitivity determinations.

Cellular esterase activity and the ability to exclude propidium iodide were examined after exposing tumor cells to anticancer agents. In general, esterase activity and the ability to exclude propidium iodide continued when cells proliferated and disappeared when proliferation was inhibited. However, with a number of preparations, drug exposure inhibited proliferation while esterase activity and propidium iodide exclusion persisted. These indications of persisting cell function or viability after drug exposure may be relevant to a potential for tumor cell recovery. When the viability of established cell lines progressively declined on days 4 and 7 following drug exposure, recovery did not occur. When proliferative recoveries occurred, viabilities remained elevated. Estimates of in vitro sensitivity by proliferation-related criteria were contrasted by persistent high viability estimates in 22% of the determinations performed with primary tumor cell preparations. The potential for recovery may explain the disappointing ability of proliferative chemosensitivity assays to predict clinical sensitivity.

Antineoplastic Agents↗
PubMed1987

The application of propidium iodide staining to the study of the macronucleus and micronuclei in the suctorian, Heliophrya sp.

Morphological changes in the macronucleus and micronuclei of the ciliated protozoon Heliophrya chapmani were investigated using the nucleic acid-specific stain propidium iodide. The fluorescence patterns of nuclei observed in propidium iodide preparations correspond well with those observed using more conventional DNA-specific methods, such as the Feulgen stain. The advantages of propidium iodide staining (minimal cell loss during staining, rapidity of the staining process, and the avoidance of cell damage during hydrolysis) make this method a quick and efficient alternative in the cytochemical study of the protozoan nucleus.

Animals↗
PubMed2005

Evaluation of plasma cell propidium-iodide and annexin-V indices: their relation to prognosis in multiple myeloma.

In a group of 117 patients with multiple myeloma (MM) examined at the time of diagnosis, i.e. excluding previous chemotherapy, we analysed the levels of propidium-iodide (proliferative) - PC-PI/CD(138) and annexin-V (apoptotic) - PC-AI/CD(138) indices of myeloma plasmocytes using the method of flow-cytometry to determine their relationship to prognosis. It was revealed that patients with high values of PC-PI/CD(138) had substantially worse overall survival than those with low values. Patients with a level of propidium-iodide index > or = 2,8 % exprimed a median survival of 13 months only in comparison with 42 months in patients with levels < 2,8 % (p = 0,0005). In the PC-AI/CD(138) index a reverse trend was registered. Patients with PC-AI/CD(138) > or = 4,0 % had long overall survival (median was not assessable at the time of evaluation), whereas patients with low apoptosis values < 4,0 % had median overall survival 16 months only (p = 0,01). Based on the sequentional graphic analysis of the curves of overall survival was found that the optimal discrimination level sequestering patients with good and poor prognosis was, in the case of PC-PI/CD(138) value 2,8 %, whereas in the case of PC-AI/CD(138) value 4,0 %. Among patients with good prognosis, there were no statistically significant differences in overall survival according to different levels of proliferative and apoptotic index. We conclude that evaluation of the propidium-iodide and annexin-V index using flow-cytometry is a quick, useful, and easily accessible method for the evaluation of plasma cell kinetics and thus prognosis of the disease, multiple myeloma.

Adult↗
PubMed1978

Effect of adriamycin and analogs on the nuclear fluorescence of propidium iodide-stained cells.

Adriamycin (ADR) and N-trifluoroacetyladriamycin-14-valerate, respectively, inhibit and enhance the nuclear fluorescence of cells stained with propidium iodide for DNA per cell estimation by flow cytometry. In cells incubated with ADR, the reduction in fluorescence is gradually manifested due to the slow intracellular drug transport. In contrast the effect of N-trifluoroacetyladriamycin-14-valerate on propidium iodide nuclear fluorescence is seen within 5 min of incubation. The effect of ADR on propidium iodide nuclear fluorescence could be detected in vivo even after 24 hr of ADR administration.

Animals↗
PubMed1996

Allosteric effects of phenyltrimethylammonium and propidium on acetylcholinesterase active site.

Different spin labelled fluorophosphates and fluorophosphonates with different chain length were investigated with respect to their sensitivity to the allosteric changes of acetylcholinesterase active site produced by phenyltrimethylammonium (Pta) or d-tubocurarine (TC); only fluorophosphates were found to be sensitive to these changes. Therefore fluorophosphates were chosen also for the study of allosteric effects of propidium. The addition of Pta and propidium to spin labelled membrane acetylcholinesterase of the Torpedo marmorata electric organ decreased maximal hyperfine splitting of the EPR spectrum, indicating that the microgeography of the acetylcholinesterase active site is usually changed in a way which increases the freedom of motion of the spin label's piperidine ring. TC alone did not change the EPR spectrum, but it prevented the influence of Pta and not that of propidium.

Acetylcholinesterase↗
PubMed1976

Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.

In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure. Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intentsity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 muM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.

Acriflavine↗
PubMed1976

Propidium diiodide-cesium chloride density gradient centrifugation for buoyant separations of duplex DNA molecules containing single-stranded regions.

At increasing dye concentrations in propidium diiodide-CsCl density gradients, the relative buoyant density shift was largest for open-circular duplex phiX174 DNA (RFII), next largest for single-stranded viral DNA, and least for closed-circular duplex DNA (RFI). These differential relative buoyant density shifts permitted discrete separations of these phiX DNA forms. Further, in propidium diiodide-CsCl density gradients, the distinctive density of single-stranded DNA permitted separations of rolling-circle intermediates with a single-stranded tail that occur during single-stranded phiX DNA synthesis. It is suggested that single-strandedness in a duplex DNA structure influences its buoyand density shift due to differential dye binding to the single-stranded region and is an additional physical basis for relative buoyant separations of DNA molecules in propidium diiodide-CsCl density gradients.

Centrifugation, Density Gradient↗
PubMed1996

Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation.

By flow cytometry, we have quantitatively evaluated HL-60, MOLT-4, and P815 apoptotic cells induced with camptothecin, staurosporine, and hyperthermia, respectively. Apoptosis was measured by two different flow cytometry techniques, using propidium iodide (PI) uptake in permeabilized and nonpermeabilized cells. Apoptosis also has been analyzed by electron microscopy and DNA cleavage by in situ nick translation and DNA gel electrophoresis. We demonstrate that supravital exposure to propidium iodide without prior permeabilization identifies apoptotic cells, and clearly distinguishes them from necrotic cells in all the cases examined. This capability is independent of the nucleosomal (180-200 bp) fragmentation of DNA (which does not take place in MOLT-4 cells), which is the basis for detection of apoptosis both as a "ladder" by DNA gel electrophoresis and as a hypodiploid peak by flow cytometry. Therefore, alterations in membrane permeability, on which PI uptake in living cells is based, allow distinction of apoptotic cells from necrotic and living cells independently of the heterogeneous biochemical patterns involved in programmed cell death, which may or may not lead to DNA oligonucleosomal fragmentation.

Animals↗
PubMed1985

Use of the fluorochrome propidium iodide for the identification of Xenopus germ plasm during immunofluorescence studies.

Addition of 0.5 microgram/ml of the red fluorescent dye propidium iodide to the penultimate wash of Xenopus early embryo serial sections during immunofluorescence studies causes germ plasm to fluoresce brightly. Germ plasm can be accurately and speedily identified in the serial sections. The use of this dye is a marked improvement over previous methods of germ plasm identification in unstained sections. Studies involving screening for germ-plasm-specific antibodies are greatly facilitated by staining germ plasm red with propidium iodide and searching for green colabelling by fluoresceinated antibodies.

Animals↗
PubMed1986

Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde.

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.

Animals↗
PubMed1994

DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay.

The alkaline comet assay is used to detect DNA single-strand breaks in individual cells embedded in agarose, lysed to denature DNA and remove proteins, and briefly exposed to an electric field to allow broken DNA to migrate. Total DNA fluorescence, measured by staining individual comets with propidium iodide, is reduced 30-40% by low doses of ionizing radiation, N-methyl-N-nitrosoguanidine (MNNG), etoposide, or hydrogen peroxide. Three possible explanations were examined: 1) these agents produce small fragments of DNA that are lost during lysis and electrophoresis, 2) the analysis of comet images is less efficient once the DNA is small enough to migrate in the electric field, and 3) DNA staining by propidium iodide is affected by changes in DNA structure caused by strand breaks and exposure to alkali. Our results indicate that the decrease in fluorescence after low doses is best explained by a change in ability of DNA, which has been denatured by alkali and subsequently renatured, to interact with fluorescent dyes. This change in fluorescence has the practical consequence of improving the ability of the alkaline comet assay to distinguish damaged from undamaged cells.

Alkalies↗
PubMed2001

Light scatter and DNA accessibility to propidium iodide of ataxia telangiectasia and fanconi anemia cells.

Cells from individuals with genetic diseases ataxia telangiectasia (AT) and Fanconi anemia (FA) exhibit hypersensitivity to ionizing radiation (AT) or DNA cross-linking agents (FA) which may be caused by multiple factors including defects in chromatin structure and DNA repair. In this study, a combination of cytometric techniques was employed to study the chromatin conformation of AT and FA cells. Nuclei of peripheral blood mononuclear cells (PBMCs) and of skin fibroblasts established from AT and FA patients were analyzed by light scattering and fluorimetric titration with the DNA-intercalating dye propidium iodide. The light scatter measurements revealed the presence of small-sized nuclei with reduced granularity in PBMCs and fibroblasts from both AT and FA patients. The fluorometric titration data could be interpreted by assuming two classes of propidium iodide binding sites with different affinities. The number of high-affinity sites in AT and FA fibroblasts was significantly larger (by 20%) than in control cells. Our findings show the applicability of cytometric techniques for the rapid assessment of chromatin conformation and also suggest the possibility to identify AT and FA carriers.

Ataxia Telangiectasia↗
PubMed1981

Propidium iodide as a probe for the study of chromatin thermal denaturation in situ.

The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturation in situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid--ethanol (1:3 v/v), treated with RNAse and submitted to different protein extraction procedures, namely, incubation with pepsin, trypsin and sodium chloride. Denaturation experiments were performed in Sörensen buffer at pH 7.4 containing 10% formamide at temperatures between 27 and 95 degrees C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 446 nm. The results indicate a higher thermostability of lymphocytes as compared to hepatocytes. The denaturation pattern suggests a certain organization complexity of chromatin, better emphasized by the derivative curves which show the presence of at least three fractions with different melting points. After protein extraction, the denaturation curves exhibit a somewhat simplified pattern, with the disappearance of the most stable peak in the derivative curves. The samples mounted in glycerine exhibit a better stability of staining with time, and an increased quantum efficiency of the fluorochrome with regard to those mounted in buffer. These data confirm the importance of protein--DNA interactions in the organization of chromatin and point to some differences, depending on the cell type and on functional activity.

Animals↗