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At least 19 recordsLinked to original sources
PubMed1992

Comparison of Hoechst 33342 and propidium iodide as fluorescent markers for sperm fusion with hamster oocytes.

Hamster oocytes were loaded with the DNA dyes Hoechst 33342 or propidium iodide. Oocytes incubated in 10 mumol Hoechst 333421(-1) showed intracellular fluorescence within 10-20 s of exposure, as did hamster and guinea-pig spermatozoa. Impaled oocytes to which acrosome-intact hamster spermatozoa were bound before injection of Hoechst 33342 showed dye transfer to adhering spermatozoa within 2 min of injection. Oocytes loaded passively with Hoechst 33342 showed dye transfer to bound, acrosome-intact hamster spermatozoa within 10 min. On ultra-structural examination, no bound, acrosome-intact hamster spermatozoa (n = 311) were found to be fused. By contrast, oocytes incubated with 10 mumol propidium iodide l-1 showed no intracellular fluorescence after 2 h, although in approximately 50% of oocytes, fluorescence developed rapidly in the first polar body. Oocytes injected with propidium iodide showed intracellular fluorescence but no dye transfer to bound, acrosome-intact hamster spermatozoa. Oocytes impaled on pipettes containing propidium iodide showed no dye transfer to unlabelled oocytes with which they were brought into contact, whereas in similar experiments using Hoechst 33342 detectable dye transfer to an adjacent oocyte occurred within 10 min. Oocytes loaded with propidium iodide transferred propidium iodide to fusion-competent guinea-pig spermatozoa during in vitro fertilization. Normally, between 20 and 40 spermatozoa bound per oocyte, and the percentage of spermatozoa showing dye transfer varied between 0 and 41%. Dye transfer occurred within 5-45 min. Only those nuclei that showed propidium iodide transfer subsequently decondensed, suggesting that dye transfer is correlated with fusion. The presence of fused spermatozoa was confirmed by ultrastructural examination of oocytes. In separate experiments, hamster and guinea-pig spermatozoa showed detectable fluorescence from propidium iodide within 20 s of osmotic rupture or membrane stripping by detergent, suggesting the lag in dye transfer to sperm nuclei during fertilization reflects a delay in sperm-oocyte fusion following adhesion. This evidence suggests that Hoechst 33342 could be an unreliable marker for sperm-oocyte fusion in fertilization because of its capacity for passive movement from oocyte to spermatozoon. This problem can be overcome using oocytes injected with propidium iodide. With this technique, it was possible to show that fusion-competent guinea-pig spermatozoa that are held in pipettes will fuse with hamster oocytes when placed mechanically against the oocyte surface.

Animals↗
PubMed1985

Poly(dA).poly(dT) exists in an unusual conformation under physiological conditions: propidium binding to poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)].

The binding of propidium to poly(dA).poly(dT) [poly(dA.dT)] and to poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]] has been compared under a variety of solution conditions by viscometric titrations, binding studies, and kinetic experiments. The binding of propidium to poly[d(A-T)2] is quite similar to its binding to calf thymus deoxyribonucleic acid (DNA). The interaction with poly(dA.dT), however, is quite unusual. The viscosity of a poly(dA.dT) solution first decreases and then increases in a titration with propidium at 18 degrees C. The viscosity of poly[d(A-T)2] shows no decrease in a similar titration. Scatchard plots for the interaction of propidium with poly(dA.dT) show the classical upward curvature for positive cooperativity. The curvature decreases as the temperature is increased in binding experiments. A van't Hoff plot of the observed binding constants yields an apparent positive enthalpy of approximately +6 kcal/mol for the propidium-poly(dA.dT) interaction. Propidium binding to poly[d(A-T)2] shows no evidence for positive cooperativity, and the enthalpy change for the reaction is approximately -9 kcal/mol. Both the magnitude of the dissociation constants and the effects of ionic strength are quite similar for the dissociation of propidium from poly(dA-T)2] and from poly[d(A-T)2], suggesting that the intercalated states are similar for the two complexes. The observed association reactions, under pseudo-first-order conditions, are quite different. Plots of the observed pseudo-first-order association rate constant vs. polymer concentration have much larger slopes for propidium binding to poly[d(A-T)2] than to poly(dA.dT).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics↗
PubMed1975

Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding.

A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.

Acetylcholinesterase↗
PubMed1985

Cytofluorescence localization of propidium iodide injected intravenously into the nervous system of the mouse.

Propidium iodide, like its analogue ethidium bromide, is a compound which can be used as a marker of nucleic acids. This substance emits a red fluorescent light after exposure to UV light and has therefore been used previously as a nuclear stain in immunofluorescence studies and in flow cytometry. The present experiments were carried out to find out if propidium iodide could be traced in sections of the nervous system after i.v. injections. Due to the general toxicity of the compound detectable amounts of propidium iodide could not be obtained by a single i.v. injection. However, multiple injections of small amounts (0.1 mg) over a period from 15 min to 8 h (total dose 0.7-1.0 mg) were tolerated without any signs of adverse effects. In such experiments propidium iodide did not extravasate into the cerebral gray or white matter, i.e., areas of the brain located within the blood-brain barrier (BBB). On the other hand, the compound spread into the choroid plexus, the circumventricular organs, the Gasserian ganglion, and sciatic nerve, i.e., regions located outside the BBB. It had a strong tendency to label the nucleus and the perikaryon of the cells in each of these territories. Perifascicular injection of propidium iodide around the sciatic nerve was followed by a marked cellular uptake not only in the epineurium but also in the endoneurium. The shape and position of the labeled nuclei strongly indicated that they were the nuclei of Schwann cells. Previous studies have shown that propidium iodide can be used as a retrograde tracer in neuroanatomic research.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.

Animals↗
PubMed1986

Intercalators as probes of DNA conformation: propidium binding to alternating and non-alternating polymers containing guanine.

Propidium iodide is used as a structural probe for alternating and non-alternating DNA polymers containing guanine and the results are compared to experiments with poly[d(A-T)2], poly(dA . dT) and random DNA sequences. Viscometric titrations indicate that propidium binds to all polymers and to DNA by intercalation. The binding constant and binding site size are quite similar for all alternating polymers, non-alternating polymers containing guanine and natural DNA. Poly(dA . dT) is unusual with a lower binding constant and positive cooperativity in its propidium binding isotherms. Poly(dA . dT) and poly(dG . dC) have similar salt effects but quite different temperature effects in propidium binding equilibria. Polymers and natural DNA have similar rate constants in their SDS driven dissociation reactions. The association rate constants are similar for the alternating polymers and poly(dG . dC) but are significantly reduced for poly(dA . dT). These results suggest that natural DNA, the alternating polymers, and non-alternating polymers containing guanine convert to an intercalated conformation with bound propidium in a very similar manner.

Chemical Phenomena↗
PubMed1987

Propidium binding to a ribonuclease-DNA complex: X-ray and fluorescence studies.

Propidium iodide, an antitumor compound, was diffused into crystals of a complex between RNase A and deoxytetraadenylate (dpA)4). This complex has four deoxyoligomers bound per protein molecule. A difference Fourier analysis at 2.9 A showed that the principal binding site for the propidium in the crystals was a hydrophobic depression on the side of RNase away from the active site and apparently involves methionine 13 and phenylalanine 8. Binding of propidium at this site produces small conformational changes that effect binding of nucleotides at the active site of the enzyme. Fluorescence titrations in the presence and absence of nucleotide inhibitors suggested that propidium iodide is a competitive inhibitor of the enzyme with a Kl of approximately 1 mM. No significant binding of propidium to the 16 nucleotides of single-stranded DNA associated with each protein molecule was observed.

Fourier Analysis↗
PubMed1977

The interaction of propidium diiodide with self-complementary dinucleoside monophosphates.

The interactions of a quinacrine derivative, methylated at both the aromatic and aliphatic nitrogens, and propidium diiodide with the dinucleoside monophosphates CpG, GpC, UpA and ApU have been investigated using 13C-NMR (for the quinacrine derivative prepared with [13C]methyl substituents and 1H-NMR and ultraviolet-visible spectroscopy. The quinacrine derivative displayed negligible interaction with the dinucleosides at concentrations up to 5 - 10(-4) M. Propidium did form complexes with dinucleosides even at concentrations as low as 10(-4) M. Propidium displayed a pyrimidine-purine binding preference and gave especially large changes in ultraviolet-visible and 1H-NMR spectra in the presence of CpG. This suggests that propidium forms an intercalated complex with a Watson-Crick hydrogen-bonded CpG dimer. At higher concentrations UpA and GpC gave similar spectral changes indicating that they could also form significant amounts of an intercalated complex with propidium under appropriate conditions. The changes caused by ApU were small under all conditions and were more similar to the effects caused by mononucleotides. These results indicate that, at least for phenanthridines, cationic side chains do not greatly inhibit complex formation with dinucleoside monophosphates, and suggest that the weak interaction of the quinacrine derivative with dinucleosides is due to weaker interactions of the acridine ring system with nucleoside bases relative to the phenanthridine ring system.

Chemical Phenomena↗
PubMed1979

Antagonism by propidium of petite induction by ethidium and ethidium azide in Saccharomyces cerevisiae.

Propidium, a phenanthridinium dye similar to ethidium, did not induce petite mutations in non-growing yeast cells in contrast to ethidium. Combined exposure to ethidium and an excess of propidium for periods up to 2 h resulted in the expected petite induction expressed after subsequent plating on growth medium. As incubation was continued with propidium, the numbers of petites declined on subsequent plating whether the drug had been added before, during, or after the mutagenic treatment by ethidium. Propidium decreased petite induction by the monoazide analog of ethidium when applied before but not after photolytic attachment of the drug.

Azides↗
PubMed1990

Temperature dependence of enthalpy changes for ethidium and propidium binding to DNA: effect of alkylamine chains.

Calorimetric titrations have been performed on the binding of ethidium and propidium to calf thymus DNA at temperatures in the 15-60 degrees C range. Enthalpy changes (delta HB) derived from these experiments performed with the new Omega reaction calorimeter have a precision of +/- 0.10 kcal/mol or less at all temperatures. For ethidium (a monocation), delta HB varies little with temperature, and the heat capacity change (delta CP) for the binding reaction derived from these parameters is 10 cal/deg/mol. In contrast, delta HB changes from -6.5 to -8.1 kcal/mol for DNA binding of propidium (a dication due to a charged amine group at the end of an alkyl chain attached to the phenanthridine ring nitrogen), and delta CP is -57 cal/deg/mol. At 21 degrees C a plot of delta HB vs mole ratio is curved downward for propidium in the 0.08-0.25 range, whereas the same plot at 45 degrees C is a straight line from 0.05 to 0.15 and sharply downward thereafter. Similar plots for ethidium follow the latter pattern between 25 and 50 degrees C. These observations and our analyses of delta HB and delta SB are consistent with the hypothesis that the location in the DNA complex and the rotational motion of the alkylamine chain change substantially over the temperature range in this study. Only near 50 degrees C is delta HB equal for the binding of these two cations to DNA, and caution must be used in analyses of enthalpic effects when the temperature dependence for delta HB is not available.

Animals↗
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↗
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↗
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↗
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↗
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↗