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PubMedDate not supplied

Quantitation of dye binding by cell monolayers in a microtiter system.

A semi-automated system has been developed for the quantitation of dye binding to cultured eukaryotic cells. It is based on staining precisely controlled numbers of cells seeded into microtiter trays. Cell-bound stain is then released using an appropriate solvent and quantitated in situ by measuring absorbance in a single beam ELISA reader with an interactive microcomputer link. In order to illustrate potential applications of this approach, the time course of dye-monolayer association and influence of cell number and stain concentration on staining has been examined for four dyes, Crystal Violet, Naphthol Yellow S, Ethyl Green and Pyronin Y. In addition, the effect of sequential and simultaneous staining was examined for Ethyl Green and Pyronin Y. The results provide evidence for the overall reliability of this approach as well as revealing several interesting features in the individual procedures examined. The combination of microtiter technology and computer link make the system particularly well suited to the efficient investigation of the permutations involved in optimizing conditions for a given staining procedure, as well as analysis of the thermodynamics of dye substrate interaction. Overall, the approach is viewed as an intermediate between artificial gel systems and microdensitometry.

Animals↗

PubMed2006

[Fluorescence investigation on interaction between artemisinin (qinghaosu) and hemin and its analytical application].

Hemin-catalytic decomposition of artemisinin (qinghaosu, QHS) was studied using pyronine B (PB) as an indicator. The interaction between hemin and QHS was an enzyme-substrate model, and the action sites were the endoperoxide moiety of QHS and the central metal ion of enzyme respectively. The kinetic catalytic constant depends upon enzyme and substrate concentrations, and the Michaelis-Menten parameters Km, Vmax and Kcat was 8.4 x 10(-5) mol x L(-1), 7.4 x 10(-6) mol x L(-1) s(-1) and 50.23 s(-1) respectively. The catalytic activity of hemin was inhibited in the presence of deactivated agents and at high temperature. Under optimal conditions, the change in fluorescence intensity (Fo-F) of pyronine B was proportional to the QHS concentration from 0.0 to 1.27 x 10(-6) mol x L(-1), and the detection limit (3sigma) was as low as 2.3 x 10(-8) mol x L(-1). The proposed method was applied to detect the concentration of QHS in the media of plasma and urine.

Artemisinins↗

PubMed1976

[Effect of physical and chemical factors on Pasteurella multocida bacteriophages].

Studied was the effect of some physical factors (temperature, pH of various values) and some chemical ones (urea, sodium citrate, sarcolysin, methylene blue, acridine orange, pyronin, crystal violet, potassium permanganate, fuchsin, tetracycline and tetraolean) on phages specifically lysing Pasteurella multocida strains, having the following signature: 3, 4, 6, 22, 32, 115, 967, 1075, and C-2. All investigated phages showed signs of inactivation at 48 degrees C with a further linear dependence, and at 65 degrees C up to 99.99 per cent of them were inactivated. With phages of group III inactivation discontinued between 50 and 60 degrees C, at 65 degrees C it being equal to that observed with I and II group phages. It was found that a broth medium of pH 7.5 was the optimum one for P. multocida phages. With altered pH values lowest percentage of survived phage particles showed I group phages, while those of II and III group proved more stable. So far as the chemical factors were concerned it was established that with the exception of pyronin and potassium permanganate which inactivated the P. multocida phages from 90 to 100 per cent, all other agents produced full inactivation (100 per cent). However urea did not inactivate I group phages, both it and sodium citrate inactivated partially II and III group phages. The variations established with the phages in terms of their physical and chemical behaviour may serve as subsidiary signs bearing upon their classification.

Acridines↗

PubMed2000

Dye-loaded zeolite L sandwiches as artificial antenna systems for light transport

The synthesis and characterization of dye loaded zeolite L sandwiches acting as artificial antenna systems for light harvesting and transport is reported. A set of experimental tools for the preparation of neutral dye-zeolite L materials ranging from low to maximum packing densities has been developed. The role of co-adsorbed water and the distribution of molecules between the inner and the outer surface were found to be the determining parameters. p-Terphenyl (pTP) turned out to be very suitable for studying these and other relevant parameters of neutral dye-zeolite L materials. We observed that pTP located in the channels of zeolite L can reversibly be displaced by water. This can be used when synthesizing such materials. We also observed that all-trans-1,6-diphenyl-1,3,5-hexatriene (DPH) which is very photolabile in solution is stable after insertion into zeolite L. By combining our extensive knowledge of these systems with ion-exchange procedures developed in an earlier study, we have realized the first bi-directional three-dye antenna. In this material the near UV absorbing compounds DPH or 1,2-bis-(5-methyl-benzoxazol-2-yl)-ethene (MBOXE) are located in the middle part of zeolite L nanocrystals followed on both sides by pyronine (Py) and then by oxonine (Ox) as acceptors. Fluorescence of the oxonine located at both ends of the cylindrical zeolite L crystals was observed upon excitation of the near UV absorber in the middle section at 353 nm, where neither oxonine nor pyronine absorb a significant amount of the excitation light.

Journal Article↗

PubMed1989

Suppression of erythroid cell differentiation in mouse embryos exposed to retinoic acid in utero.

Differentiation of circulating erythrocytes was inhibited by all-trans-retinoic acid in 8- and 9-day-old mouse embryos exposed in utero on day 8 of gestation. Histochemical, light, and electron microscopic examinations revealed that all-trans-retinoic acid first decreased the numbers of polychromatic erythroblasts and then increased them. Simultaneously, immature erythroblasts proliferated, and differentiation of mature primitive erythrocytes was suppressed. Electron microscopy of these immature erythroblasts revealed monosomes rather than the usual polyribosomes in the cytoplasm. RNA histochemistry revealed a greater number of intermediate differentiating erythroblasts with pyronin-positive cytoplasm than those in the controls and revealed also a dose-dependent relationship between the number of polychromatic erythroblasts with positive pyronin staining and the controls. These findings suggest that all-trans-retinoic acid destroys the messenger RNA system, resulting in an inability to produce hemoglobin.

Animals↗

PubMed1981

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization.

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.

Animals↗

PubMed1983

Inhibition of an oligomycin-sensitive ATPase by cationic dyes, some of which are atypical uncouplers of intact mitochondria.

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.

Adenosine Triphosphatases↗

PubMed2006

Diagnosis of chronic endometritis in biopsies with stromal breakdown.

Plasma cells are the hallmark of chronic endometritis but are not specific for upper tract infection. Plasma cells have also been noted in hormonally mediated endometrial disorders in association with gland architectural changes ("disordered proliferative" and "anovulatory" patterns), and stromal breakdown. We reviewed benign endometrial biopsies diagnosed at Beth Israel Deaconess Medical Center over a 2-year period described as disordered/anovulatory patterns +/- stromal breakdown. Cases were excluded if tissue was not available; women were younger than 50 years where most diagnoses were atrophic or cancer; or diagnoses were secretory, menstrual endometrium, or polyps. The remaining 61 cases were compared to 33 samples of unremarkable proliferative endometrium. Plasma cells were quantified on hematoxylin and eosin-stained sections and using a histochemical stain methyl green pyronin. The indication for biopsy was an abnormal pattern of bleeding in 34 cases, infertility workup in 7, incidental part of workup for pain, or other findings in 5. The majority of disordered proliferative endometrium had plasma cells (61% grade 1, 17% grade 2) all seen on methyl green pyronin staining only. Two thirds of proliferative endometrium with breakdown showed plasma cells (19% grade 1, 39% grade 2, 10 % grade 3). Plasma cells were rare in inactive endometrium and noted in only 18% of unremarkable proliferative endometrium, all grade 1. Plasma cells are commonly present in the endometrium of women with dysfunctional uterine bleeding and focal stromal breakdown. Given the lack of clinical evidence for infection, the inflammation likely represents a physiologic process.

Adult↗

PubMed1976

Thin-layer chromatography of some cationic dyes commonly used in histology.

A number of cationic dyes commonly used in histology may be conveniently and effectively analysed using the thin-layer chromatographic system of Marshall and Lewis. Commercial samples of Acridine Orange, Crystal Violet, Janus Green B,Methyl Violet, Neutral Red, Pyronin B, Pyronin Y(G), Safranin, Victoria Blue B and Victoria Blue 4R have been analysed with this system. All have been found to be complex mixtures of coloured components. Chromatographic data on these components are presented.

Chromatography, Thin Layer↗

PubMed1989

Platinum-dye complexes inhibit repair of potentially lethal damage following bleomycin treatment.

Several new complexes of platinum with positively charged cellular dyes have been synthesised in an effort to find chemotherapeutic drugs with increased antitumour cytotoxicity. As part of this effort, the direct cytotoxicities of some of these complexes as well as their ability to inhibit bleomycin potentially lethal damage repair (PLDR) was studied in vitro in a squamous cancer cell line of human origin (SCC-25). All of the new agents were more cytotoxic against exponentially growing than against plateau phase cell cultures. Exposure of cells to non-lethal drug concentrations for between 1 and 6 h led to measurable inhibition of bleomycin PLDR in the case of each drug tested. In order of decreasing ability to inhibit bleomycin PLDR, Pt(fast black)2, Pt(thioflavin)2 and Pt(thionin)2 were more effective than CDDP, while Pt(methylene blue)2, Pt(Rh-123)2 and Pt(pyronin Y)2 were less effective. The most directly cytotoxic agents were Pt(thioflavin)2, Pt(pyronin Y)2 and Pt(Rh-123)2 which also proved to be the least selectively toxic drugs towards exponential versus plateau phase cells. These results indicate that several of the new platinum complexes may be effective cytotoxic agents as well as effective inhibitors of DNA repair process following exposure of cells to other DNA interactive modalities.

Bleomycin↗

PubMed2000

Cerebellar basket cells of Creutzfeldt-Jakob disease: immunohistochemical and ultrastructural study.

To elucidate possible abnormalities of cerebellar basket cells of Creutzfeldt-Jakob disease (CJD), seven sporadic cases were examined neuropathologically. Recently, parvalbumin-positive, GABAergic cerebral interneurons have been demonstrated to show early, selective loss in CJD, and the phenomenon is postulated as a cause of characteristic neurological symptoms of CJD. In this study, however, we demonstrated that the basket cells, cerebellar counterparts, were resistant even in patients with severe brain atrophy, and their processes showed intense argyrophilia and immunopositivity to phosphorylated neurofilament. They can newly be listed as CJD-resistant neurons similar to those of the hippocampus and brainstem nuclei. The mechanism to escape cell loss is of great interest, and there might be unknown factors modulating susceptibility within parvalbumin-positive neuronal subgroups. Furthermore, one case showed abnormal positivity with hematoxylin, crystal violet and pyronin in the basket cells. The pyronin positivity was reduced after ribonuclease digestion, suggesting that the causative substance was composed of RNA. Ultrastructurally, the fibers contained free ribosomes and amorphous electron-dense deposits. To our knowledge, such a finding has also not been previously reported.

Aged↗

PubMed1969

Transient changes in extrinsic fluorescence of nerve produced by electric stimulation.

NERVES STAINED WITH ONE OF THE FOLLOWING FLUOROCHROMES WERE FOUND TO RESPOND TO ELECTRIC STIMULATION WITH TRANSIENT CHANGES IN THE FLUORESCENT LIGHT: acridine orange, acridine yellow, 8-anilinonaphthalene-1-sulfonate, auramine O, 1-dimethylaminonaphthalene-5-sulfonyl chloride, fluorescein isothiocyanate, LSD, pyronin B, pyronin Y, rhodamine B, rhodamine G, rose bengal, and 2-toluidinylnaphthalene sulfonate. The properties of covalently bonded fluorochromes in the nerve and the behavior of fluorochromes introduced into the axon interior were studied. By measuring fluorescence polarization, direct evidence was obtained showing a decrease in intramembrane viscosity during the action potential.

Acridines↗

PubMed2003

Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry.

Bcl-x(L) and Bcl-2 inhibit both apoptosis and proliferation. In investigating the relationship between these two functions of Bcl-x(L) and Bcl-2, an analysis of 24 Bcl-x(L) and Bcl-2 mutant alleles, including substitutions at residue Y28 previously reported to selectively abolish the cell cycle activity, showed that cell cycle delay and anti-apoptosis co-segregated in all cases. In determining whether Bcl-2 and Bcl-x(L) act in G(0) or G(1), forward scatter and pyronin Y fluorescence measurements indicated that Bcl-2 and Bcl-x(L) cells arrested more effectively in G(0) than controls, and were delayed in G(0)-G(1) transition. The cell cycle effects of Bcl-2 and Bcl-x(L) were reversed by Bad, a molecule that counters the survival function of Bcl-2 and Bcl-x(L). When control and Bcl-x(L) cells of equivalent size and pyronin Y fluorescence were compared, the kinetics of cell cycle entry were similar, demonstrating that the ability of Bcl-x(L) and Bcl-2 cells to enhance G(0) arrest contributes significantly to cell cycle delay. Our data suggest that cell cycle effects and increased survival both result from intrinsic functions of Bcl-2 and Bcl-x(L).

Alleles↗

PubMed1966

Ribonucleic acid content of Burkitt tumour cells.

Methyl-green-pyronin-Y staining was performed on 57 biopsies of Burkitt's tumour and 62 biopsies from other types of malignant lymphoma. The specificity of the pyronin staining for ribonucleic acid (RNA) was controlled by staining duplicate slides previously digested in ribonuclease. Burkitt's tumour cells have a higher cytoplasmic content of RNA than the cells of most other malignant lymphomas. The results of acridine orange staining of a smaller number of biopsies support these findings.

Aniline Compounds↗

PubMed1998

Morphological and cytochemical changes during nuclear maturation in Bufo arenarum oocytes.

The morphological and cytochemical changes undergone by the oocyte during progesterone-induced nuclear maturation were analyzed in fully grown follicle oocytes of Bufo arenarum during the breeding period. The distribution and the staining properties of the nuclear material and of the perinuclear cytoplasm were studied. Our observations indicate that, during this complex process, the germinal vesicle (GV) undergoes migration and dissolution. The nuclear sap, which in the control has an homogeneous aspect, during maturation adopts a fibrillar appearance in the basal zone, showing basophilia and positive reaction to PAS, Feulgen and Methyl green-Pyronin staining. When the GV breakdown occurs, the nuclear contents become mixed with the cortical cytoplasm, developing a different cytoplasmic mass that disappears about 12 h after the beginning of incubation. The perinuclear cytoplasm exhibits metachromasia and orthochromasia with Toluidine Blue at pH 5.6 and a strong basophilia with PAS, suggesting the existence of acid compounds. The presence of nucleic acids such as RNA and DNA is a strong possibility because of the positive reaction to Methyl green-Pyronin, Feulgen and DAPI. This cytoplasmic mass remains until the end of the meiotic resumption process.

Age Factors↗

PubMed2001

Comparison of historic Grübler dyes with modern counterparts.

Seventeen Grübler dyes produced in Germany between 1880 and 1939 were examined in this study. These dyes were: fuchsin-bacillus, diamond fuchsin, fuchsin S acid, rubin S, safranin O water soluble, safranin yellowish water soluble, methyl eosin, Sudan III, scarlet R, auramine, orange G, aniline blue, pyronin, carmine, lithium carmine, hematein and aurantia. Spectrophotometry and staining characteristics were used to determine the maximum absorbance and efficacy of each dye in common staining techniques. The spectral curves and staining characteristics of these dyes compared well with modern dyes used as controls. Fuchsin bacillus and diamond fuchsin are synonyms for basic fuchsin. Fuchsin S acid and rubin S are synonyms for acid fuchsin. The scarlet R sample was the same as the Sudan III. The two safranins were the same. The basic fuchsin samples were unsuitable for preparation of Schiff's reagent. Both basic fuchsin and pyronin samples were less concentrated than modern counterparts. It is noteworthy that the dyes worked well after up to 100 years in storage, and this observation indicates that dyes can have a long shelf life when stored in cool, dry, air-tight conditions.

Bacteria↗

PubMed2002

[Reperfusion injury in the isolated rat liver after hypothermic preservation].

Histological changes which appear as a result of reperfusion injury of cold-preserved rat liver were studied at intervals of 0 hr, 3 hr, 24 hr and 48 hr of cold storage. The isolated livers were stored in a UW solution (University of Wisconsin), which is used in human liver transplantations. Computer image analysis of light microscopic sections (methyl green-pyronin stained) was used for the study and quantification of injured cells. The method of TUNEL was performed to prove possible apoptosis of sinusoidal endothelial cells and heptocytes. Bile production during reperfusion and ALT, AST, LDH and ACP were measured in the reperfusion medium at the end of the 90 min reperfusion. It has been confirmed that prolongation of the cold storage of liver results in extensive changes in the liver structure and increased injury of liver cells. Sinusoidal endothelial cells were damaged more and earlier than hepatocytes. It has been shown that methyl green-pyronin stained sections are advantageous for the study of these morphological changes, allowing the strongest view of these changes. The appearance of TUNEL positive cells and an increase in the levels of biochemical parameters, e.g. AST or ALT, indicate earlier cell injury. The methodology described in this article can be used for the study of reperfusion injury of the liver and for the study of this phenomenon in other experiments.

Animals↗

PubMed1988

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks.

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.

Anaplasma↗