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Biomedical subjects

R M Zucker

Publications and source records attributed to R M Zucker.

At least 55 records · Page 3Linked to original sources
PubMed1988

Metal-induced alteration of the cell membrane/cytoplasm complex studied by flow cytometry and detergent lysis.

Flow cytometric analysis of the cell cycle is most effectively accomplished with membrane-/cytoplasm-free ("clean") nuclei. Non-ionic detergents (e.g. NP40 or Triton X-100) commonly are employed to solubilize cell membranes/cytoplasm to produce "clean" nuclei. Treatment of murine erythroleukemic cells (MELC) with tri-n-butyltin methoxide, cadmium acetate, zinc sulfate, or lead acetate alters the properties of the cell membrane/cytoplasm complex making it resistant to NP40 dissolution. On a molar basis, the organotin compound was more effective in inducing resistance to detergent-mediated dissolution than the inorganic metal compounds. Resistance to NP40-mediated dissolution was manifested as an increase in the flow cytometric parameters 90 degrees scatter and fluorescein isothiocyanate (FITC) fluorescence and was confirmed by light microscopy.

Acetates↗
PubMed1988

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter.

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.

Animals↗
PubMed1988

Effects of tributyltin on biomembranes: alteration of flow cytometric parameters and inhibition of Na+, K+-ATPase two-dimensional crystallization.

Carboxyfluorescein diacetate (CFDA) is a lipophilic nonfluorescent molecule that readily crosses the cell membrane. In the cytoplasm, it is hydrolyzed by nonspecific esterases to carboxyfluorescein (CF), a negatively charged fluorescent molecule, which is retained incompletely by cells with an intact plasma membrane. Exposure (4 hr) of the murine erythroleukemic cell (MELC) to micromolar quantities (0.1 to 5.0 microM) of tributyltin (TBT) results in increased cellular CF fluorescence. The increase occurs within a range below a critical value of the product (CPV) of the concentration (C) of TBT X duration (T) of exposure to TBT. Fluorescence increase is a sensitive indicator of the interaction of TBT with the cell: it is observed following exposure to 0.1 microM TBT for 4 hr at 37 degrees C. In the range above the CPV, cellular CF fluorescence is reduced apparently resulting from perturbation of membrane structure. For example, exposure of MELC to 2.5 microM TBT for 4 hr at 37 degrees C produces resistance to detergent-mediated cytolysis and inhibition of vanadate-mediated two-dimensional crystallization of Na+, K+-ATPase molecules in porcine renal microsomal membrane preparations, a process requiring molecular mobility within the membrane. Taken together, the increased cellular CF fluorescence and resistance of the MELC to cytolysis along with the inhibition of Na+, K+-ATPase crystallization in the microsomal membrane preparations suggest fixation (protein denaturation, cross-linking, etc.) at the level of the plasma membrane as a mode of toxic action of TBT.

Animals↗
PubMed1988

Flow cytometric analysis of the cellular toxicity of tributyltin.

Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose-dependent and time-dependent manner. The flow cytometric parameter axial light loss, a measure of cell volume, decreases in cells exposed to 5 microM TBT relative to control cells or cells exposed to 50 microM TBT. The flow cytometric parameter 90 degrees light scatter, a function of refractive index and a measure of protein content, increases as a function of TBT concentration above 0.5 microM. Following exposure to TBT concentrations greater than 0.5 microM, but less than 50 microM, DNA distribution across the cell cycle cannot be resolved adequately by flow cytometry. Also, the cells become resistant to solubilization of the cell membrane/cytoplasm complex by nonionic detergents. Relative to logarithmically growing cells, MELC in the stationary phase of the growth cycle and butyric acid-differentiated cells exhibit decreased plasma membrane permeability resulting in increased carboxyfluorescein (CF) retention derived from the intracellular hydrolysis of carboxyfluorescein diacetate (CFDA). Similarly, cells exposed to TBT concentrations below 50 microM exhibit increased cellular CF retention. Viability in terms of CFDA hydrolysis/CF retention and propidium iodide (PI) exclusion is not decreased by exposure to TBT concentrations below 1 microM. At doses between 5 and 50 microM, however, cells exhibit both CF and PI fluorescence simultaneously and are programmed for death. At TBT concentrations greater than 1.0 microM, MELC plasma membrane potential, measured with the cyanine dye, 3,3'-dihexyloxacarbocyanine iodide (DiOC6) decreases at the same time that the uptake of PI is observed. In conjunction with other data, the concentration-dependent increase in CF fluorescence, resistance to detergent-mediated solubilization of the plasma membrane/cytoplasm complex, and increase in 90 degrees light scatter suggest fixation (protein denaturation, cross-linking, etc.) as a mechanism of the toxic action of TBT.

Animals↗
PubMed1988

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry.

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.

Animals↗
PubMed1987

Differential hemoglobin synthesis in murine erythroleukemic cells: hemin effects.

The addition of butyric acid to murine erythroleukemic cells (clone T3Cl2) induced the cells to differentiate, producing adult hemoglobin (A, alpha 2,beta 2) and an embryonic hemoglobin (E2, alpha 2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four-fold and the synthesis of embryonic hemoglobin two-fold; the relative synthesis of the alpha and beta globins increased more than the y globin. The embryonic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.

Animals↗
PubMed1985

Synchronization of rapid globin expression in murine erythroleukemic cells.

The addition of butyric acid (BA) to murine erythroleukemia cells (MELC) produces the expression of primarily A and E2 hemoglobins while DMSO incubation produces the expression of primarily A hemoglobin. Preincubation of MELC with DMSO followed by BA induction accelerates the expression of hemoglobins in a synchronous manner, yielding the preferential synthesis of alpha and y globins, and E2 hemoglobin.

Animals↗
PubMed1983

5-Azacytidine increases the synthesis of embryonic hemoglobin (E2) in murine erythroleukemic cells.

The addition of 5-azacytidine to erythroleukemic cells which were induced to differentiate with DMSO or BA altered the expression of the hemoglobins. After the addition of 5-azacytidine there was an increase in hemoglobin synthesis especially in the embryonic E2 band. The beta-globin increased in synthesis after 5-azacytidine treatment. The level of hemoglobin synthesis in DMSO-induced cells is less than BA-induced cells while the effect of the 5-azacytidine stimulation was greater with DMSO induction than with BA induction.

Animals↗
PubMed1983

The effect of glutathione on HL-60 treated with dimethylsulfoxide, butyric acid or 12-O-tetradecanoylphorbol-13-acetate.

HL-60 promyelocytic leukemic cells can be induced to differentiate into granulocytes or macrophages. Reduced glutathione lyses undifferentiated HL-60 cells but has minimal effect on their differentiated counterparts. The addition of reduced glutathione to HL-60 promyelocytic leukemic cells retards cell growth and lyses cells. HL-60 cells can be induced to differentiate into granulocytes with dimethylsulfoxide butyric acid or into macrophages with 12-O-tetradecanoylphorbol-13-acetate. After treatment of HL-60 cells with these inducing agents the HL-60 cells become unresponsive to the effects of glutathione.

Butyrates↗
PubMed1983

Differentiation of HL-60 cells: cell volume and cell cycle changes.

HL-60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). After the addition of an inducer, the HL-60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation. Within 3 hours after the addition of TPA to HL-60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.

Butyrates↗
PubMed1983

Glutathione mediated lysis of HL-60 cells.

Various concentrations of glutathione (GSH), between 50-2000 micrograms/ml, were shown to retard the proliferation of HL-60 cells, with the optimum level being 200 micrograms/ml. Using electronic cell volume, GSH was shown to increase the percentage of small size cells and debris, and decrease the population cell volume and the percentage of large size cells. This data combined with chromium-51 release, trypan blue exclusion, and morphology data suggested that GSH was lysing HL-60 cells. Catalase eliminated the GSH stimulated cell lysis suggesting a H2O2 mediated mechanism was involved.

Cell Count↗
PubMed1983

Embryonic hemoglobins in mouse erythroleukemic cells.

Multiple hemoglobins are synthesized in the T3C12 murine erythroleukemic cells after induction with butyric acid and hemin. The pI values determined by IEF and globin composition of these hemoglobins suggests that A, E2, and E3 hemoglobins are synthesized in T3C12 cells. The embryonic mouse globins x, y, z and alpha from the yolk sac cells could be separated by acid urea triton x-100 electrophoresis. The mobility of the globins are the following alpha greater than z greater than y greater than x.

Animals↗
PubMed1983

Measurements of iron absorption from prenatal multivitamin--mineral supplements.

Although prenatal multivitamin--mineral supplements containing 60 to 65 mg of iron, taken once daily, are used widely to assure that pregnant women absorb the approximately 3.5 mg of supplemental iron per day that they require, there have been no studies concerning the absorption of iron from these preparations. Using cross-over studies in groups of normal nonpregnant women of childbearing age, such iron absorption was assessed using a technique in which absorption is calculated from the measured increase in serum iron after the oral ingestion of iron in various forms. With each of 4 different brands of prenatal supplements, mean iron absorption was less than the required 3.5 mg and ranged from 1.8 to 3.0 mg. These values were significantly less (P less than .01) than the 8.1 mg that was absorbed from 65 mg of iron alone. Decreased iron absorption in the prenatal supplements was shown to be due to inhibition by calcium carbonate and magnesium oxide and, in some cases, to poor iron release. When one of the 4 brands was reformulated to contain less calcium carbonate and less magnesium oxide, mean iron absorption increased to 4.5 mg. It is concluded that the amount of iron absorbed from many prenatal multivitamin--mineral supplements is significantly less than with standard forms of iron in nonpregnant women and that bioavailability studies should be performed on pregnant patients to determine whether these commercial preparations provide adequate amounts of iron during pregnancy.

Absorption↗
PubMed1982

Mouse hemoglobin during gestation. Absence of fetal hemoglobin.

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.

Animals↗
PubMed1981

Cell cycle dependency of tumor promoter on murine erythroleukemic cells.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) affected the cell cycle of murine erythroleukemic cells. In logarithmically growing cells a transient G1 block lasting approximately 6 hr developed after the addition of TPA. To investigate this cell cycle effect of TPA, cells were synchronized either by a double thymidine block or by centrifugal elutriation and were analyzed by flow cytometry and electronic nuclear volume. Using double thymidine blocked synchronized cells or elutriated separated fractions containing cells other than G1, a transient G1 block occurred as the cells entered early G1. The G1 elutriated cell population was blocked in the G1 period by TPA before cell cycle traverse. This transient block in G1 was not observed after the next cell cycle traverse. Addition of TPA to butyric acid or dimethylsulfoxide induced-cells resulted in a transient G1 block in addition to inhibition of differentiation. TPA was more inhibiting on dimethylsulfoxide-induced cells than butyric acid-cells.

Animals↗
PubMed1981

A search for the photoreceptor in the photosensitive irises of normally pigmented and albino hamsters (Mesocricetus auratus).

Hamsters are among the mammalian species whose irises are directly sensitive to light, responding with a constriction even when completely isolated from the rest of the hamster. Examination of the ultrastructure of the irises of both normally pigmented and albino hamsters disclosed a typical vertebrate iris, containing a sphincter and dilator pupillae, and anterior stromal layer, and two layers of pigment epithelial cells. No structures obviously specialized for photoreception were found. We examined the possibility that the pigment granules in the cells of the sphincter pupillae are photosensitive, and that their electron-dense melanin masks some photosensitive structure. However, this was ruled out by the observation that albino irises, which are photosensitive, are devoid of even rudimentary pigment granules. These results are consistent with the hypothesis that the sarcolemmas of the smooth muscle cells of the sphincter pupillae are photosensitive in this species.

Albinism↗