Ethanol directly stimulates dihydrotestosterone conversion to 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol in rat liver

The present results demonstrate for the first time in rat liver, that low ethanol concentrations (2.2 and 22 mM) directly stimulate dihydrotestosterone conversion to 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol. Because this effect was blocked by 4-methylpyrazole, an alcohol dehydrogenase inhibitor, or by the addition of a saturating NADH concentration, this action probably is mediated by hepatic alcohol dehydrogenase activity through elevation of the NADH/NAD+ ratio. It remains to be determined whether this effect of ethanol actually reduces circulating and/or target tissue dihydrotestosterone levels; nevertheless, it is tempting to speculate that this action, in part, is responsible for the reported adverse effects of alcohol on male reproductive functions.     

The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure:

with Fuc present on only about 50% of the oligosaccharides.     

Studies on the induction and expression of T-cell-mediated immunity. XIII. Membrane-associated antigens of cytotoxic T lymphocytes involved in cytotoxicity

An xenogeneic rat anti-mouse T-cell serum, designated RAT*, has been shown to block the cytolytic activity of cytotoxic T lymphocytes (CTL) at a postbinding step. RAT* serum or the IgG fraction was extensively absorbed with the target cell, P815, a DBA mastocytoma, and used with or without further absorption to immunoprecipitate specific molecules from radiolabeled membrane extracts of CTL derived from either in vivo-allosensitized mice or from cytotoxic clones maintained in in vitro cultures. Cell surface sialic acid residues were labeled by oxidation with sodium periodate (NaIO4) and reduction with tritiated sodium borohydride ([3H]NaBH4). Alternatively, cell surface proteins were labeled with 125I by lactoperoxidase-catalyzed iodination. Nonidet P-40 (NP-40)-solubilized radiolabeled membranes were then immunoprecipitated with RAT* serum and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Three membrane-associated molecules of 95,000, 140,000 and 180,000 Mr were found by such analysis. The sensitivity of these three molecules to trypsinization and their susceptibility to labeling with [3H]NaBH4 suggested that they are glycoproteins. Moreover, when RAT* serum or the IgG fraction was absorbed with various cell types, its ability to immunoprecipitate the three molecules correlated with its ability to block cytolysis. Adsorption of RAT* serum with CTL, but not with nonimmune thymocytes, significantly reduced the ability of RAT* serum to inhibit cytotoxicity and to immunoprecipitate the 95k, 140k, and 180k molecules. Thus, these findings suggest that one or more of these cell surface molecules of CTL may be involved in the cytolytic process.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Injected mitotic extracts induce condensation of interphase chromatin

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.     

Prolactin increases the density of striatal dopamine receptors in normal and hypophysectomized male rats

Administration of prolactin to adult male rats, by s.c. injection, significantly increases the density of the striatal dopamine (DA) receptors, without altering the apparent affinity of the receptors for [³H]spiroperidol. Larger doses of prolactin are required to increase the density of the striatal DA receptors in hypophysectomized rates compared to normal rats. These results suggest that prolactin might be the common mediator of the increase in striatal DA receptor density produced by either estrogen or haloperidol administration. Monitoring and/or altering prolactin levels might be informative in neurologic or psychiatric disorders involving striatal DA neurotransmission.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Activated T lymphocytes resist the toxic effects of the adenosine deaminase inhibitor, 2-deoxycoformycin

Tonsil lymphocytes from three adults and three children were examined for immunoglobulin (Ig) production before and after Epstein-Barr virus (EBV) transformation. T-cell depletion was required to obtain cell lines from EBV-seropositive individuals. Cytoplasmic Ig was mainly IgG in adult lymphocytes before and after transformation; IgA and IgM were more prominent after than before. IgM and IgG predominated in lymphocytes of children before and after transformation; IgA was more prominent after than before. Cytoplasmic Ig of peripheral blood lymphocytes from these individuals was mainly IgM. Secreted Ig from tonsil lymphocytes was mainly IgA or IgG; after transformation IgM predominated with adult cell lines, and IgG or IgM with cell lines from children. IgE was consistently sparse in spite of ragweed and/or grass allergies of the adults.     

cAMP-dependent protein kinases, their subunits, and cAMP-binding protein from four sources: a comparison of physical characteristics determined by polyacrylamide gel electrophoresis

The physical characteristics of cAMP-dependent protein kinases and their, regulatory subunits from calf uterus, human uterus, human mammary tumor, and rat pituitary and of cAMP-binding protein from calf uterus were determined by quantitative polyacrylamide gel electrophoresis in buffers containing the detergent, Triton X-100. In the four tissues, protein kinases of either type A¹, with molecular weight (M_r) = 200,000, or type B, of M_r = 80,000, or both, previously described were found. Trivial charge isomerism, or size isomerism, exists within each of the two classes, Protein Kinase A and B. The protein kinase recombined from the regulatory and catalytic subunits is not significantly different from the crude or isolated protein kinase. Protein Kinases A and B exist each in either one of the isozyme forms I and II but these are not reflected in polyacrylamide gel electrophoresis at pH 10.2. Protein Kinase B appears to be a product of the partial proteolysis of Protein Kinase A. The regulatory subunits of Protein Kinases A from the four tissues are distinct from those of Protein Kinases B. No physical distinction exists between regulatory subunits derived from isozyme forms I and II. cAMP-Binding Proteins A and B are physically indistinguishable, by polyacrylamide gel electrophoresis at pH 10.2, from the regulatory subunits of Protein Kinases A and B, respectively.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.