Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.     

Analgesic and hypnosis potentiation effect of some 1-(2-benzothiazolyl)-1-aryl-3-phenyl-4-aryl guanidines.

1-(2-benzothiazolyl)-1-aryl-3-phenyl-4-arylguanidines (I-X) were prepared by oxidation of 1,3-diarylthioureas. The compounds were screened for their analgesic and hypnotic activities in rats. Of these, p-methyl group substituted compound of the series was the most potent analgesic as compared to other compounds of the series. In hypnotic test all the compounds potentiated pentobarbitone-induced hypnosis.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Efficient preparation of steroidal 5,7-dienes of high purity.

Protected forms of dehydroepiandrosterone, delta 5 cholenic acid, (25R)-26-hydroxycholesterol and diosgenin were converted to the corresponding delta 5,7 dienes by successive treatment with 1,3-dibromo-5,5-dimethylhydantoin (dibromantin), tetrabutylammonium bromide and tetrabutylammonium fluoride. The crude products, which contained the delta 5,7 species contaminated by minor amounts of the delta 5 and delta 4,6 steroids, were purified by silica gel-AgNO3 chromatography to give the following steroids in approximately 99% purity and at least 50% yield: 3 beta-acetoxyandrosta-5,7-dien-17-one, methyl 3 beta-acetoxychola-5,7-dien-24-oate, (25R)-3 beta,26-diacetoxycholesta-5,7-diene and (25R)-3 beta-acetoxyspirosta-5,7-diene. Analogous treatment of acetate derivatives of pregnenolone and stigmasterol gave 3 beta-acetoxypregna-5,7-dien-20-one and 3 beta-acetoxystigmasta-5,7,22-triene in approximately 50% yield but of lower purity. Full 1H and 13C NMR assignments are given for seven delta 5,7 steroid acetates and the corresponding delta 5 starting materials. Coupling constants for rings A, B and C of delta 5,7 steroids are presented and stereochemical assignments have been made for the following 1H NMR signals: the C-11 protons of delta 5,7 steroids, the C-16 protons of sterols and bile acids, the C-22 and C-23 protons of bile acid esters and the C-28 protons of stigmasterol derivatives.     

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.     

Modulation of cortisol receptors in embryonic retina cells by changes in cell-cell contacts: Correlations with induction of glutamine synthetase

Cortisol induces glutamine synthetase (GS) in neural retina tissue of chick embryos. GS induction represents a characteristic feature of embryonic retina differentiation. However, if the tissue is dissociated into single cells, the dispersed cells are not inducible for GS. We report that cell dispersion results in a rapid and marked reduction in the level of cortisol-binding cytoplasmic receptors. This reduction persists if the cells are maintained in a dispersed state. However, if the cells are reaggregated and they reconstruct tissue-like contacts and architecture, the level of cortisol receptors increases, and so does inducibility for GS. The results indicate that, in the embryonic neural retina histotypic cell contacts and interactions are involved in regulating the level of cortisol receptors. We propose that cell contact-dependent signals from the cell surface may modulate levels of cytoplasmic cortisol receptors necessary for GS induction.     

Studies on the lipid metabolism of the metacercariae of Clinostomum complanatum (Trematoda: Digenea)

Metacercariae of Clinostomum complanatum possess considerable amounts of lipids. Fractionation of the lipids shows triglycerides and phospholipids as the major components whereas cholesterol and free fatty acids are minor components. Furthermore phospholipid fractions by thin layer chromatography reveal lecithin and cephalin as the major polar lipids whereas lysolecithin and lysocephalin are present in small fractions. The specific activity of lipase (E.C. 3.1.1.3) is 150.8 μg free fatty acids liberated/mg protein/h. Epinephrine, testosterone, insulin, sodium fluoride and iodoacetate stimulated, but 2-propanol inhibited, the lipase activity.     

Nutritional and hormonal regulation of the translatable levels of malic enzyme and albumin mRNAs in avian liver cells in vivo and in culture

Relative synthesis of malic enzyme is stimulated 25-to 100-fold by feeding neonatal ducklings or by incubating embryonic chick hepatocytes in culture with triiodothyronine. Synthesis of the enzyme is almost completely blocked when fed birds are starved or when triiodothyronine-treated hepatocytes in culture are also treated with glucagon. Cytoplasmic poly(A)+ RNA was isolated from livers of intact ducklings or hepatocytes in culture treated as described above and translated in an mRNA-dependent rabbit reticulocyte lysate. The identity of malic enzyme synthesized in the cell-free system was confirmed by virtue of its antigenicity, subunit molecular weight, and proteolytic peptide pattern. Translatable levels of malic enzyme mRNA paralleled changes in relative synthesis of malic enzyme in vivo and in hepatocytes in culture. Translatable levels od albumin mRNA were either unaffected or changed in a direction opposite to that of malic enzyme mRNA. Thus, both nutritional and hormonal regulation of malic enzyme synthesis involves regulation of cytoplasmic translatable malic enzyme mRNA levels. The hepatocyte culture system is ideally suited for future studies on the regulation of malic enzyme mRNA synthesis and/or degradation by thyroid hormone and glucagon.