Immunochemical detection of the alpha-subunit of the G-protein, GZ, in membranes and cytosols of mammalian cells

An antiserum (AS 98) was raised against a synthetic peptide deduced from published cDNA sequences of the alpha-subunit of the putative G-protein, GZ (Fong et al. Proc. Natl. Acad. Sci. USA 85, 3066-3070, 1988; Matsuoka et al. Proc. Natl. Acad. Sci. USA 85, 5384-5388, 1988). In membrane and cytosol preparations of many but not all tested mammalian tissues, AS 98 predominantly recognized two proteins of 40 and 43 kDa Mr. Whereas high levels of a 40 kDa GZ alpha-subunit were found in rat liver membranes and in brain cytosol, AS 98 failed to detect the alpha-subunit of GZ in brain membranes.     

Preparation of raf-oncogene-specific antiserum with raf protein produced in E. coli

In this report we describe the expression of v-raf protein in E. coli using a tryptophan-promoter-driven expression vector and its immunological characterization by anti-peptide sera. The purified recombinant protein was used to produce raf-specific antibodies which are suitable for studying v-raf and c-raf proteins in vitro and in vivo in a variety of species ranging from mouse to man.     

Characterization of SV40 chromatin by mass determination on STEM

Direct mass determination of purified SV40 minichromosomes was obtained by scanning transmission electron microscopy. Twenty to thirty percent of the minichromosomes were found with an Mr of 6.9±0.4×10⁶. The rest of the molecules formed a spread Mr distribution ranging from 7.3×10⁶ to 9.5×10⁶ due possibly to different contents of the virus-coded proteins, mainly VP1. The apparent mass histogram of individual SV40 nucleosomes presents three maxima at Mr 2.1×10⁵, 2.6×10⁵ and 3.1×10⁵ that could correspond to partially unravelled nucleosomes, complete nucleosomes and complete nucleosomes with the addition of VP1. Beaded structures with a higher mass were also measured; some were found at either side of the open nucleosome-free region.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Conservation of Chi cutting activity in terrestrial and marine enteric bacteria

Homologous recombination in Escherichia coli occurs at increased frequency near Chi sites, 5'G-C-T-G-G-T-G-G3'. Cutting of DNA close to the Chi sequence by the E. coli RecBC enzyme is essential to Chi's stimulation of recombination. We have detected Chi-dependent cutting activity in extracts of several genera of terrestrial enteric bacteria (family Enterobacteriaceae) and of two genera of marine enteric bacteria (family Vibrionaceae). More distantly related bacteria had no detectable Chi-dependent cutting activity. These results support the view that recognition of this specific nucleotide sequence as a signal activating recombination has been maintained during the evolution of certain groups of bacteria. We discuss the possibility that other sequences play a similar role in other groups of bacteria.     

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.     

Adenylate cyclase activity in zona-free mouse oocytes

Zona-free mouse oocytes, prepared by either chemical or enzymatic treatment, possess adenylate cyclase activity, since forskolin elevated cAMP to similar levels in either these oocytes or in oocytes with intact zonae. In addition, it was shown that oocyte isolation conditions can affect 'basal' cAMP levels.     

Extraction of the adenylate cyclase-activating factor of bovine sperm and its identification as a trypsin-like protease

We previously reported the activation of adenylate cyclases from rat brain (Johnson, R. A., Awad, J. A., Jakobs, K. H., and Schultz, G., (1983) FEBS Lett. 152, 11-16) and from human platelets (Jakobs, K. H., Johnson, R. A., and Schultz, G. (1983) Biochim. Biophys. Acta 756, 369-375) by a factor derived from bovine sperm. In this report we describe the conditions for the extraction of the factor from bovine sperm and characteristics of its effects on adenylate cyclase which are consistent with its being a protease. The activating capacity of sperm particles was extracted from previously washed and frozen sperm into a 30,000 X g supernatant fraction by various salts, but not by the nonionic detergent Lubrol-PX. The amount of extracted factor: (a) was greatest with NH4HCO3 greater than NaCl greater than Na acetate; (b) was optimal with 0.5 M salt; (c) was not appreciably affected by the pH of the extraction buffer between pH 5.0 and 8.5; and (d) exhibited the greatest specific activity at the lower pH. The extracted sperm factor could be concentrated without loss by ultrafiltration on Amicon PM-10 membranes. The effect on adenylate cyclase of concentrated and desalted sperm extracted was inhibited 50% by various salts at 10 to 30 mM. The effects of the sperm factor to activate platelet adenylate cyclase, to block its inhibition via the alpha-adrenoceptor, and to block inhibition of stimulated forms of the enzyme by stable guanine nucleotides were prevented by protease inhibitors. A 50% reduction in the sperm factor's activation of platelet adenylate cyclase was caused by 30 nM soybean trypsin inhibitor, 30 nM alpha 2-macroglobulin, 300 nM leupeptin, 1 microM antipain, 15 microM aprotinin, and 100 microM benzamidine. Up to 3 mM phenylmethanesulfonyl fluoride was without effect on activation of the platelet cyclase by the sperm factor. The effects of the sperm factor persisted after its removal by the washing of pretreated platelet membranes and after its inactivation by the subsequent addition of leupeptin. The data strongly support the conclusion that the bovine sperm factor is a trypsin-like protease. alpha-Chymotrypsin, trypsin, and sperm acrosin were comparably effective in stimulating the platelet adenylate cyclase 5- to 8-fold, with concentrations eliciting maximal stimulation being: 200 ng trypsin/ml; 2 micrograms alpha-chymotrypsin/ml; and 2 micrograms acrosin/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

The acute effects of cigarette smoke exposure on experimental skin flaps

Random vascular patterned caudally based McFarlane-type skin flaps were elevated in groups of Fischer 344 rats. Groups of rats were then acutely exposed on an intermittent basis to smoke generated from well-characterized research filter cigarettes. Previously developed smoke inhalation exposure protocols were employed using a Maddox-ORNL inhalation exposure system. Rats that continued smoke exposure following surgery showed a significantly greater mean percent area of flap necrosis compared with sham-exposed groups or control groups not exposed. The possible pathogenesis of this observation as well as considerations and correlations with chronic human smokers are discussed. Increased risks of flap necrosis by smoking in the perioperative period are suggested by this study.