Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver

The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction.     

Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.     

Synthesis and biologic activity of 3 beta-thiovitamin D3

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.     

A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants

A simple method was developed for the controlled cleavage of protein disulfide bonds and the simultaneous blockage of the free sulfhydryl groups in the absence of a denaturant. The disulfide bonds of bovine serum albumin were cleaved unsymmetrically at pH 7.0 using 0.1 M sulfite in 0.1 M phosphate buffer and the free sulfhydryl groups formed were sulfonated in an oxidation-reduction cycle using molecular oxygen and 400 microM cupric sulfate as a catalyst. The reaction was affected by cupric ion concentration, sulfite concentration, reaction pH and temperature. The standardized method was successfully used to cleave the disulfide bonds of other proteins pepsin, trypsin, and chymotrypsin. The method is reliable and can be used for achieving progressive cleavage of disulfide bonds in proteins without employing a denaturant.     

IgA nephropathy in adults: immunohistologic findings and clinical course

Laboratory examination of specimens from 123 consecutive renal biopsies performed at Victoria General Hospital, Halifax revealed six cases of mesangial deposition, predominantly of IgA, unassociated with systemic disorders. Immunohistologic examination showed deposits of only IgA in one specimen, IgA and IgG in two and IgA, IgG and IgM in three. Glomerular deposits of C3 were seen in five of the specimens, and properdin was seen in three. Glomeruli in all the specimens showed increased matrix and increased numbers of cells in the mesangium. Electron microscopy revealed deposits in the mesangium or capillary wall in all five of the specimens so studied. All six patients had proteinuria, four had microscopic hematuria, and three had hypertension; in one patient the disease progressed to renal failure.     

Isolation of a hydroxyproline-secreting pigment-deficient mutant ofNostoc sp. by metronidazole selection

Summary A pigment-deficient mutant ofNostoc sp. was induced by N-methyl-N-nitro-N-nitrosoguanidine and selected in a medium containing metronidazole. It formed chains of heterocysts, grew more slowly than the control, and excreted hydroxyproline into the medium, imparting a pinkish-brown colour to the cultures.

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Aislamiento de un mutante do Nostoc sp. deficiente en pigmentos y secretor de hidroxiprolina en un medio selectivo con metronidazol

Resumen Un mutante pigmento-deficiente deNostoc sp. fue inducido mediante N-metil-N-nitro-N-nitrosoguanidina y seleccionado en un medio conteniendo metronidazol. La cepa mutante creció más lentamente que la control formando cadenas de heterocistos y excretando hidroxiprolina al medio lo cual confirió una coloración rosa-parduzca a los cultivos.

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Isolement par sélection avec le métronidazole d'un mutant de Nostoc sp. excrétant de l'hydroxyproline et déficient en pigment

Résumé Un mutant deNostoc sp. déficient en pigment a été induit par la N-méthyl-N-nitro-N-nitrosoguanidine et sélectionné dans un milieu contenant du métronidazole. Ce mutant forme des chaînes d'hétérocystes, pousse plus lentement que le contrôle, et excréte de l'hydroxyproline dans le milieu, conférant aux cultures une coloration brun rosâtre.

     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Isolation of the cDNA for human prostaglandin H synthase

Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin synthesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500,000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones were isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large approximately 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenase, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level.