Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

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.     

Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

A rapid method for preparation of plasma membrane fromAcanthamoeba culbertsoni involving toluene treatment followed by lithium bromide extraction is described. In the plasma membrane preparation, 5′-nucleotidase, Na⁺ + K⁺ -ATPase, Mg²⁺ -ATPase and glucose-6-phosphatase activities were enriched. The membrane preparation was free from nucleic acid, cytochrome P-450 and cytochrome b5. Amino acid (¹⁴C-Ieucine) was not incorporated in the plasma membrane in 2 min. Succinic dehydrogenase was not detectable in the plasma membrane preparation. The molar ratio of cholesterol and phospholipids was 0.95 which is characteristics for plasma membranes. Under electronmicroscopy the preparation was homogenous without any other component of the cell. Plasma membrane proteins and glycoproteins were separated on acrylamide gel electrophoresis     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.     

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.

     

Has treatment for childhood gastroenteritis changed?

Because so many children with gastroenteritis in our area were being treated with drugs, which are potentially harmful, we assessed the extent of treatment before admission to hospital of 288 children. Sixty four had been treated: 45 with antibiotic, antidiarrhoeal, or antiemetic drugs and 34 had been given glucose-electrolyte solution, 15 of those had also been given drugs; 119 had had no treatment. Since 1979 there has been a decrease in the use of drugs for gastroenteritis, but glucose-electrolyte mixtures are still underused.     

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.