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Biomedical subjects

J Orr

Publications and source records attributed to J Orr.

At least 73 records · Page 4Linked to original sources
PubMed1984

Mucin synthesis. The action of pig gastric mucosal UDP-GlcNAc:Gal beta 1-3(R1)GalNAc-R2 (GlcNAc to Gal) beta 3-N-acetylglucosaminyltransferase on high molecular weight substrates.

Membrane preparations from pig gastric mucosa were shown to transfer [14C]GlcNAc from UDP-[14C]GlcNAc to blood group A-negative porcine submaxillary mucin previously subjected to mild acid hydrolysis to remove terminal sialyl and fucosyl residues. O-Glycosyl oligosaccharides were removed from enzyme product by alkaline borohydride treatment and, after purification, were subjected to high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Two trisaccharide products were detected: [14C]GlcNAc beta 1-3Gal beta 1-3GalNAcOH and Gal beta 1-3[( 14C]GlcNAc beta 1-6)GalNAcOH. We have previously reported the in vitro synthesis of the latter compound, a branched trisaccharide, by UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from canine submaxillary glands. However, this is the first report of the in vitro synthesis of the linear trisaccharide GlcNAc beta 1-3Gal beta 1-3GalNAc. Pig gastric mucosal beta 3-N-acetylglucosaminyltransferase catalyzed the formation of this trisaccharide by incorporation of GlcNAc into the terminal Gal of Gal beta 1-3GalNAc-alpha-R when R was a polypeptide from either mucin or antifreeze glycoprotein, but not when R was o-nitrophenyl. We have previously reported the in vitro synthesis by pig gastric mucosa of the tetrasaccharide GlcNAc beta 1-3Gal beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-R when R was o-nitrophenyl or benzyl. We show in this report that pig gastric mucosa can synthesize this tetrasaccharide in vitro when R is a polypeptide from either porcine submaxillary mucin or antifreeze glycoprotein. Pig gastric mucosa therefore contains a beta 6-N-acetylglucosaminyltransferase capable of converting Gal beta 1-3GalNAc-alpha-R to Gal beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-R and one or more beta 3-N-acetylglucosaminyltransferases which can add GlcNAc in beta 1-3 linkage to a terminal Gal residue to form either GlcNAc beta 1-3Gal beta 1-3GalNAc-alpha-R or GlcNAc beta 1-3Gal beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-R where R is the polypeptide backbone of either mucin or antifreeze glycoprotein.

Animals↗
PubMed1983

Mucin synthesis. III. UDP-GlcNAc:Gal beta 1-3(GlcNAc beta 1-6)GalNAc-R (GlcNAc to Gal) beta 3-N-acetylglucosaminyltransferase, an enzyme in porcine gastric mucosa involved in the elongation of mucin-type oligosaccharides.

Pig gastric mucosa microsomes have been shown to catalyze the following reaction: UDP-GlcNAc + Gal beta 1-3(GlcNAc beta 1-6)-GalNAc-alpha-R----GlcNAc beta 1-3Gal beta 1-3 (GlcNAc beta 1-6)GalNAc-alpha-R + UDP, where R is o-nitrophenyl or benzyl. The enzyme catalyzing this reaction has been named UDP-GlcNAc:Gal beta 1-3(GlcNAc beta 1-6)GalNAc-R (GlcNAc-R (GlcNAc to Gal) beta 3-N-acetylglucosaminyltransferase. The beta 3-GlcNAc-transferase does not act on Gal beta 1-3GalNAc-alpha-o-nitrophenyl. The beta 3-GlcNAc-transferase requires Mn2+ and Triton X-100 for optimal activity. The Vmax for the microsomal enzyme is 8.7 nmol/mg protein per hour and the Km values are 1.6, 0.9, and 1.2 mM for UDP-GlcNAc and the alpha-o-nitrophenyl and alpha-benzyl derivatives of Gal beta 1-3(GlcNAc beta 1-6)GalNAc, respectively. Pig gastric mucosa microsomes catalyze the transfer of GlcNAc to lactose to form GlcNAc beta 1-3Gal beta 1-4Glc, but fail to transfer GlcNAc to lactosyl ceramide, Gal beta 1-4GlcNAc, or Gal beta 1-4GlcNAc-beta-benzyl.

Animals↗
PubMed1982

Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives.

A highly sensitive ion monitoring method for the determination of valproic acid in saliva and in serum has been developed based on the gas chromatographic--mass spectrometric analysis of the tert.-butyldimethylsilyl derivatives. Extraction methods are simple and the techniques for derivatization are rapid and convenient. Selected ion monitoring was carried out using electron ionization conditions and a common ion m/z 201 (M+--57) present in valproic acid and the internal standard octanoic acid. The lower limit of sensitivity that has acceptable precision for assay purposes is 0.1 mg/l based on a 200-microliter sample size. The ion monitoring method (derivatized) was compared to a gas chromatographic method (underivatized) for serum valproate assays and found to be essentially identical. The assay methodology was used in a kinetic study of valproic acid in two normal subjects. Saliva levels of drug were found to give reasonably good correlations with serum total and with serum free concentrations of drug in both individuals.U

Administration, Oral↗
PubMed1982

Regulation of glutamine synthetase activity and synthesis in free-living and symbiotic Anabaena spp.

Regulation of the synthesis and activity of glutamine synthetase (GS) in the cyanobacterium Anabaena sp. strain 7120 was studied by determining GS transferase activity and GS antigen concentration under a variety of conditions. Extracts prepared from cells growing exponentially on a medium supplemented with combined nitrogen had a GS activity of 17 mumol of gamma-glutamyl transferase activity per min per mg of protein at 37 degrees C. This activity doubled in 12 h after transfer of cells to a nitrogen-free medium, corresponding to the time required for heterocyst differentiation and the start of nitrogen fixation. Addition of NH3 to a culture 11 h after an inducing transfer immediately blocked the increase in GS activity. In the Enterobacteriaceae, addition of NH3 after induction results in the covalent modification of GS by adenylylation. The GS of Anabaena is not adenylylated by such a protocol, as shown by the resistance of the transferase activity of the enzyme to inhibition by Mg2+ and by the failure of the enzyme to incorporate 32P after NH3 upshift. Methionine sulfoximine inhibited Anabaena GS activity rapidly and irreversibly in vivo. After the addition of methionine sulfoximine to Anabaena, the level of GS antigen neither increased nor decreased, indicating that Glutamine cannot be the only small molecule capable of regulating GS synthesis. Methionine sulfoximine permitted heterocyst differentiation and nitrogenase induction to escape repression by NH3. Nitrogen-fixing cultures treated with methionine sulfoximine excreted NH3. The fern Azolla caroliniana contains an Anabaena species living in symbiotic association. The Anabaena species carries out nitrogen fixation sufficient to satisfy all of the combined nitrogen requirements of the host fern. Experiments by other workers have shown that the activity of GS in the symbiont is significantly lower than the activity of GS in free-living Anabaena. Using a sensitive radioimmune assay and a normalization procedure based on the content of diaminopimelic acid, a component unique to the symbiont, we found that the level of GS antigen in the symbiont was about 5% of the level in free-living Anabaena cells. Thus, the host fern appears to repress synthesis of Anabaena GS in the symbiotic association.

Cyanobacteria↗
PubMed1981

Purification, physical characterization, and NH2-terminal sequence of glutamine synthetase from the cyanobacterium Anabaena 7120.

A procedure for the complete purification of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from the cyanobacterium (blue-green alga) Anabaena 7120 is described. The enzyme has structural characteristics in common with glutamine synthetases from other sources: a subunit molecular weight of approximately 50,000 and a native structure, determined by low dose exposure electron microscopy, consisting of a two-layered regular hexagon made up of 12 subunits. Sequence analysis suggests that the subunits are identical. There was no indication that the enzyme from cyanobacteria is adenylylated, a feature shared with the glutamine synthetase from Bacillus subtilis but not with the enzyme from Escherichia coli. NH2-terminal sequence analysis and the predicted conformation of the NH2-terminal regions showed definite homology among the enzymes from all three sources. The limited analysis suggested a stronger structural homology between the enzyme from Anabaena 7120 and that from E. coli.

Amino Acid Sequence↗
PubMed1981

Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120.

A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined. Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 mM, respectively; that for ammonia was found to be below 20 microM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase.

Amino Acids↗
PubMed1976

Relation between drug-induced central nervous system effects and plasma levels of diazepam in man.

Pharmacodynamic effects and plasma levels of diazepam were studied in healthy male volunteers at different dose levels. Responses to diazepam were quantified, using instruments which measured body sway (statometry) and psychomotor performance (stressalyser tests). High dose-related correlations were obtained between drug-induced changes in test parameters and drug plasma levels, both with regard to stimulant and depressive effects. Techniques were devised for evaluating and comparing the efficacy and usefulness of different types of tests, taking into account critical thresholds, slopes and error estimates, correcting for changes in predrug levels and control (nondrug) trials.

Adolescent↗
PubMed1976

Steroid metabolism in rat submaxillary glands.

The ability of rat submaxillary gland to metabolize C21 steroids was studied by culturing explants with labeled progesterone, pregnenolone and 17 alpha-hydroxyprogesterone. The metabolites isolated were identified by paper chromatography, thin layer chromatography and combined gas liquid chromatography mass spectrometry. The radiochemical purity of certain metabolites was confirmed by recrystallization to constant specific activity. The capability of this gland to reduce C21 steroids was confirmed with the isolation of 3 alpha-hydroxy-5 alpha-pregnan-20-one and 5 alpha-pregnane-3 alpha, 20 beta-diol. The presence of delta 5, 3 beta hydroxy steroid dehydrogenase was shown by the conversion of pregnenolone to progesterone. Only in the incubations with 17 alpha-hydroxyprogesterone a compound with a molecular weight 290 was detected.

Animals↗