Hugh D. Braymer

Differential expression of insulin receptor tyrosine kinase inhibitor (fetuin) gene in a model of diet-induced obesity

The Differential Display technique has been used to identify differences in mRNA expression in adipose tissue after the introduction of a high fat diet to two strains of rat (OM and S5B/PI) that differ in their susceptibility to develop obesity on this diet. The insulin receptor tyrosine kinase inhibitor protein (fetuin) was shown to be differentially expressed in OM but not S5B/PI rats. This circulating protein may play a role in the development of peripheral insulin resistance associated with high fat diets.

Ultrastructure and Invertase Secretion of the Slime Mutant of Neurospora crassa

SUMMARY: Electron micrographs of thin sections of cells of the slime mutant of Neurospora crassa showed that it, like artificially prepared protoplasts, possessed no cell wall. Invertase was produced by the mutant and appeared to be serologically identical to wild-type N. crassa invertase. The mutant secreted over 95 % of the invertase into the medium whereas cells of the wild-type retained almost all the invertase. The slime mutant hence resembles wild-type protoplasts in both structure and invertase secretion.

Purification and characterization of the extracellular proteinase of Serratia marcescens

The extracellular proteinase produced by a depressed strain of Serratia marcescens ATCC 25419 was purified and characterized. This produces more than 10-times the amount of extracellular proteinase produced by other strains of Serratia tested. The purified enzyme was prepared from the culture supernatant by (NH4)2SO4 fractionation and DEAE-cellulose chromatography. The purified enzyme has an so20,w of 3.95 and is a monomer of molecular weight 51,900. The proteinase has a broad pH optimum in the alkaline range with a maximum at pH 9.5. The enzyme will utilize a number of proteins as substrate. The products of digestion are primarily in the size range of tripeptides to hexapeptides. Peptides containing a sensitive bond and a minimum size of size amino acids are hydrolyzed. The proteinase is inhibited by chelating agents but unaffected by sulfhydryl or serine reagents and is devoid of esterase activity.

Isosucrose. Definitive structural assignment by spectral correlation to α,β- and α,α-sucrose octaacetates

Abstract Three nonreducing disaccharides containing D -glucosyl and D -fructosyl groups, namely, “isosucrose” octaacetate ( 3 ), sucrose octaacetate ( 4 ), and “α,α-sucrose” octaacetate ( 5 ), give qualitatively identical mass spectra and are, therefore, related as diastereoisomers. Spin-coupling values in the 300-MHz n.m.r. spectra of 3 , 4 , and 5 establish that the D -glucopyranosyl group is present in the 4 C 1 ( D ) conformation, and that the anomeric configuration of this group is α in 4 and 5 , and β in 3 . In the n.m.r. spectra, the H-4 atom of the D -fructofuranosyl group of 3 and 5 resonates 0.5 p.p.m. upfield of the corresponding signal of 4 , from which it was deduced that both 3 and 5 are α- D -fructofuranosides.

Purification, Characterization, and Quantitation of the Antigen Employed in the Immunodiffusion Test for Diagnosis of Equine Infectious Anemia

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.

Neurospora crassa invertase: A study of amino acids at the active center

1. The effects on Neurospora crassa invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) of a variety of group specific reagnets and other potential inhibitors were determined during a search for an irreversible inhibitor of the enzyme. Aniline, pyridoxal, enzyme substrate and products did not inactivate invertase under reducing conditions. Bromoacetic acid, iodoacetic acid, iodoacetamide, p-chloromercuribenzoate, hydroxylamine and 2-hydroxy-5-nitrobenzyl bromide were also ineffective. Iodine was the only reagent which irreversibly inhibited invertase. 2. Invertase was rapidly inactivated by low concentrations of iodine, indicating specific inhibition. However, the enzyme could not be protected from this inactivation by substrate. It was not reactivated by mercaptoethanol or cysteine. 3. Experiments on the uptake of radioactive iodine demonstrated that invertase is not iodinated under the conditions of iodine inactivation. 4. The sedimentation (S20,w) value of invertase was not altered by iodine inactivation. One-dimensional electrophoresis and finger-printing of tryptic digests revealed no differences between iodine treated and untreated invertase. There was no loss of carbohydrate from this glycoprotein during iodine inactivation. 5. Standard amino acid analyses of iodine-inactivated invertase showed some loss of tyrosine and a trace amount of methionine sulfone. Attempts to demonstrate oxidation of methionine to the sulfone, through modification of the procedure for preparation of samples for analysis, were unsuccessful. However, oxidation of half-cystine was indicated and further loss of tyrosine noted. A hypothesis is advanced that half-cystine is oxidized by iodine to a normally unstable oxidation state which is maintained and protected by its protein invironment and that loss of tyrosine may be an artifact caused by the presence of this residue during acid hydrolysis.

Purification of the Protein Employed in an Immunodiffusion Test to Diagnose Infectious Bovine Rhinotracheitis

The antigen used in an immunodiffusion test to diagnose infectious bovine rhinotracheitis has been purified by affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A So20,w of 0.749 was determined and a molecular weight of 8900 was calculated from sedimentation equilibrium analysis. The purified antigen formed precipitin lines of identity with crude diagnostic antigen. Purified antigen remained serologically active in the immunodiffusion test after lyophilization and subsequent reconstitution.