Activity of 11β-hydroxysteroid dehydrogenase of rat kidney and liver in inherited stress-induced arterial hypertension

11β-hydroxysteroid dehydrogenase (11β-HSD) catalyzing the interconversion of corticosterone and 11-dehydrocorticosterone is the key enzyme of glucocorticoid metabolism in rats. The activity of 11β-HSD in kidney of rats with inherited stress-induced arterial hypertension (ISIAH) was significantly (p < 0.05) higher than that in WAG rats. The opposite was observed in activity of liver 11β-HSD. No changes in the kidney 11β-HSD activity of both strains were observed under stress condition, however, the liver 11β-HSD activity in ISIAH rats was significantly (p < 0.05) higher as compared to basal level and stressed WAG rats. It is possible that the features of the 11β-HSD activity in ISIAH rats may reflect their hypertensive status.     

Structural studies of glycans isolated from rat plasma hemopexin

After exhaustive pronase digestion, purification by gel filtration and affinity chromatography on concanavalin A, three glycopeptide fractions were obtained from rat hemopexin. Two fractions (I and II) were concanavalin A non-reactive and one (III) was concanavalin A reactive. On the basis of carbohydrate composition, methylation analysis and proton nuclear magnetic resonance spectroscopy, the primary structure of the glycan in fraction III is proposed as being a mixture of mono- and di-sialo-diantennae of the N-glycosidic, N- acetyllactosamine type. Hydrazinolysis of glycopeptides not binding to concanavalin A yielded mixtures of oligosaccharides for both fractions. These oligosaccharides were separated by HPLC; the molar composition of each of them is given. These data suggest that rat hemopexin contains, among others, a diantennary structure bearing three sialic acid residues.     

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

A computationally efficient Monte-Carlo model for biomedical Raman spectroscopy

Monte Carlo (MC) modeling is a valuable tool to gain fundamental understanding of light-tissue interactions, provide guidance and assessment to optical instrument designs, and help analyze experimental data. It has been a major challenge to efficiently extend MC towards modeling of bulk-tissue Raman spectroscopy (RS) due to the wide spectral range, relatively sharp spectral features, and presence of background autofluorescence. Here, we report a computationally efficient MC approach for RS by adapting the massively-parallel Monte Carlo eXtreme (MCX) simulator. Simulation efficiency is achieved through “isoweight,” a novel approach that combines the statistical generation of Raman scattered and Fluorescence emission with a lookup-table-based technique well-suited for parallelization. The MC model uses a graphics processor to produce dense Raman and fluorescence spectra over a range of 800 − 2000 cm⁻¹ with an approximately 100× increase in speed over prior RS Monte Carlo methods. The simulated RS signals are compared against experimentally collected spectra from gelatin phantoms, showing a strong correlation.     

Formation of a stable l-ascorbic acid α-glucoside by mammalian α-glucosidase-catalyzed transglucosylation

Enzymatic transglucosylation from maltose to l-ascorbic acid (AA) with mammalian tissue homogenates was determined by a high-performance liquid chromatography method and compared with the reaction catalyzed by α-glucosidase from Aspergillus niger. The homogenates of small intestine and kidney had a high transglucosylase activity to form a new type of glucosylated AA, which was associated with α-glucosidase activity. The new compound was demonstrated to be an equimolar conjugate of AA and glucose by the spectral and quantitative analyses. In particular, it showed a high stability in a neutral solution and no reducing activity toward cytochrome c and a dye. These properties were very different from those of AA and l-ascorbic acid α-glucoside formed with α-glucosidase form A. niger, but they were consistent with those of l-ascorbic acid 2-O-phosphate and l-ascorbic acid 2-O-sulfate. Moreover, it exhibited a reducing power associated with AA after mild acid hydrolysis or treatment with rat intestinal α-glucosidase. These results indicate that it should be assigned the 2-O-α-glucoside structure. Consequently, i should be assigned the 2-O-α-glucoside structure. Consequently, it is concluded that mammalian α-glucosidase is able to form a very stable and nonreducing form of glucosylated AA through a specific transglucosylation reaction distinct from that of microbial α-glucosidase.     

Optical Configuration Analysis of Hydroxy Fatty Acids in Bacterial Lipids by Chiral Column High-Performance Liquid Chromatography

Through the adoption of a chiral stationary phase in high-performance liquid chromatography and a simple derivatization method for hydroxy fatty acids, it became easy to separate and identify the optical isomers of 2- and 3-hydroxy fatty acids composing several kinds of microbial lipids. The 2- and 3-hydroxy fatty acids were converted with dinitrophenyl isocyanate to their 3, 5-dinitrophenyl urethane derivatives (DU-derivatives), which were analyzable by HPLC using a chiral column. By varying the composition of an eluent, separation of the DU-derivatives of hydroxy fatty acids differing in optical configuration, chain length and position of hydroxyl group was achieved. The general elution orders of these DU-derivatives were determined with authentic 2- and 3-hydroxy fatty acids. Small amounts (~300 μg) of ornithine-containing lipids isolated from the Serratia marcescens strains were examined by this method to identify 3-hydroxy fatty acids of the lipids as D isomers.