Intracellular retention of the extracellular domain of the (pro)renin receptor in mammalian cells

As a component of the renin-angiotensin system, the (pro)renin receptor [(P)RR] activates prorenin along with intracellular signaling pathways. In this study, the glutathione S-transferase-fused extracellular domain of (P)RR expressed in mammalian cells was recovered in the detergent phase in detergent-based two-phase separation experiments, and intracellular localization was observed by immunocytochemistry, suggesting retention inside the cell through stable membrane association.     

Spray-Dried Gliadin Powders Inclusive of Linoleic Acid (Microcapsules): Their Preservability,Digestibility and Application to Bread Making

An aqueous-alcohol solution containing gliadin (or zein) and linoleic acid was pulverized by spray-drying and the powders were examined for their stability, digestibility and application. Linoleic acid in the powders was kept stable during three months’ storage in each case of low, moderate and high water activity. When the spray-dried powders were treated with such digestive enzymes as pepsin and pancreatin, linoleic acid was effectively changed into the hexane-extractable form concomitantly with the digestion of gliadin. The powders were administered to rats, instead of gluten and essential fatty acid in a semi-purified diet, and there was no abnormal excretion of fatty acid into the faeces. These facts indicate that two components of the powders could be digested and absorbed in the gut as usual. On the other hand, supplementation of bread with gliadin and linoleic acid in the form of spray-dried powders led to a considerable increase in bulk after baking, without affecting the taste and flavor. Linoleic acid in a loaf of bread seemed to be fairly resistlant to peroxidation during baking in an oven and during storage under aerobic conditions for a week.     

Tyramine,as an Active Factor for the Production of Phenolsulphatase by Aerobacter aerogenes

An active factor for the production of phenolsulphatase by some strains of Aerobacter aerogenes was separated as pure crystals from a commercial peptone preparation by ion exchange chromatographic techniques. Properties of this substance were studied, and it was found to be identical with tyramine. Even when pure tyramine in a concentration of 1 × 10^?5M was added to the culture medium, its effect on the level of enzymatic activity could be observed. In addition to tyramine, hordenine was also found effective but this, only in the case of A. aerogenes strain 9621. This specific activity of tyramine appears to be an induction of enzyme production.     

Enzymatic Studies on the Conversion of Citric Acid Fermentation to Polyol Fermentation in Hydrocarbon-assimilating Yeast Candida zeylanoides

Production of polyols such as erythritol, d-mannitol and d-arabitol by citric acid-producing yeasts occurred only when the medium-pH was controlled at acidic pH, as described in the previous papers.

In order to elucidate the conversion mechanism of citric acid fermentation to polyol fermentation, the effect of pH on the activities of enzymes involved in polyol synthesis and tricarboxylic acid cycle was studied. Shifting down of the medium-pH from 5.5 to 3.5 led immediately to the change of intracellular pH, from 6.5~6.7 to 5.5~5.7. Such the change affected remarkably on the activities of intracellular enzymes. Citrate synthase was significantly depressed at pH 5.7, but isocitrate lyase and phosphoenolpyruvate carboxykinase were reversely stimulated at this pH.

In some yeast strains incapable of polyol production, the change of medium-pH reflected directly on intracellular pH, whereby almost all enzymes were inhibited.

From these results, the conversion of citric acid production to polyol production was explained by the change in the enzyme activities caused by lowering of intracellular pH.     

Studies on Mold Proteases

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with that of several acid proteases from various sources. The peptide bonds susceptible to the action of Rhiz. acid protease were found to be mainly those involving the amino group of bulky amino acids. The enzyme split the B-chain of oxidized insulin at twelve sites of the peptide linkages and a certain similarity in the specificity was observed among the three acid proteases, Rhiz. protease, rennin and pepsin, all of which were known to show potent milk clotting activities.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.     

Glycerol monomycolate, a latent tuberculosis-associated mycobacterial lipid, induces eosinophilic hypersensitivity responses in guinea pigs

Dynamic changes in the lipid composition of the cell wall occur in pathogenic mycobacteria that are often intended for adaptation to the host environment. Dormant mycobacteria should have evolved efficient maneuvers for cohabitation, allowing the microbes to persist for years within the host. Glycerol monomycolate (GroMM) has been implicated as a specific immune target in human individuals with latent, but not active, tuberculosis, but the in vivo response to GroMM and the relevance of it to latent infection remain poorly understood. Here, we immunized guinea pigs with bacillus Calmette–Guerin (BCG) expressing high levels of GroMM and then, monitored skin reactions at the site of challenge with GroMM-containing liposome. We found that BCG-immunized guinea pigs mounted enhanced skin reactions to GroMM with prominent local infiltration by eosinophils. Consistent with this, GroMM-stimulated lymph node cells upregulated the expression of T helper (Th)2-type cytokines, such as interleukin (IL)-5 and IL-10, that could potentially counteract the microbe-eliminating Th1-type cytokine response. On the basis of these observations, we predict that the host response to GroMM produced by dormant mycobacteria would contribute to their long-term survival in the host.     

Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity

The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.