Fusao Tomita

Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase.

Staurosporine, microbial alkaloid which has been known to have antifungal activity was found to inhibit markedly phospholipid/Ca++dependent protein kinase (protein kinase C) from rat brain, with an IC50 value of 2.7 nM. However, it had little effect on the binding of 3H-phorbol-12, 13-dibutyrate (PDBu) to protein kinase C. The inhibition of protein kinase C was not competitive with phospholipid. This compound also showed the strong cytotoxic effect on the growth of HeLa S3 cells, with an IC50 value of 4 X 10(-12)M under the condition of 72 hr-exposure.

Induction and catabolite repression mechanisms of cellulase in fungi

Cellulases are induced in most of fungi only when cellulose or an inducer exists. In Hypocrea jecorina and Penicillium purpurogenum, the respective inducers are sophorose and gentiobiose, which do not have beta-1,4 linkages though cellobiose, which has this linkage, is an inducer in other fungi. beta-Glucosidase, which catalyzes transglucosylation, is the key enzyme in converting cello-oligosaccharides to the inducers for cellulase induction in H. jecorina and P. purpurogenum. There are three states in the regulation of cellulase at the transcriptional level in fungi: expression at a basal level, mass secretion of cellulases induced by inducers, and glucose or catabolite repression. Expression at a basal level allows a small amount of cellulase to hydrolyze cellulose to soluble oligosaccharides or to an inducer if cellulose exists near the mycelia. Once the inducer enters the cell, it triggers full-scale transcription of the cellulase gene mediated by activator proteins and activating elements. After cellulose is degraded a large amount of glucose is liberated, which causes catabolite repression.

Difructose Anhydrides: Their Mass-Production and Physiological Functions

Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two fructose residues, and are expected to have novel physiological functions from their unique structures and properties. For mass-production of α-D-fructofuranose-β-D- fructofuranose-2′,1:2,3′-dianhydride (DFA III) and β-D-fructofuranose-β-D-fructofuranose-2′,6:2,6′-dianhydride (DFA IV), Arthrobacter sp. H65-7 and A. nicotinovorans GS-9 were selected as the best producers of inulase II, which produced DFA III from inulin and LFTase, which produced DFA IV from levan. The enzymes were purified and their genes were subsequently cloned and expressed in E. coli at higher levels than in the original bacteria. Thus, it became possible to provide a large amount of DFA III and DFA IV for evaluating their physiological properties. DFA III and DFA IV have half the sweetness of sucrose, but cannot be digested by the digestive system of rats. Their use by the intestinal microorganisms was observed in vivo even though their assimilation could not be detected in vitro. This implied that they were degraded by an unknown system in the intestine. It was also found that they affected calcium absorption mainly in the small intestine through mechanisms different from the known stimulants such as fructooligosaccharides and raffinose.

Production of inulin fructotransferase (depolymerizing) by Arthrobacter sp. H65-7 and preparation of DFA III from inulin by the enzyme

Abstract A bacterial strain H65-7 isolated from soil as an inulin-assimilating microorganism produces inulin fructotransferase (inulase II) which converts inulin into di- d -fructofuranose-1,2′:2,3′-dianhydride (DFA III). This strain was classified as Arthrobacter sp. The inulase II production was induced by inulin, and markedly enhanced by the addition of yeast extract. Under optimal conditions, the enzyme activity in the culture supernatant reached 90 units/ml after cultivation for 18 h. The optimum pH and temperature for the enzyme reaction were 5.5 and 60°C, respectively. The enzyme was stable within a pH range of 4.5 to 9.0 and at up to 75°C. Using this crude enzyme, 300 mg/ml of inulin was converted into 237 mg/ml of DFA III after incubation for 4 h.

Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485

A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid aas the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5 g/l pyruvic acid was obtained from 50 g/l glucose after the culture for 32–40 h at pH6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermentor culture was discussed.

Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve

ABSTRACT To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.

Purification and Properties of an Inulin Fructotransferase (Depolymerizing) from Arthrobacter sp. H65-7

Abstract Arthrobacter sp. H65-7 produced inulin fructotransferase (depolymerizing) which converts inulin into di- d -fructofuranose 1, 2′ : 2, 3′ dianhydride (DFA III) and small amounts of oligosaccharides. The enzyme was purified 8-fold with a yield of 13% from a culture supernatant by ammonium sulfate precipitation followed by DEAE-Toyopearl 650 M column chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 49,000 by SDS-polyacrylamide gel electrophoresis, and 100,000 by gel filtration. The isoelectric point of the enzyme was determined to be pH 4.7. The optimal pH and temperature for the enzyme reaction were 5.5, and 60°C, respectively. The enzyme was stable with a pH range of 4.5 to 9.0, and at up to 70°C. After exhaustive digestion of inulin by this enzyme, nystose and 1-F-fructofuranosyl-nystose were produced in addition to DFA III.

Asaccharobacter celatus gen. nov., sp. nov., isolated from rat caecum

An obligately anaerobic and equol-producing bacterium, designated strain do03T, was isolated from the caecal content of a rat. Cells were Gram-positive, non-spore-forming rods. The results from a phylogenetic analysis based on 16S rRNA gene sequences showed that strain do03T formed a separate line of descent in the phylogenetic cluster of the family Coriobacteriaceae. The strain was unable to metabolize glucose or other carbohydrates as sole carbon sources; growth was enhanced in the presence of arginine. The cell wall contained meso-diaminopimelic acid. The major fatty acid was C18 : 1cis9 (54.0 %). The strain had one unidentified predominant (91.9 %) quinone that was not menaquinone, methylmenaquinone, demethylmenaquinone, ubiquinone or rhodoquinone. The DNA G+C content was 63 mol%. The data presented in this work show that strain do03T differs from members of the related recognized genera Eggerthella and Denitrobacterium at both the phylogenetic and phenotypic level. Therefore, the strain constitutes a novel genus and species, for which the name Asaccharobacter celatus gen. nov., sp. nov. is proposed. The type strain of the type species is do03T (=JCM 14811T=DSM 18785T=AHU 1763T).

Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 years

ABSTRACT Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.