Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions.

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.

Hepatoprotective effects of a concentrate and components of sake against galactosamine (GalN)-induced liver injury in mice.

We investigated the hepatoprotective effects of a concentrate of sake (CS) and its components against D-galactosamine (GalN)-induced liver injury by measuring the plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in mice. CS significantly suppressed the GalN-induced elevation of ALT and AST activities. Each of four concentrated fractions extracted from sake (respectively consisting mainly of basic amino acids, neutral and acidic amino acids, organic acids and sugars) suppressed the GalN-induced elevation of ALT and AST activities. We focused on the sugar fraction containing glucose and ethyl alpha-D-glucoside (alpha-EG), which is a sake-specific sugar, as the major components and demonstrated that only alpha-EG showed significant suppression of the GalN-induced elevation of ALT and AST activities. We compared the effects of the alpha-EG analogues, methyl alpha-D-glucoside and ethyl beta-D-glucoside, on GalN-induced liver injury and confirmed that only alpha-EG significantly suppressed both the ALT and AST activities. Moreover, CS and alpha-EG suppressed the GalN-induced production of interleukin 6 (IL-6) and liver DNA fragmentation. Together these results show that CS and its component, alpha-EG, suppressed GalN-induced liver injury by inhibiting IL-6 production.

S-adenosylmethionine (SAM)-accumulating sake yeast suppresses acute alcohol-induced liver injury in mice.

The suppressive effects on acute alcoholic liver injury of S-adenosylmethionine (SAM) and the sake yeast, Saccharomyces cerevisiae Kyokai No. 9, have been shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed on a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed on the SAM yeast, the ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, the SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by the SAM yeast. These results suggest that the SAM yeast had a stronger effect suppressing acute alcoholic liver injury in mice than the sake yeast.

Sake yeast suppresses acute alcohol-induced liver injury in mice.

Brewer's and baker's yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.

Application of EST Data of Siderophore Regulation Protein-like Gene of Aspergillus oryzae RIB40 / 吉林大学学报(医学版)

Objective: To acquire the new clear color phenotype of Aspergillus oryzae b y th e antisense strategy of siderophore regulation protein (SREP)-like gene. Method s: Construct the cDNA library of Aspergillus oryzae RIB40 and amplify the fr agme nt ac7336f from the every EST clone, which had high homology with SREP gene of o ther species, then construct the eukaryotic expression vector with SREP-like ge ne using antisense strategy. Results: The sequence of this SREP-like gene was a cquired, the vector was successfully constructed. Conclusion: The deduced amino acid sequence of SREP-like gene of Aspergillus oryzae indicated that there is t he high homology with those of SREP genes of Penicillium chrysogenum, Neur ospora crassa and Schizosaccharomyces pombe.

Subtractive cloning of cDNA from Aspergillus oryzae differentially regulated between solid-state culture and liquid (submerged) culture.

In solid-state cultures (SC), Aspergillus oryzae shows characteristics such as high-level production and secretion of enzymes and hyphal differentiation with asexual development which are absent in liquid (submerged) culture (LC). It was predicted that many of the genes involved in the characteristics of A. oryzae in SC are differentially expressed between SC and LC. We generated two subtracted cDNA libraries with bi-directional cDNA subtractive hybridizations to isolate and identify such genes. Among them, we identified genes upregulated in or specific to SC, such as the AOS ( A. oryzae SC-specific gene) series, and those downregulated or not expressed in SC, such as the AOL ( A. oryzae LC-specific) series. Sequencing analyses revealed that the AOS series and the AOL series contain genes encoding extra- and intracellular enzymes and transport proteins. However, half were functionally unclassified by nucleotide sequences. Also, by expression profile, the AOS series comprised two groups. These gene products' molecular functions and physiological roles in SC await further investigation.