Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site.     

Predictive factors of psychological disorder development during recovery following SARS outbreak.

Objective: To investigate strategies for broad mass isolation during outbreaks of infectious diseases. Design: A survey using a self-administered questionnaire was conducted on 300 printing company workers in Beijing, China, which was under mass isolation following the 2003 SARS outbreak, in the 7-8 months after the isolation was lifted. Main Outcome Measures: Individuals with psychological disorders were classified on the basis of scores on the 30-item General Health Questionnaire during the recovery period. Psychological disorders were observed in 49 of 187 respondents (26.2%; 95% CI = 20.2, 32.7). Results: The predicting factor with the highest correlation was income reduction, with an odds ratio of 25.0. Other items obtained were gender, range of activities, eating restrictions, restrictions in going out, disinfection of clothing, and infection control, with odds ratios of 3.2, 5.5, 3.9, 3.2, 0.2, and 0.1, respectively, and the contribution ratio was 87.7%. Conclusion: Securing income is suggested to be important in future strategies. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Production of lepidimoide by an endophytic fungus from polysaccharide extracted from Abelmoschus sp.: identification of the product and the organism producing it

A highly potent allelopathic factor, lepidimoide, was initially extracted from mucilage of germinated cress seeds. Polysaccharide extracted from okra (Abelmoschus esculentum Moench) is considered to have a similar structure to lepidimoide as its repeating unit. We therefore initiated the screening of enzymes capable of degrading okra polysaccharide into lepidimoide from endophytes. We discovered an endophytic fungal strain AHU9748 isolated from Coleus galeatus, which produced an oligosaccharide having similar properties to lepidimoide on thin layer chromatography. The physico-chemical data from ESI-MS, NMR spectra and other analyses also showed the purified product to be identical to lepidimoide. The strain AHU9748 was identified as a fungus belonging to the coelomycetes, closely related to the genus Colletotrichum, based on morphological characteristics and sequence analysis of the 18S rDNA and ITS region.     

A novel assay method for glycosphingolipid deacylase by enzyme-linked immunochemical detection of lysoglycosphingolipid

Lysoglycosphingolipids consist of a sphingoid long-chain base and monosaccharide or complex sugar, and they lack the fatty acyl group present in native glycosphingolipids. Less than 1 pmol of lyso-Forssman glycolipid and lysoganglioside GM1 were detected on a thin-layer chromatogram by an enzyme-linked immunochemical coloration method with anti-Forssman glycolipid antibody (FOM-1) and cholera toxin B subunit, respectively. Each spot between 1 and 100 pmol lyso-Forssman glycolipid was immunostained as densely as that of the same amount of native Forssman glycolipid. The density of the lyso-Forssman glycolipid spots increased proportionally with increment in the amount of lysoglycolipid. The density of spots of 0.2–100 pmol lysoganglioside GM1 was also proportional to the amount of each lyso-GM1 spot. These results indicated that less than 1 to 100 pmol of deacylated glycosphingolipid was quantifiable by the immunochemical coloration method with sugar chain-specific antibodies. Glycosphingolipid deacylase, which cleaved an amide bond between the sphingoid long-chain base and fatty acyl chain in ceramide of glycosphingolipid, was assayed by detecting the lyso-Forssman glycolipid produced. Lipophilic compounds, recovered from an aliquot of the reaction mixture of Forssman glycolipid and crude enzyme at appropriate times, were analyzed by thin-layer chromatography. It was found that lyso-Forssman glycolipid was produced in the first 1–2 h by the enzyme and production increased with incubation time. This coloration method is more sensitive and specific than the visualization method with a non-specific reagent such as orcinol-sulfuric acid reagent.     

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein,rimM, for detection and enumeration of Streptococcus thermophilus in dairy products

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein, rimM, for specific detection of Streptococcus thermophilus was developed. The designed real-time PCR primers and probe were specific for S. thermophilus JCM20026, LMG6896, LMG18311, OJT101, OJT102 but not Enteroccocus spp., Lactococcus lactis subsp. lactis, and Streptococcus salivarius which are phylogenetically closely related to S. thermophilus and are difficult to identify using culture-based methods. The linear range of the developed real-time PCR method was from 2.7 to 8.6 log CFU ml^?1 with an amplification efficiency of 96%. Minor differences (about 0.4 log CFU ml^?1) were observed between counts of S. thermophilus obtained by culture and real-time PCR method in plain yoghurt and yoghurt containing fruits. Therefore, the developed real-time PCR method could be of potential application in specific detection and accurate enumeration of S. thermophilus in a wide range of dairy products.     

An ecological perspective on smoking among Asian American college students: The roles of social smoking and smoking motives.

Using electronic diaries, the present study examined the roles of social smoking and smoking motives in relation to cigarette use patterns among Asian American college smokers. Multilevel modeling results showed that participants smoked more cigarettes when smoking with peers than when smoking alone. Participants' coping (but not social) motives moderated the within-person associations between smoking with peers and the cigarettes smoked during a smoking episode. The findings support the utility of an ecological perspective in examining the dynamic interaction between smoking motives and the social settings of cigarette use, and call for further research on the social smoking behaviors in diverse populations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Induced albumin secretion from HepG2 spheroids prepared using poly(ethylene glycol) derivative with oleyl groups

We developed a poly(ethylene glycol) (PEG) derivative with oleyl groups, so-called “cell adhesive”, for the promotion of human hepatocellular carcinoma HepG2 cell spheroids. Our approach was based on crosslinking of the cell membrane with a cell adhesive via a hydrophobic interaction. A cell adhesive, PEG derivative with hydrophobic oleyl groups at both ends was synthesized and characterized. HepG2 spheroids formed when the adhesive was added to cell suspensions. The size of the spheroids increased with time in culture. In addition, Ammonia elimination of HepG2 spheroid with cell adhesive was 3.4 times higher than that without cell adhesive. Furthermore, albumin secretion from HeG2 spheroids grown with the cell adhesive for 7 days was 3.3 times that from HepG2 spheroids grown without cell adhesive. Fluorescence microscopy showed greater albumin staining in spheroids grown with cell adhesive compared with spheroids grown without adhesive. This cell adhesive may be useful not only for single type of cells but also for multi types of cells to form artificial organs. This cell adhesive will be a key material for liver tissue engineering when it will apply to primary hepatocytes.     

Formation of Aluminosilicate Glasses Containing Rare-Earth Oxides

Formation of aluminosilicate glasses containing oxides of rare-earth elements, e.g. Sc, Ce, Pr, Nd, Eu, Gd, Tb, Dy, Ho, Er, and Yb, was studied by melting at 1550°C and air quenching. The upper limit of the amount of rare-earth oxide which can be incorporated into the aluminosilicate glasses decreases according to the lanthanide contraction.