4-O-Methyl Modifications of Glucuronic Acids in Xylans Are Indispensable for Substrate Discrimination by GH67 α-Glucuronidase from Bacillus halodurans C-125

A GH67 α-glucuronidase gene derived from Bacillus halodurans C-125 was expressed in E. coli to obtain a recombinant enzyme (BhGlcA67). Using the purified enzyme, the enzymatic properties and substrate specificities of the enzyme were investigated. BhGlcA67 showed maximum activity at pH 5.4 and 45 °C. When BhGlcA67 was incubated with birchwood, oat spelts, and cotton seed xylan, the enzyme did not release any glucuronic acid or 4-O-methyl-glucuronic acid from these substrates. BhGlcA67 acted only on 4-O-methyl-α-D-glucuronopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (MeGlcA³Xyl₃), which has a glucuronic acid side chain with a 4-O-methyl group located at its non-reducing end, but did not on β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronopyranosyl-(l→2)]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylop- yranose (MeGlcA³Xyl₄) and α-D-glucuronopyranosyl-(l→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (GlcA³Xyl₃). The environment for recognizing the 4-O-methyl group of glucuronic acid was observed in all the crystal structures of reported GH67 glucuronidases, and the amino acids for discriminating the 4-O-methyl group of glucuronic acid were widely conserved in the primary sequences of the GH67 family, suggesting that the 4-O-methyl group is critical for the activities of the GH67 family.     

Effects of aspect ratio on mixed convection in a horizontal rectangular duct with heated and cooled side walls

This paper describes the effect of aspect ratio on mixed convection in a horizontal rectangular duct with heated and cooled side walls numerically and experimentally. In the numerical analysis, fluid flow and temperature distributions for Ri=1.61, Pr=6.99, Re=100 and aspect ratio, Ar=0.2–10, were obtained by solving dimensionless governing equations using the SIMPLE procedure. The QUICK scheme was applied to the convective term of these equations. In the experimental analysis, the flow behavior for A_r=0.5–2 was visualized by the dye‐injection method. Numerical results showed that the swirl flow was generated along the flow direction, and its pitch length was influenced by A_r. The pitch length was the shortest when A_r=0.5–1, and this tendency was the same in numerical results and experimental results. The heat transfer behavior was also discussed corresponding to the flow, and the heat transfer ratio was highest at A_r=1 in 0.2 ≤ A_r ≤ 10. © 2012 Wiley Periodicals, Inc. Heat Trans Asian Res; Published online in Wiley Online Library ( wileyonlinelibrary.com/journal/htj ). DOI 10.1002/htj.20391     

Species Identification and PCR-RFLP Analysis of Cytochrome b Gene in Cod Fish (Order Gadiformes) Products

ABSTRACT: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiles using the PCR product tRNA^Glu-cytochrome b of 9 standard cod fish, all of which are imported into Japan under the name "cod fish," were investigated as a means of rapid species identification of imported cod fish products. Endonuclease digestion using 4 restriction enzymes ( Alu I, Fok I, Mbo I, and Mse I) generated different digestion patterns for the standards, enabling species identification in imported cod fish products by comparison to the standards'RFLP profiles. Additionally, we conducted a more strict check by phylogenetic analysis on imported cod fish products with 13 standards (including 4 additional cod species from GenBank) using the mitochondrial cytochrome b gene (385 nt). Consequently, it was found that the PCR-RFLP method was sufficient for rapidly screening cod products, and in cases that require conclusive identification, phylogenetic analysis was the most suitable.     

Deciphering cellular functions of protein phosphatases by comparison of gene expression profiles in Saccharomyces cerevisiae

Expression profiles of protein phosphatase (PPase) disruptants were analyzed by use of Pearson's correlation coefficient to find profiles that correlated with those of 316 Reference Gene (RG) disruptants harboring deletions in genes with known functions. Twenty-six Δppase disruptants exhibited either a positive or negative correlation with 94 RG disruptants when the p value for Pearson's correlation coefficient was > 0.2. Some of the predictions that arose from this analysis were tested experimentally and several new Δppase phenotypes were found. Notably, Δsit4 and Δsiw14 disruptants exhibited hygromycin B sensitivity, Δsit4 and Δptc1 disruptants grew slowly on glycerol medium, the Δptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Δppg1 disruptant was found to be sensitive to congo red. Because on-going analysis of expression profiles of Saccharomyces cerevisiae disruptants is rapidly generating new data, we suggest that the approach used in the present study to explore PPase function is also applicable to other genes.     

Effects of long-term ingestion of difructose anhydride III (DFA III) on intestinal bacteria and bile acid metabolism in humans

Changes in the intestinal microbiota of 10 human subjects with long-term ingestion of 3 g/d difructose anhydride III (DFA III; 4 persons, 2 months; 3 persons, 6 months; and 3 persons, 12 months) were examined by denaturing gradient gel electrophoresis (DGGE). According to the answers to questionnaires, the subjects were divided into two groups (constipated and normal). The DGGE profile was different for every individual and each subject had unique profiles of intestinal microbiota. In the DGGE profiles of constipated subjects, the intensities of bands related to Bacteroides spp. increased. Moreover, the DFA III-assimilating bacteria, Ruminococcus sp. were isolated from subjects who ingested DFA III for 12 months. These strains showed 95% similarity of their 16S rDNA sequences with that of Ruminococcus obeum ATCC 29174(T) (X85101) and produced large amounts of acetic acid. DFA III ingestion for 2 months tended to increase total organic acids in feces, and tended to decrease fecal pH and the secondary bile acid (SBA) ratio in total bile acids. The SBA ratio in total bile acids corresponded to fecal pH. The production of SBA was decreased by low pH in vitro. These results indicated that DFA III ingestion in humans tended to lower intestinal pH, inhibited bile acid 7alpha-dehydroxylation activities and also tended to decrease the SBA ratios in total bile acids. Moreover, as another cause for the decrease in the SBA ratio in total bile acids, it was suggested that the number of bile acid 7alpha-dehydroxylating bacteria were decreased by DFA III ingestion.     

Perfluorinated contaminants in sediments and aquatic organisms collected from shallow water and tidal flat areas of the Ariake Sea, Japan: environmental fate of perfluorooctane sulfonate in aquatic ecosystems

Perfluorinated compounds (PFCs), such as perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorohexane sulfonate (PFHS), and perfluorooctane sulfonamide (PFOSA) are widely distributed in aquatic ecosystems. Despite studies reporting the occurrence of PFCs in aquatic organisms, the fate of PFCs in tidal flat and marine coastal ecosystems is not known. In this study, we determined concentrations of PFOS, PFOA, PFNA, PFHS, and PFOSA in sediments; benthic organisms, including lugworm, mussel, crab, clam, oyster, and mudskipper fish from tidal flat; and shallow water species, such as filefish, bream, flounder, shark, finless porpoise, gull, and mallard collected from the Ariake Sea, Japan. PFOS and PFOA were detected in most of the samples analyzed, followed by PFNA, PFOSA, and PFHS. In shallow water species, PFOS was the dominant contaminant, and elevated concentrations were found in higher trophic level species, such as marine mammals and omnivorous birds. These results suggest biomagnification of PFOS through the coastal food chain. In contrast, PFOA was the most abundant compound in tidal flat organisms and sediments. PFOA concentrations in sediments, lugworms, and omnivorous mudskippers in tidal flat were approximately 1 order of magnitude greater than the levels of PFOS. This indicates differences in exposure pattern and bioavailability of PFOS and PFOA between shallow water and tidal flat organisms. The accumulation profiles of PFCs were compared with those of organochlorines (polychlorinated biphenyls, PCB), organotin (tributyltin,TBT), and polycyclic aromatic hydrocarbons (PAHs) in tidal flat and shallow water samples collected from the Ariake Sea. Concentrations of PFCs in sediments and in tidal flat organisms were significantly lower than that found for PCBs, TBT, and PAHs. Nevertheless, PFOS concentrations in shallow water species were comparable to and/or significantly greater than those of other classes of contaminants. This implies that the aqueous phase is a major sink for PFCs, which is different from what was observed for nonpolar organic pollutants.     

Refined method for the genomic integration of complex synthetic circuits

Genetic reconstruction of regulatory gene circuits is currently applied in systematic dynamics and structure–function studies of intact cellular networks in systems biology. We present a modified procedure for the integration of complex genetic circuits into the Escherichia coli genome, to provide an efficient synthetic approach for stochastic study and the artificial engineering of genetic networks. Linear artificial sequences of various lengths were easily integrated into the bacterial genome at one time. Comparison of the cellular concentrations of proteins encoded by genes carried on plasmids or the genome indicated that genome recombination could minimize the copy number noise in the genetic circuit, allowing precise design and interpretation of the cellular network. The refined recombination procedure allowed efficient construction of a single copy of a complex genetic circuit in cells, and the resultant reduced fluctuation in copy number led to accurate phenotypic behaviour of the genome-integrated synthetic switch corresponding to the design principle. The availability of long-fragment insertions makes the reconstruction of complex networks easy on the genome, and provides a powerful tool for precise engineering in synthetic and systems biology.     

Mating-induced cumulus-oocyte maturation in the shrew, Suncus murinus

The musk shrew, Suncus murinus, is an induced ovulator, the cumulus oophorus of which is unusual in several respects: it has no matrix, it always induces the acrosome reaction and it appears to be essential for fertilization. The present study documents distinctive features of the cumulus oophorus before and after ovulation, and of copulation-induced maturation of the ovulatory follicle, which has no antrum. In unmated females, potentially responsive ovarian follicles are distinguishable from large secondary follicles by differentiation of the granulosa into outer and inner cell layers, the latter being characterized particularly by intracellular glycogen deposits. The average number of responsive follicles equates with the number that ovulate. By about 10 h after mating, meiosis has reached metaphase II, with extrusion of the first polar body. Coincidentally, a cavity has developed between the inner and outer follicular layers, demarcating the smaller cells of the granulosa from the glycogen-rich cells of the cumulus oophorus. Subsequently, the glycogen becomes restricted primarily to the inner cumulus, and the corona cells began to retreat from the zona pellucida surface to form an unusual very distinct perizonal space that is clearly evident at the time of ovulation. The cumulus is stabilized by gap and tight junctions, and presents a smooth external surface that appears to initiate the acrosome reaction. After fertilization, at which time the zona pellucida becomes more resistant to both trypsin and dithiothreitol, the cumulus develops intercellular lacunae, and is eventually discarded about 15 h after ovulation.