Molecular characterization and secreted production of basidiomycetous cell-bound β-glycosidases applicable to production of galactooligosaccharides

Cell-bound β-glycosidases of basidiomycetous yeasts show promise as biocatalysts in galactooligosaccharide (GOS) production. Using degenerated primers designed from Hamamotoa singularis (Hs) bglA gene, we newly identified three genes that encode cell-bound β-glycosidase from Sirobasidium magnum (Sm), Rhodotorula minuta (Rm), and Sterigmatomyces elviae (Se). These three genes, also named bglA, encoded family 1 glycosyl hydrolases with molecular masses of 67‒77 kDa. The BglA enzymes were approximately 44% identical to the Hs-BglA enzyme and possessed a unique domain at the N-terminus comprising 110 or 210 amino acids. The Sm-, Rm-, and Se-BglA enzymes as well as the Hs-BglA enzyme were successfully produced by recombinant Aspergillus oryzae, and all enzymes were entirely secreted to the supernatants. Furthermore, addition of some nonionic detergents (e.g. 0.4% [v/v] Triton-X) increased the production, especially of the Hs- or Se-BglA enzyme. Out of the BglA enzymes, the Se-BglA enzyme showed remarkable thermostability (∼70°C). Additionally, the Sm- and Se-BglA enzymes had better GOS yields, so there was less residual lactose than in others. Accordingly, the basidiomycetous BglA enzymes produced by recombinant A. oryzae would be applicable to GOS production, and the Se-BglA enzyme appeared to be the most promising enzyme for industrial uses.     

Heparin-binding EGF-like growth factor in the human prostate: Synthesis predominantly by interstitial and vascular smooth muscle cells and action as a carcinoma cell mitogen

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth. J. Cell. Biochem. 68:328-338, 1998. © 1998 Wiley-Liss, Inc.     

Effect of breakwater restoration work following the subsidence caused by the 2011 Tohoku Earthquake on the subtidal kelp population

We conducted a 5‐year monitoring survey of a population of Eisenia bicyclis kelp at a coastal area that suffered subsidence from the 2011 earthquake and the subsequent breakwater restoration work. Due to subsidence, the former intertidal area sank to the subtidal zone, therefore, the density of E. bicyclis increased because of many recruits. However, after the restoration work, the area harboring the recruits was destroyed and landfilled. In an area that sank below the pre‐subsidence border of the kelp distribution, recruits were rare. Furthermore, an indirect effect of the restoration work was also observed on recruitment in the intact offshore kelp zone. Our results showed that the breakwater restoration work following subsidence had negative effects, not only to the density but also to the age structure of the E. bicyclis population.     

Maintenance of Complex Life Cycles Via Cryptic Differences In The Ecophysiology Of Haploid And Diploid Spores Of An Isomorphic Red Alga1

Recognition of the wide diversity of organisms that maintain complex haploid–diploid life cycles has generated interest in understanding the evolution and persistence of such life cycles. We empirically tested the model where complex haploid–diploid life cycles may be maintained by subtle/cryptic differences in the vital rates of isomorphic haploid–diploids, by examining the ecophysiology of haploid tetraspores and diploid carpospores of the isomorphic red alga Chondrus verrucosus. While tetraspores and carpospores of this species did not differ in size or autofluorescence, concentrations of phycobiliproteins of carpospores were greater than that of tetraspores. However, tetraspores were more photosynthetically competent than carpospores over a broader range of photosynthetic photon flux densities (PPFDs) and at PPFDs found at both the depth that C. verrucosus is found at high tide and in surface waters in which planktonic propagules might disperse. These results suggest potential differences in dispersal potential and reproductive success of haploid and diploid spores. Moreover, these cryptic differences in ecological niche partitioning of haploid and diploid spores contribute to our understanding of some of the differences between these ploidy stages that may ultimately lead to the maintenance of the complex haploid–diploid life cycle in this isomorphic red alga.     

Elucidation of gastrointestinal dysfunction in response to irradiation using metabolomics

Gastrointestinal toxicity is frequently observed secondary to accidental or therapeutic radiation exposure. However, the variation in the intestinal metabolites after abdominal radiation exposure remains ambiguous. In the present study, C57BL/6 mice were exposed to 0, 2, and 20 Gy irradiation dose. The Head and chest of each mouse were covered with a lead shield before x-ray irradiation. 24 h post-irradiation treatment, intestinal tissue of each mouse was excised and prepared for metabolites measurement using gas chromatography-mass spectrometry (GC-MS). Our comprehensive analysis of metabolites in the intestinal tissues detected 44 metabolites after irradiation, including amino acids, carbohydrates, organic acids, and sugars. Amino acid levels in the intestinal tissue gradually rose, dependent on the radiation dose, perhaps as an indication of oxidative stress. Our findings raise the possibility that amino acid metabolism may be a potential target for the development of treatments to alleviate or mitigate the harmful effects of oxidative stress-related gastrointestinal toxicity due to radiation exposure.     

Tooth Size Variation in Pinniped Dentitions

It is contentious whether size variation among mammalian teeth is heterogeneous or homogeneous, whether the coefficient of variation is reliable, and whether the standard deviation of log-transformed data and the residual of standard deviation on mean variable size are useful replacements for the coefficient of variation. Most studies of tooth size variation have been on mammals with complex-crowned teeth, with relatively little attention paid to taxa with simple-crowned teeth, such as Pinnipedia. To fill this gap in knowledge and to resolve the existing controversies, we explored the variation of linear size variables (length and width) for all teeth from complete permanent dentitions of four pinniped species, two phocids (Histriophoca fasciata, Phoca largha) and two otariids (Callorhinus ursinus, Eumetopias jubatus). Size variation among these teeth was mostly heterogeneous both along the toothrow and among species. The incisors, canines, and mesial and distal postcanines were often relatively highly variable. The levels of overall dental size variation ranged from relatively low as in land carnivorans (Phoca largha and both otariids) to high (Histriophoca fasciata). Sexual size dimorphism varied among teeth and among species, with teeth being, on average, larger in males than in females. This dimorphism was more pronounced, and the canines were larger and more dimorphic relative to other teeth in the otariids than in the phocids. The coefficient of variation quantified variation reliably in most cases. The standard deviation of log-transformed data was redundant with the coefficient of variation. The residual of standard deviation on mean variable size was inaccurate when size variation was considerably heterogeneous among the compared variables, and was incomparable between species and between sexes. The existing hypotheses invoking developmental fields, occlusal complexity, and the relative timing of tooth formation and sexually dimorphic hormonal activity do not adequately explain the differential size variation along the pinniped toothrow.     

Synthesis of guanine nucleosides by fusion,and a mechanistic aspect of the reaction

N²-Acetylguanine (1) was condensed by fusion with the fully acetylated derivatives of the following sugars: β-D-ribofuranose (2), β-D-ribopyranose (3), α-D-xylopyranose (4), β-D-xylopyranose (5), α-D-glucopyranose (6), and β-D-gluco-pyranose (7). The reaction of 1 with either 2 or 3 gave a mixture of 7-β, 9-α, and 9-β isomers, whereas only the 7-β and 9-β isomers, and virtually no 9-α isomer, were obtained when 4, 5, 6, and 7 were used. When each isomeric acetylated ribofuranosylguanine was heated in the presence of an acidic catalyst, a mixture of 7-β, 9-α, and 9-β nucleosides was formed. Close examination of the product ratios showed that the ratio of 7:9 isomers remained unchanged throughout the reactions, but the anomeric nature of the 9-substituted nucleoside was dependent on the sugar used.     

Immunological identification of soluble carbonyl reductase with testosterone 17β-dehydrogenase (NADP+) in guinea pig liver and kidney

Antiserum was produced against one of two carbonyl reductases purified from guinea pig liver cytosol to identify the enzymes as testosterone 17β-dehydrogenase isozymes. Immunoelectrophoresis and immunoprecipitation with the antiserum indicated that the two reductases had common antigenic sites. The antiserum inhibited most of both carbonyl reductase and testosterone 17β-dehydrogenase activities in the purified reductases and in cytosols of liver and kidney.