Immunohistochemical, in situ hybridization and biochemical studies on endogenous cellulase of Corbicula japonica

We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.     

Intraperitoneal fluid accumulation induced by Clostridium perfringens alpha-toxin (phospholipase C)

We report that the intraperitoneal injection of Clostridium perfringens alpha-toxin into mice induces ascites. This phenomenon was monitored by measuring fluid volume and analyzing hematologic data. The mouse toxicity test provides a simple and useful model for examining C. perfringens alpha-toxin-induced vascular permeability.     

Mycolyltransferase-mediated glycolipid exchange in Mycobacteria

Trehalose dimycolate (TDM), also known as cord factor, is a major surface glycolipid of the cell wall of mycobacteria. Because of its potent biological functions in models of infection, adjuvancy, and immunotherapy, it is important to determine how its biosynthesis is regulated. Here we show that glucose, a host-derived product that is not readily available in the environment, causes Mycobacterium avium to down-regulate TDM expression while up-regulating production of another major glycolipid with immunological roles in T cell activation, glucose monomycolate (GMM). In vitro, the mechanism of reciprocal regulation of TDM and GMM involves competitive substrate selection by antigen 85A. The switch from TDM to GMM biosynthesis occurs near the physiological concentration of glucose present in mammalian hosts. We further demonstrate that GMM is produced in vivo by mycobacteria growing in mouse lung. These results establish an enzymatic pathway for GMM production. More generally, these observations provide a specific enzymatic mechanism for dynamic alterations of cell wall glycolipid remodeling in response to the transition from noncellular to cellular growth environments, including factors that are monitored by the host immune system.     

The chondroitin polymerase K4CP and the molecular mechanism of selective bindings of donor substrates to two active sites

Bacterial chondroitin polymerase K4CP is a multifunctional enzyme with two active sites. K4CP catalyzes alternative transfers of glucoronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to elongate a chain consisting of the repeated disaccharide sequence GlcAbeta1-3GalNAcbeta1-4. Unlike the polymerization reactions of DNA and RNA and polypeptide synthesis, which depend upon templates, the monosaccharide polymerization by K4CP does not. To investigate the catalytic mechanism of this reaction, we have used isothermal titration calorimetry to determine the binding of the donor substrates UDP-GlcA and UDP-GalNAc to purified K4CP protein and its mutants. Only one donor molecule bound to one molecule of K4CP at a time. UDP-GlcA bound only to the C-terminal active site at a high affinity (K(d)=6.81 microm), thus initiating the polymerization reaction. UDP-GalNAc could bind to either the N-terminal or C-terminal active sites at a low affinity (K(d)=266-283 microm) but not to both sites at the same time. The binding affinity of UDP-GalNAc to a K4CP N-terminal fragment (residues 58-357) was profoundly decreased, yielding the average K(d) value of 23.77 microm, closer to the previously reported K(m) value for the UDP-GalNAc transfer reaction that takes place at the N-terminal active site. Thus, the first step of the reaction appears to be the binding of UDP-GlcA to the C-terminal active site, whereas the second step involves the C-terminal region of the K4CP molecule regulating the binding of UDP-GalNAc to only the N-terminal active site. Alternation of these two specific bindings advances the polymerization reaction by K4CP.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses

Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml⁻¹ in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

Effect of vitamin C depletion on age-related hearing loss in SMP30/GNL knockout mice

Using senescence marker protein 30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), we examined whether modulating VC level affects age-related hearing loss (AHL). KO and wild-type (WT) C57BL/6 mice were given water containing 1.5 g/L VC [VC(+)] or 37.5 mg/L VC [VC(−)]. At 10 months of age, KO VC(−) mice showed significant reduction in VC level in the inner ear, plasma, and liver, increase in auditory brainstem response (ABR) thresholds, and decrease in the number of spiral ganglion cells compared to WT VC(−), WT VC(+), and KO VC(+) mice. There were no differences in VC level in the inner ear, ABR thresholds, or the number of spiral ganglion cells among WT VC(−), WT VC(+), and KO VC(+) mice. These findings suggest that VC depletion can accelerate AHL but that supplementing VC may not increase VC level in the inner ear or slow AHL in mice.