Structure of Novel Enzyme in Mannan Biodegradation Process 4-O-β-d-Mannosyl-d-Glucose Phosphorylase MGP

The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-d-mannosyl-d-glucose and phosphate, and the product d-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Transient and permanent gene transfer into the brain of the teleost fish medaka (Oryzias latipes) using human adenovirus and the Cre-loxP system

In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the β-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae.     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Oxidative modification of proteasome: identification of an oxidation-sensitive subunit in 26 S proteasome

Reactive oxygen species (ROS) have the potential to damage cellular components, such as protein, resulting in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress, J. Biol. Chem. 276, 12076-12083]. In the present study, to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we analyzed the protein carbonylation on SH-SY5Y cells when these cells were submitted to an endogenous inducer of ROS production. Upon exposure of SH-SY5Y cells to this endogenous electrophile, we observed significant accumulation of protein carbonyls within the cells. Proteomic analysis of oxidation-sensitive proteins showed that the major intracellular target of protein carbonylation was one of the regulatory subunits in 26 S proteasome, S6 ATPase. Accompanied by a dramatic increase in protein carbonyls within S6 ATPase, the electrophile-induced oxidative stress exerted a significant decrease in the S6 ATPase activities and a decreased ability of the 26 S proteasome to degrade substrates. Moreover, in vitro oxidation of 26 S proteasome with a metal-catalyzed oxidation system also confirmed that S6 ATPase represents the most oxidation-sensitive subunit in the proteasome. These and the observation that down-regulation of S6 ATPase by RNA interference resulted in the enhanced accumulation of ubiquitinated proteins suggest that S6 ATPase is a molecular target of ROS under conditions of electrophile-induced oxidative stress and that oxidative modification of this regulatory subunit of proteasome may be functionally associated with the altered recognition and degradation of proteasomal substrates in the cells.     

TMPRSS13, a type II transmembrane serine protease, is inhibited by hepatocyte growth factor activator inhibitor type 1 and activates pro-hepatocyte growth factor

Type II transmembrane serine proteases (TTSPs) are structurally defined by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain. The human TTSP family consists of 17 members. Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigenesis and viral infections. The activities of TTSPs are regulated by endogenous protease inhibitors. However, protease inhibitors of most TTSPs have not yet been identified. In this study, we investigated the inhibitory effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family. We found that the protease activity of a member, TMPRSS13, was inhibited by HAI-1. A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2). In addition, an in vitro protein binding assay showed that NK1 formed complexes with TMPRSS13, but NK1LK2 did not. TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited by NK1. The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2. These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1.     

Computational analyses reveal spatial relationships between nuclear pore complexes and specific lamins

Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D–structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna^−/− and Lmnb1^−/− fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.