Bifunctional properties and characterization of a novel sialidase with esterase activity from Bifidobacterium bifidum

ABSTRACT

Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189 kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity.

Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose     

ATP-induced calcium oscillations and change of P2Y subtypes with culture conditions in HeLa cells

ATP, UTP, ADP and UDP induced intracellular Ca(2+) responses and oscillations in HeLa cells that sometimes lasted over 1 h. The response is due to the activation of P2Ys, G-protein coupled ATP receptors, because the oscillations persisted for several minutes even in Ca(2+)-free solution, and suramin and PPADS, antagonists of ATP receptors, partially inhibited the response. The potency of these nucleotides varied with the culture or cell conditions, i.e. UTP was generally most potent but in some cases UDP was more potent; responses to UDP were variable while those to ATP were constant. In addition, Ca(2+) responses to ATP and UDP were additive. These findings suggested the existence of two or more subtypes of P2Ys in HeLa cells. RT-PCR experiments revealed the existence of P2Y(2), P2Y(4) and P2Y(6). Recovery from starvation (culture in FBS-free medium overnight and re-addition of FBS) increased the responses to UTP and UDP but not to ATP, suggesting that the number or activity of P2Y(6) and/or P2Y(4) receptors may increase with cell proliferation in HeLa cells.     

铜绿假单胞菌PIC－N萘降解基因的研究

铜绿假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉｎｏｓａ）ＰＩＣ－Ｎ对萘、邻苯二甲酸、水杨酸等有较强的氧化能力。发现该菌株以禁为底物可诱导产生芳香烃分解酶系。菌株中存在一个５７．４ｋｂ的质粒,经限制性内切酶ＨｉｎｄⅢ处理可产生７个片段,用限制性内切酶ＥｃｏＲⅠ处理可产生８个片段。将以限制性内切酶ＨｉｎｄⅢ部分酶切的片段克隆至大肠杆菌质粒ｐＭＦＹ４３上,获得２９个克隆株。通过对含有菌株ＰＩＣ－Ｎ质粒ＨｉｎｄⅢ片段的７个重组质粒进行限制酶分析,绘出了该质粒ＨｉｎｄⅢ内切酶７个切点的酶切图谱。     

Higher plant RecA-like protein is homologous to RadA

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.     

The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides.The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.     

Preparation of human immune effector T cells containing iron-oxide nanoparticles

Preparation of human immune T cells containing iron-oxide nanoparticles was carried out for the development of magnetically mediated immunotherapy. Peripheral blood lymphocytes (PBLs) after the incubation with magnetite nanoparticles were found to contain measurable ferric ions, which suggested the incorporation of magnetite nanoparticles. Transmission electron microscopic (TEM) study indicated that the incorporation of magnetite nanoparticles was mediated by endocytosis of PBLs. Furthermore, the effects of dosages and diameter of magnetite nanoparticles on the magnetite incorporation were investigated, and it was demonstrated that the increase in dosage promoted the incorporation of nanoparticles and the uptake into PBLs was more effective for magnetite nanoparticles, which formed smaller aggregations in medium. Finally, the demonstration of magnetite incorporation into enriched T cells and tumor antigen-specific cytotoxic T lymphocyte (CTL) line promises the achievement of magnetically mediated immunotherapy with tumor-specific CTLs containing magnetic nanoparticles.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Exotic species of freshwater decapod crustaceans in the state of São Paulo,Brazil: records and possible causes of their introduction

Based on recent surveys of the freshwater decapod fauna, distributional data of five exotic species of freshwater decapod crustaceans for the hydrographic basins of the state of São Paulo are presented, as part of a large initiative for a comprehensive survey of the state’s biodiversity (BIOTA-FAPESP Program). These species are the North American crayfish Procambarus clarkii (Girard) (Cambaridae), the crab Dilocarcinus pagei Stimpson (Trichodactylidae) from the Amazon and Paraguay/lower Paraná River Basins, and the palaemonid shrimps Macrobrachium rosenbergii (De Man), from the Indo-Pacific region, Macrobrachium amazonicum (Heller) and Macrobrachium jelskii (Miers), both from the Orinoco, Amazon and the Paraguay/lower Paraná River Basins. Possible modes by which their introduction might have occurred are commented upon and potential consequences are discussed.     

A social network analysis of treatment discoveries in cancer

Controlled clinical trials are widely considered to be the vehicle to treatment discovery in cancer that leads to significant improvements in health outcomes including an increase in life expectancy. We have previously shown that the pattern of therapeutic discovery in randomized controlled trials (RCTs) can be described by a power law distribution. However, the mechanism generating this pattern is unknown. Here, we propose an explanation in terms of the social relations between researchers in RCTs. We use social network analysis to study the impact of interactions between RCTs on treatment success. Our dataset consists of 280 phase III RCTs conducted by the NCI from 1955 to 2006. The RCT networks are formed through trial interactions formed i) at random, ii) based on common characteristics, or iii) based on treatment success. We analyze treatment success in terms of survival hazard ratio as a function of the network structures. Our results show that the discovery process displays power law if there are preferential interactions between trials that may stem from researchers' tendency to interact selectively with established and successful peers. Furthermore, the RCT networks are "small worlds": trials are connected through a small number of ties, yet there is much clustering among subsets of trials. We also find that treatment success (improved survival) is proportional to the network centrality measures of closeness and betweenness. Negative correlation exists between survival and the extent to which trials operate within a limited scope of information. Finally, the trials testing curative treatments in solid tumors showed the highest centrality and the most influential group was the ECOG. We conclude that the chances of discovering life-saving treatments are directly related to the richness of social interactions between researchers inherent in a preferential interaction model.     

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.