Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg²⁺ and Ca²⁺ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.     

Nesfatin-1 Induces the Phosphorylation Levels of cAMP Response Element-Binding Protein for Intracellular Signaling in a Neural Cell Line

Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30) and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE) reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB), without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK) kinase inhibitor and an L-type Calcium (Ca²⁺) channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that ¹²⁵I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons.     

Inhibition of epsilon-poly-L-lysine biosynthesis in Streptomycetaceae bacteria by short-chain polyols

Antimicrobial epsilon-poly-L-lysine (ePL) is secreted by Streptomycetaceae bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). In this study, by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.     

The Pinguiophyceae classis nova, a new class of photosynthetic stramenopiles whose members produce large amounts of omega-3 fatty acids

The Pinguiophyceae class. nov., a new class of photo‐synthetic stramenopiles (chromophytes), is described. The class includes five monotypic genera, Glossomastix, Phaeomonas, Pinguiochrysis (type genus), Pinguio‐coccus and Polypodochrysis. These algae have an unusually high percentage of polyunsaturated fatty acids, especially 20:5 (n‐3)(EPA, eicosapentaenoic acid). These fatty acids are the basis for choosing the Latin noun ‘Pingue’ (= fat, grease) as the root for the class name. Analyses of nuclear‐encoded 18S rRNA and chloroplast‐encoded rbcL gene sequence data showed that these algae formed a monophyletic group that could not be placed in any other class. Morphologically, the species are all single‐celled microalgae from picoplanktonic size to over 40 urn in length. Each cell has one (or two) typical chloroplast(s) with a girdle lamella and a surrounding chloroplast endoplasmic reticulum. Pyrenoids occur within the chloroplast, varying from embedded to stalked, and membranes penetrate into the pyrenoid in all five genera. Phaeomonas has motile cells with two flagella, and the forward‐directed flagellum bears mastigonemes (tripartite flagellar hairs). Two other genera (Glossomastix, Polypodochrysis) produce zoospores that possess only one smooth flagellum (no mastigonemes), and this flagellum apparently is the mature flagellum, a feature previously unknown in the photosynthetic stramenopiles. The major carotenoid pigments in the pinguiophytes are fucoxanthin, violaxanthin, zeaxanthin and P‐carotene, as well as chlorophyll a and chlorophyll c‐related pigment(s). These features support recognition of the Pinguiophyceae class. nov. as a unique group of algae.     

Tumor-derived microvesicles induce proangiogenic phenotype in endothelial cells via endocytosis

Background

Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear.

Methodology/Principal Findings

Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.

Conclusion

We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: Analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.     

Exogenously Hypercholesterolemic Rats, Compared with Their Progenitor Sprague-Dawley Rats, Have Altered mRNAs for Cholesterol 7α-Hydroxylase and Low-Density-Lipoprotein Receptor and Activities of Cholesterol 7α-Hydroxylase and Acyl-CoA:Cholesterol Acyltransferase in the Liver in Response to Dietary Cholesterol

Exogenously hypercholesterolemic (ExHC) rats promptly increase serum cholesterol concentration in response to dietary cholesterol. To examine underlying mechanism(s) for this susceptibility, responses of mRNAs for cholesterol metabolism-related proteins and their activities in the liver to dietary cholesterol were compared between ExHC rats and their progenitor Sprague-Dawley rats. ExHC rats slightly decreased the abundance of low-density-lipoprotein (LDL) receptor mRNA in response to dietary cholesterol, although the amount of LDL receptor was not influenced. The abundance of cholesterol 7α-hydroxylase mRNA and the enzyme activity in response to dietary cholesterol were greater in ExHC rats, but the fecal excretion of bile acid was comparable between the strain. Dietary cholesterol-dependent elevation of acyl-CoA:cholesterol acyltransferase activity was greater in ExHC rats. The concentration of liver triacylglycerols was markedly lower in ExHC rats. These results suggest that ExHC rats may increase serum cholesterol by increasing hepatic secretion of cholesteryl ester-rich particles.     

A Fine Physical Map of Arabidopsis thaliana Chromosome 5: Construction of a Sequence-ready Contig Map

A fine physical map of Arabidopsis thaliana chromosome 5 wasconstructed by ordering the clones from YAC, P1, TAC and BAClibraries of the genome using the sequences of a variety ofgenetic and EST markers and terminal sequences of clones. Themarkers used were 88 genetic markers, 13 EST markers, 87 YACend probes, 100 YAC subclone end probes, and 390 end probesof P1, TAC and BAC clones. The entire genome of chromosome 5,except for the centromeric and telomeric regions, was coveredby two large contigs 11.6 Mb and 14.2 Mb long separated by thecentromeric region. The minimum tiling path of the chromosomewas constituted by a total of 430 P1, TAC and BAC clones. Themap information is available at the Web site http://www.kazusa.or.jp/arabi/.