Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

Biosynthesis of dothistromin

Dothistromin is a mycotoxin that is remarkably similar in structure to versicolorin B, a precursor of both aflatoxin and sterigmatocystin. Dothistromin-producing fungi also produce related compounds, including some aflatoxin precursors as well as alternative forms of dothistromin. Dothistromin is synthesized by pathogenic species of Dothistroma in the red bands of pine needles associated with needle blight, but is also made in culture where it is strongly secreted into the surrounding medium. Orthologs of aflatoxin and sterigmatocystin biosynthetic genes have been found that are required for the biosynthesis of dothistromin, along with others that are speculated to be involved in the same pathway on the basis of their sequence similarity to aflatoxin genes. An epoxide hydrolase gene that has no homolog in the aflatoxin or sterigmatocystin gene clusters is also clustered with the dothistromin genes, and all these genes appear to be located on a minichromosome in Dothistroma septosporum. The dothistromin genes are expressed at an early stage of growth, suggesting a role in the first stages of plant invasion by the fungus. Future studies are expected to reveal more about the role of dothistromin in needle blight and about the genomic organization and expression of dothistromin genes: these studies will provide for interesting comparisons with these aspects of aflatoxin and sterigmatocystin biosynthesis.     

The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases.

A method to characterize plant cell wall polysaccharides is presented. The complexity of the polymer structures and the large number of different charged and uncharged monosaccharides that make up plant polysaccharides have previously made analysis technically demanding and laborious. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars and oligosaccharides with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. We show that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations. In combination with specific hydrolases, it can be used to analyze the structure of polysaccharides. Moreover, the specificity and kinetics of the plant polysaccharide hydrolases themselves can be quickly and effectively studied. PACE can detect as little as 500 fmol of monosaccharides and 100 fmol of oligosaccharides, and it is fast and quantitative.     

蜜环菌糖苷水解酶家族基因在菌索与菌丝间的差异表达分析

蜜环菌是一种兼性腐生和寄生的真菌,通过降解伴栽基质并为药用植物(天麻)或菌物(猪苓)提供营养物质,而糖苷水解酶是这一过程的主要酶类.本研究从蜜环菌Armillaria mellea 541菌株转录数据库中共获得糖苷水解酶家族基因170个,分布于39个亚家族.进一步分析发现,这些家族基因编码的糖苷水解酶家族蛋白(glyc...     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

栽植生姜对不同种植模式下紫色土微生物生物量及水解酶活性的影响

研究了岷江下游紫色丘陵区玉米+红薯间作、大豆单作、生姜连作、水稻-紫云英轮作等4个典型种植模式下栽植生姜后土壤微生物生物量碳、氮、磷含量和水解酶活性的变化特征.结果表明： 栽植生姜显著降低了4个种植模式下土壤微生物生物量碳、氮和磷含量,但各种植模式之间存在较大差异.其中,玉米+红薯间作和水稻-紫云英轮作模式下土壤微生物生物量碳、氮的下降幅度明显低于大豆单作与生姜连作模式,但土壤微生物生物量磷下降幅度明显较高.栽植生姜显著降低了土壤酸性磷酸酶活性,其下降幅度以玉米+红薯间作模式最大,水稻-紫云英轮作模式最小;土壤转化酶活性在生姜连作模式下显著降低;土壤脲酶活性在大豆单作、生姜连作和水稻-紫云英轮作模式下均显著降低.相对于其他模式,栽植生姜使玉米+红薯间作模式下的土壤维持了较高的转化酶和脲酶活性.     

HER-2/neu-mediated down-regulation of biglycan associated with altered growth properties

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-β(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ～5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.