Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.     

麻蝇幼虫肠道蛋白酶BGP的分离纯化及性质

棕尾别麻蝇幼虫肠液经ＳＤＳ－ＰＡＧＥ后,Ｘ光片显影,呈现两条蛋白酶活性带．ＩＥＦ后,两条蛋白酶活性带的等电点分别为ｐＨ７．７和６．８．麻蝇幼虫肠液经５５％～７５％硫酸铵沉淀,以及连续两次制备等电聚焦,分离纯化出等电点约为ｐＨ７．７,分子量约为３５ｋＤ的蛋白酶ＢＧＰ．该酶能分解酪蛋白和类胰蛋白酶专一底物Ｂｚ－Ｐｈｅ－Ｖａｌ－ＡｒｇＮＡ,不能分解弹性蛋白酶专一底物ｅｌａｓｔｉｎ－ＣｏｎｇｏＲｅｄ和类胰凝乳蛋白酶专一底物Ｓｕｃ（Ａｌａ）２Ｐｒｏ－ＰｈｅＮＡ．ＳＢＢＩ,Ｌｅｕｐｅｐｔｉｎ和ＰＭＳＦ能强烈抑制其活性．专一底物和抑制剂的结果表明,ＢＧＰ是一种类胰蛋白酶．其最适反应温度为５０℃,最适作用ｐＨ为８．５．不耐高温,５０℃保温３０ｍｉｎ活性急剧下降．Ｈｇ２＋,Ｚｎ２＋和Ｃｕ２＋能抑制酶活性．Ｃａ２＋,Ｍｇ２＋对酶无激活作用,ＥＤＴＡ无抑制作用．     

Purification and partial characterization of a lysine-specific protease of Porphyromonas gingivalis

Abstract A lysine-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion-exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and the p I value was 7.3. The enzyme hydrolysed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Abstract Porphyromonas gingivalis culture supernate was found to induce hemotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of aglutination induced by the secretagogues PMA and FMLP. Lipopolysaccaharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors of PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity fot P. gingivalis cells but when PMN were treated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutinatin or enhanced adherence could also lead to decreased phagocytic capacity of the adherant or agglutinated cells.     

西北干旱区不同海拔高度地区气温变化对春小麦生长的影响

基于1981—2006年西北干旱区高海拔地（民乐站）和低海拔地（张掖站）农业气象观测站的观测资料,对民乐站和张掖站的气温变化及其对春小麦生长发育和产量的影响进行了研究.结果表明：研究期间,民乐站、张掖站的气温均呈增加趋势,且高海拔地区的增温幅度大于低海拔地区;民乐站春小麦对气温变暖的响〖JP2〗应表现为生育期缩短、产量增加,张掖站春小麦对气温变暖的响应表现为生育期缩短、产量下降;春小麦生育期内日均气温每升高1 ℃,民乐站和张掖站春小麦生育期分别缩短8.3 d和3.8 d.民乐站春小麦生育期内最高气温低于30.4 ℃时,春小麦生育期和产量均小幅增加,当超过30.4 ℃时,春小麦生育期缩短、产量下降.     

P38 mitogen-activated protein kinase pathways are involved in the hypertrophy and apoptosis of cardiomyocytes induced by Porphyromonas gingivalis conditioned medium

The surrounding medium of periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of p38 pathway in P. gingivalis conditioned medium-induced H9c2 myocardial cell hypertrophy and apoptosis. DNA fragmentation, cellular morphology, nuclear condensation, p38 protein products, and mitochondrial-dependent apoptotic related proteins in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, immunofluorescence, DAPI, and western blotting following P. gingivalis conditioned medium and/or pre-administration of SB203580 (p38 inhibitor). The p38 protein products and associated activities in H9c2 cells were both upregulated by P. gingivalis conditioned medium. P. gingivalis conditioned medium increased cellular sizes, DNA fragmentation, nuclear condensation, mitochondrial Bcl2-associated death promoter (Bad), cytosolic cytochrome c (cyt c), and the activated form of caspase-9 proteins in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, Bad, cyt c, and caspase-9 activities of H9c2 cells treated with P. gingivalis conditioned medium were all significantly reduced after pre-administration of SB203580. Our findings suggest that the activity of p38 signal pathway may be initiated by P. gingivalis conditioned medium and further activate mitochondrial-dependent apoptotic pathways leading to cell death in cultured H9c2 myocardial cells.     

咸水灌溉条件下土壤水盐分布特征

通过设置3种灌水量水平（100%ET_c、80%ET_c、60%ET_c）和3种灌水水质水平（0.7、3和6 g·L^-1）,研究了咸水灌溉条件下春小麦120 cm土层内水分动态和盐分累积特征.结果表明：水分在农田土壤中的分布主要受灌水量和土壤质地的影响,充分灌溉使水分存贮在较深土层中,而非充分灌溉则使水分存贮在表层;在相同灌水量的条件下,土体内的盐分积累程度随着灌溉水矿化度的增大而加剧;在相同矿化度条件下,土体内的盐分含量及积盐深度随着灌水量的增加而增大.在作物整个生育期内,连续使用咸水灌溉将导致土壤积盐,且非充分灌溉较充分灌溉更易使土壤表层积盐.     

好气条件下不同形态外源砷在土壤中的转化

在35%的田间持水量下,通过模拟试验研究了外源二甲基砷酸盐（DMA）、一甲基砷酸盐（MMA）、五价无机砷［As(V)］在土壤中的形态转化.结果表明: 外源砷进入土壤后,其含量均有随时间推延而逐渐下降的趋势,两种不同形态的有机砷DMA和MMA在土壤中主要发生脱甲基化过程,经150 d的恒温恒湿培养,其在土壤中主要转化为As(V),DMA处理仅在120 d时检测到少量MMA,而MMA处理则在7～60 d内均有少量的DMA生成.培养结束时土壤中DMA和MMA含量均显著降低(P<0.01),降幅分别为99.5%、94.3%,而两者的主要转化产物As(V)的含量则分别显著增加了4.61和5.15倍.表明外源有机态砷在土壤中基本上被转化为无机形态;与有机态外源砷相比,外源As(V)进入土壤后其形态基本上没有发生转化.     

Structure of the lysine specific protease Kgp from Porphyromonas gingivalis,a target for improved oral health

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell‐surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X‐ray crystal structure of the lysine‐specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.