Hybrid penicillin-binding proteins in penicillin-resistant strains of Neisseria gonorrhoeae

Benzylpenicillin has been used extensively for approximately 40 years in the treatment of gonorrhoea. The intense selective pressures resulting from the continual exposure of Neisseria gonorrhoeae to penicillin have resulted in the emergence of resistant strains that produce altered forms of penicillin-binding proteins (PBPs) with decreased affinity for the antibiotic. A comparison of the sequences of the PBP-2 genes from penicillin-sensitive and penicillin-resistant strains, suggests that penicillin-resistant forms of PBP 2 may have arisen both by amino-acid substitutions and insertions, and by the exchange of a region encoding part of the penicillin-sensitive transpeptidase domain with the homologous region from a closely related species.     

Reassortment of pilin genes in Neisseria gonorrhoeae occurs by two distinct mechanisms

Phase and antigenic variation of pilin expression in Neisseria gonorrhoeae result from recombination events in which variant sequences from one of the silent loci (pilS) are transferred to the expression locus (pilE). Such rearrangements were originally thought to be gene conversions, but findings showing that phase variation is partially inhibited by DNase I, that piliated (P+) cells are highly competent for DNA uptake and that gonococci readily undergo autolysis in culture, led to the suggestion that pilin variation occurs through transformation by exogenous DNA. We have developed a simple method for the selection of non-piliated (P-) cells and have evaluated naturally occurring P+ to P- transitions. Two primary pathways of pilin variation can be distinguished--transformation-mediated recombination, which is influenced by culture conditions and inhibited by DNase I, and intragenomic reciprocal recombination, which is unaffected by DNase I. Furthermore, we demonstrate that both piliated and revertible P- cells are competent for DNA uptake, an essential prerequisite of the first pathway.     

生长抑素对胰岛素分泌的调控机制研究

生长抑素(Somatostatins,SST)对胰腺β细胞胰岛素的分泌有重要的调节作用,这一调节作用与细胞内钙离子浓度变化相偶联.以大鼠胰腺β细胞为研究对象,采用显微荧光测钙技术和膜片钳技术,研究了胞外SST对胞内钙离子信号的影响,初步探讨了其作用机制.结果表明:在细胞外液有钙时,胞外SST可减少由去极化产生的胞外钙离子内流;而在细胞外液无钙时,胞外SST通过动员胞内钙库释放而引起胞浆内钙离子浓度显著增高,并触发胰岛素的分泌.     

Crystal structure of a retroviral protease proves relationship to aspartic protease family

Retroviral gag, pol and env gene products are translated as precursor polyproteins, which are cleaved by virus-encoded proteases to produce the mature proteins found in virions. On the basis of the conserved Asp-Thr/Ser-Gly sequence at the putative protease active sites, and other biochemical evidence, retroviral proteases have been predicted to be in the family of pepsin-like aspartic proteases. It has been suggested that aspartic proteases evolved from a smaller, dimeric ancestral protein, and a recent model of the human immunodeficiency virus (HIV) protease postulated that a symmetric dimer of this enzyme is equivalent to a pepsin-like aspartic protease. We have now determined the crystal structure of Rous sarcoma virus (RSV) protease at 3-A resolution and find it is dimeric and has a structure similar to aspartic proteases. This structure should provide a useful basis for the modelling of the structures of other retroviral proteases, such as that of HIV, and also for the rational design of protease inhibitors as potential antiviral drugs.     

Crystal structure of a rhomboid family intramembrane protease

Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and gamma-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 A resolution crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water molecules are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.     

COD质量浓度和水流剪切力对胞外聚合物分泌的影响

试验使用悬浮载体生物膜反应器,研究了COD质量浓度和水流剪切力对生物膜上中胞外多糖和胞外蛋白的影响.结果表明,胞外多糖和胞外蛋白的分泌量随着进水COD质量浓度的波动而变化,基质质量浓度的增加使生物膜上EPS的分泌增多.水流速度的提高促进了水流剪切力的增加;当水流速度从0.032 m/s,增加到0.056 m/s,然后在增加到0.089 m/s时,水流剪切力的提高促进了胞外多糖的分泌,微生物在经历一个短暂的适应阶段后胞外多糖的分泌量开始升高.生物膜重新形成和达到稳定后胞外多糖的分泌量又逐渐回落到一个稳定的水平.回落所用时间的长短和水流剪切力的大小有关.水流剪切力越大,回复所用的时间越长.水流剪切力的增加对胞外蛋白的影响较小.高的水流剪切力能够引起更多的生物膜脱落.     

Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.     

Crystal structure of the tricorn protease reveals a protein disassembly line.

The degradation of cytosolic proteins is carried out predominantly by the proteasome, which generates peptides of 7-9 amino acids long. These products need further processing. Recently, a proteolytic system was identified in the model organism Thermoplasma acidophilum that performs this processing. The hexameric core protein of this modular system, referred to as tricorn protease, is a 720K protease that is able to assemble further into a giant icosahedral capsid, as determined by electron microscopy. Here, we present the crystal structure of the tricorn protease at 2.0 A resolution. The structure reveals a complex mosaic protein whereby five domains combine to form one of six subunits, which further assemble to form the 3-2-symmetric core protein. The structure shows how the individual domains coordinate the specific steps of substrate processing, including channelling of the substrate to, and the product from, the catalytic site. Moreover, the structure shows how accessory protein components might contribute to an even more complex protein machinery that efficiently collects the tricorn-released products.     

Relationship among the secretion of extracellular polymeric substances,heat resistance,and bioleaching ability of Metallosphaera sedula

This paper describes the investigation of the secretion of extracellular polymeric substances(EPS) by an extremely thermoacidophilic archaea, Metallosphaera sedula(M. sedula), during the bioleaching of pyrite under different temperatures and discusses the relationship among the EPS secretion, its heat resistance, and its ability to bioleach pyrite. The investigation results indicate that the amount of extracellular proteins is significantly higher than the amount of extracellular polysaccharides in the extracted EPS whether free cells or attached cells; these results are quite different from the behavior of mesophilic Acidithiobacillus ferrooxidans. Although the growth of M. sedula is inhibited at 80℃, the bioleaching ability of M. sedula is only slightly lower than that at the optimum growth temperature of 72℃ because of the heat resistance mechanism based on EPS secretion. The secretion of more extracellular proteins is an important heat resistance mechanism of M. sedula.     

半知菌曲霉族真菌胞外多糖的研究Ⅱ:爪哇正青霉胞外多糖化学结构的研究

 经形态鉴定和18srRNA基因组序列分析,编号Ym31794菌株的分类学地位属于半知菌类丛梗孢科曲霉族青霉属丝状真菌,种名爪哇正青霉(Eupenicillium javanicum).该菌发酵液经离心弃菌体,除脂类及乙醇分级提取,分离、纯化的胞外多糖相对分子量为2.6×10⁴,糖的质量分数为89%.真菌胞外多糖经红外光谱(IR),簿层层析(TLC),色质联用(GC-MS),核磁共振(¹³C-NMR,¹H-NMR)等分析可知爪哇正青霉菌胞外多糖为1-6连接的葡萄糖和1-2连接半乳糖构成主干,侧链由1-3连接的甘露糖和1-2连接的葡萄糖构成,结构应为吡喃糖.     

Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.

The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibres more susceptible to necrosis.     

Relationship among the secretion of extracellular polymeric substances,heat resistance,and bioleaching ability of Metallosphaera sedula

This paper describes the investigation of the secretion of extracellular polymeric substances (EPS) by an extremely thermoacidophilic archaea, Metallosphaera sedula (M. sedula), during the bioleaching of pyrite under different temperatures and discusses the relationship among the EPS secretion, its heat resistance, and its ability to bioleach pyrite. The investigation results indicate that the amount of extracellular proteins is significantly higher than the amount of extracellular polysaccharides in the extracted EPS whether free cells or attached cells; these results are quite different from the behavior of mesophilic Acidithiobacillus ferrooxidans. Although the growth of M. sedula is inhibited at 80℃, the bioleaching ability of M. sedula is only slightly lower than that at the optimum growth temperature of 72℃ because of the heat resistance mechanism based on EPS secretion. The secretion of more extracellular proteins is an important heat resistance mechanism of M. sedula.     

Genomic structure of metabotropic glutamate receptor 7 and comparison of genomic structures of extracellular domains of mGluR family

Metabotropic glutamate receptor 7, coupled with a chemical neurotransmitter L-glutamate, plays an important role in the development of many psychiatric and neurological disorders. To study the biological and genetic mechanism of the mGluR7-related diseases, a physical map covering the full-length mGluR7 genomic sequence has been constructed through seed clone screening and fingerprinting database searching. These BAC clones in the physical map have been sequenced with shotgun strategy and assembled by Phred-Phrap-Consed software; the error rate of the final genomic sequence is less than 0.01%. mGluR7 spans 880 kb genomic region, the GC content and repeat content of mGluR7 genomic sequence are 38% and 37.5% respectively. mGluR7 has a typical “house-keeping” promoter and consists of 11 exons, with introns ranging from 6 kb to 285 kb. mGhiR7a and mGluR7b are two known alternatively splicing variants. Comparing the genomic structures of extracellular domains of mGluR family, their genomic structures can be subdivided into three groups, which are consistent with that of proteins. Although the genomic organization of mGluR7’s group is conserved, the majority of introns in the extracellular segments vary dramatically. It is an obvious trend of the increasing intron size inverse proportion to phylogenetic time. Variation of genomic structure is higher than that of protein, which is attributed to the species characteristic regulation of gene expression.     

离子对深海适冷菌Pseudoalteromonas sp. SM9913胞外蛋白酶分泌的影响

以海水产酶发酵培养基为对照,研究了多种离子对P. sp. SM9913 胞外蛋白酶分泌的影响. 结果表明,淡水培养基添加NaCl浓度越高,胞外蛋白酶的酶活也越高. K+、Ca2+、磷酸盐的协同作用促进胞外蛋白酶的分泌. Mg2+能够明显抑制胞外蛋白酶的分泌. Fe3＋对胞外蛋白酶分泌影响不明显.Sr2+、F-、B-这3种离子单独作用时都能促进胞外蛋白酶的分泌;但是这3种离子协同作用劣于单独作用. 实验浓度下上述多种离子协同作用可明显促进胞外蛋白酶的分泌. 该研究为深海适冷菌P. sp. SM9913 的淡水化培养和进一步研究胞外蛋白酶的分泌机制奠定了基础.     

砂引草泌盐腺的结构与泌盐的关系

利用石蜡切片法研究了砂引草(Messerschmidia sibirica)茎叶的解剖结构,尤其是对泌盐腺的位置、泌盐腺的形态结构及泌盐腺的发生发育过程进行了系统的研究,结果表明:砂引草的盐腺由茎叶的表皮细胞发育而成,通过观察发现在砂引草的表皮上存在着一种主要的盐腺结构,它是由多个细胞组成,包括收集细胞和分泌细胞等.有时分泌细胞旁边的表皮细胞也参与盐分的收集,属于收集细胞.这些细胞的细胞质稠密有明显的细胞核,但是没有中央液泡,其泌盐过程主要是靠细胞的破裂来完成.     

高活力碱性蛋白酶产生菌的选育与发酵放大

为了选育高活力碱性蛋白酶产生菌株.采用复合诱变(紫外照射、NTG、离子注入)方法,结合平板初筛与摇瓶复筛.获得一株高产突变株HAPN-169,其酶活力为3.5×10 u/mL.高产菌株HAPN-169根据气液接触中体积传氧系数kLa相同的原则发酵放大(7L,30L,200L),其发酵参数为:温度34℃,装液系数0.7,接种量5%,使溶氧维持在20～30%,控制发酵过程pH不低于7.0.在7L发酵罐,发酵时间为54小时,酶活力为3.6×104u/mL;在30L发酵罐,发酵时间为50小时,酶活力为3.86×104u/mL;在200L发酵罐,发酵时间为36小时,酶活力为4.2×104u/mL.发酵液pH在7.5左右时,酶活力在峰值,发酵终点一到,必须马上下罐.该结果对为我围碱性蛋白酶工业发展提供一定的基础.