生殖支原体脂质相关膜蛋白激活核因子κB诱导人单核细胞表达前炎症细胞因子及凋亡

为了解生殖支原体(Mg)潜在的致病性及其脂质相关膜蛋白(LAMPs)诱导人单核细胞(THP-1)凋亡及表达前炎症细胞因子(CKs)的分子机制,用Mg提取的LAMPs刺激THP-1细胞,以ELISA法和RT-PCR方法分析CKs产生和其mRNA的表达。不同试实验组的细胞经AnnexinV联合PI染色后通过流式细胞仪检测细胞凋亡。采用EMSA方法检测LAMPs处理的THP-1细胞中核转录因子kappaB(NF-κB)的激活,并分析NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocoarbamate,PDTC)对LAMPs处理的THP-1细胞产生CKs的量和其mRNA表达及细胞凋亡的影响。LAMPs能以时间和剂量依赖方式刺激THP-1细胞产生TNF-α、IL-1β和IL-6,且能激活NF-κB诱导THP-1细胞表达CKs的mRNA及发生凋亡,PDTC能显著抑制CKs的mRNA表达水平和细胞凋亡。由于LAMPs能激活NF-κB诱导THP-1细胞表达CKs及产生细胞凋亡,因而可能是一个重要的致病因素。     

Target selection and deselection at the Berkeley Structural Genomics Center

At the Berkeley Structural Genomics Center (BSGC), our goal is to obtain a near-complete structural complement of proteins in the minimal organisms Mycoplasma genitalium and M. pneumoniae, two closely related pathogens. Current targets for structure determination have been selected in six major stages, starting with those predicted to be most tractable to high throughput study and likely to yield new structural information. We report on the process used to select these proteins, as well as our target deselection procedure. Target deselection reduces experimental effort by eliminating targets similar to those recently solved by the structural biology community or other centers. We measure the impact of the 69 structures solved at the BSGC as of July 2004 on structure prediction coverage of the M. pneumoniae and M. genitalium proteomes. The number of Mycoplasma proteins for which the fold could first be reliably assigned based on structures solved at the BSGC (24 M. pneumoniae and 21 M. genitalium) is approximately 25% of the total resulting from work at all structural genomics centers and the worldwide structural biology community (94 M. pneumoniae and 86 M. genitalium) during the same period. As the number of structures contributed by the BSGC during that period is less than 1% of the total worldwide output, the benefits of a focused target selection strategy are apparent. If the structures of all current targets were solved, the percentage of M. pneumoniae proteins for which folds could be reliably assigned would increase from approximately 57% (391 of 687) at present to around 80% (550 of 687), and the percentage of the proteome that could be accurately modeled would increase from around 37% (254 of 687) to about 64% (438 of 687). In M. genitalium, the percentage of the proteome that could be structurally annotated based on structures of our remaining targets would rise from 72% (348 of 486) to around 76% (371 of 486), with the percentage of accurately modeled proteins would rise from 50% (243 of 486) to 58% (283 of 486). Sequences and data on experimental progress on our targets are available in the public databases TargetDB and PEPCdb.     

Investigation on silent bacterial infections in specimens from pregnant women affected by spontaneous miscarriage

Miscarriage is one of the main complications occurring in pregnancy. The association between adverse pregnancy outcomes and silent bacterial infections has been poorly investigated. Ureaplasma parvum and urealiticum, Mycoplasma genitalium and hominis and Chlamydia trachomatis DNA sequences have been investigated by polymerase chain reaction (PCR) methods in chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from females with spontaneous abortion (SA, n = 100) and females who underwent voluntary interruption of pregnancy (VI, n = 100). U. parvum DNA was detected in 14% and 15% of SA and VI, respectively, with a mean of bacterial DNA load of 1.3 × 10⁻¹ copy/cell in SA and 2.8 × 10 ⁻³ copy/cell in VI; U. urealiticum DNA was detected in 3% and 2% of SA and VI specimens, respectively, with a mean DNA load of 3.3 × 10⁻³ copy/cell in SA and 1.6 × 10⁻³ copy/cell in VI; M. hominis DNA was detected in 5% of SA specimens with a DNA load of 1.3 × 10⁻⁴ copy/cell and in 6% of VI specimens with a DNA load of 1.4 × 10⁻⁴ copy/cell; C. trachomatis DNA was detected in 3% of SA specimens with a DNA load of 1.5 × 10⁻⁴ copy/cell and in 4% of VI specimens with a mean DNA load of 1.4 × 10⁻⁴ copy/cell. In PBMCs from the SA and VI groups, Ureaplasma spp, Mycoplasma spp and C. trachomatis DNAs were detected with a prevalence of 1%–3%. Bacteria were investigated, for the first time, by quantitative real-time PCR (qPCR) in chorionic villi tissues and PBMCs from women affected by SA and VI. These data may help to understand the role and our knowledge of the silent infections in SA.     

Building Structural Models of a Whole Mycoplasma Cell

Building structural models of entire cells has been a long-standing cross-discipline challenge for the research community, as it requires an unprecedented level of integration between multiple sources of biological data and enhanced methods for computational modeling and visualization. Here, we present the first 3D structural models of an entire Mycoplasma genitalium (MG) cell, built using the CellPACK suite of computational modeling tools. Our model recapitulates the data described in recent whole-cell system biology simulations and provides a structural representation for all MG proteins, DNA and RNA molecules, obtained by combining experimental and homology-modeled structures and lattice-based models of the genome. We establish a framework for gathering, curating and evaluating these structures, exposing current weaknesses of modeling methods and the boundaries of MG structural knowledge, and visualization methods to explore functional characteristics of the genome and proteome. We compare two approaches for data gathering, a manually-curated workflow and an automated workflow that uses homologous structures, both of which are appropriate for the analysis of mesoscale properties such as crowding and volume occupancy. Analysis of model quality provides estimates of the regularization that will be required when these models are used as starting points for atomic molecular dynamics simulations.     

Association of Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitalium and Ureaplasma species infection and organism load with cervicitis in north Indian population

Cervicitis is predominantly caused by Neisseria gonorrhoeae and Chlamydia trachomatis, which accounts for almost half of all the cases of cervicitis. The role of newer organisms like Mycoplasma genitalium and Ureaplasma sp. and association of bacterial load with cervicitis are also not well established. So the study aimed to determine the relative frequency of these organisms and their load in association with cervicitis cases from north India. A case–control study involving 300 women was conducted using quantitative real-time PCR from endocervical swabs for identification of organisms and quantification of bacterial load. Among 150 cervicitis cases, C. trachomatis, N. gonorrhoeae, M. genitalium and Ureaplasma parvum were detected in 5 (3·3%), 10 (6·6%), 37(24·6%) and 47 (31·3%) respectively. Old age (<0·001, chi-squared test) and irregular menstrual cycles (<0·001, chi-squared test) were significantly associated with cervicitis. M genitalium was the only organism to be associated significantly with cervicitis with regard to age (<0·031) and symptoms like discharge (P < 0·033, chi-squared test) and dysuria (P < 0·044, chi-squared test) in multivariate analysis. Our finding suggests that the bacterial load of these organisms is not significantly associated with cervicitis. However, we found significant association of M. genitalium infection with clinical characteristics of cervicitis cases.     

Development and large scale benchmark testing of the PROSPECTOR_3 threading algorithm

This article describes the PROSPECTOR_3 threading algorithm, which combines various scoring functions designed to match structurally related target/template pairs. Each variant described was found to have a Z-score above which most identified templates have good structural (threading) alignments, Z(struct) (Z(good)). 'Easy' targets with accurate threading alignments are identified as single templates with Z > Z(good) or two templates, each with Z > Z(struct), having a good consensus structure in mutually aligned regions. 'Medium' targets have a pair of templates lacking a consensus structure, or a single template for which Z(struct) < Z < Z(good). PROSPECTOR_3 was applied to a comprehensive Protein Data Bank (PDB) benchmark composed of 1491 single domain proteins, 41-200 residues long and no more than 30% identical to any threading template. Of the proteins, 878 were found to be easy targets, with 761 having a root mean square deviation (RMSD) from native of less than 6.5 A. The average contact prediction accuracy was 46%, and on average 17.6 residue continuous fragments were predicted with RMSD values of 2.0 A. There were 606 medium targets identified, 87% (31%) of which had good structural (threading) alignments. On average, 9.1 residue, continuous fragments with RMSD of 2.5 A were predicted. Combining easy and medium sets, 63% (91%) of the targets had good threading (structural) alignments compared to native; the average target/template sequence identity was 22%. Only nine targets lacked matched templates. Moreover, PROSPECTOR_3 consistently outperforms PSIBLAST. Similar results were predicted for open reading frames (ORFS) < or =200 residues in the M. genitalium, E. coli and S. cerevisiae genomes. Thus, progress has been made in identification of weakly homologous/analogous proteins, with very high alignment coverage, both in a comprehensive PDB benchmark as well as in genomes.     

Evolutionary Lability of Context-Dependent Codon Bias in Bacteria

In bacteria, synonymous codon usage can be considerably affected by base composition at neighboring sites. Such context-dependent biases may be caused by either selection against specific nucleotide motifs or context-dependent mutation biases. Here we consider the evolutionary conservation of context-dependent codon bias across 11 completely sequenced bacterial genomes. In particular, we focus on two contextual biases previously identified in Escherichia coli; the avoidance of out-of-frame stop codons and AGG motifs. By identifying homologues of E. coli genes, we also investigate the effect of gene expression level in Haemophilus influenzae and Mycoplasma genitalium. We find that while context-dependent codon biases are widespread in bacteria, few are conserved across all species considered. Avoidance of out-of-frame stop codons does not apply to all stop codons or amino acids in E. coli, does not hold for different species, does not increase with gene expression level, and is not relaxed in Mycoplasma spp., in which the canonical stop codon, TGA, is recognized as tryptophan. Avoidance of AGG motifs shows some evolutionary conservation and increases with gene expression level in E. coli, suggestive of the action of selection, but the cause of the bias differs between species. These results demonstrate that strong context-dependent forces, both selective and mutational, operate on synonymous codon usage but that these differ considerably between genomes. Received: 6 May 1999 / Accepted: 29 October 1999     

Building the blueprint of life

With recent breakthroughs in experimental microbiology making it possible to synthesize and implant an entire genome to create a living cell, the challenge of constructing a working blueprint for the first truly minimal synthetic organism is more important than ever. Here we review the significant progress made in the design and creation of a minimal organism. We discuss how comparative genomes, gene essentiality data, naturally small genomes, and metabolic modeling are all being applied to produce a catalogue of the biological functions essential for life. We compare the minimal gene sets from three published sources with functions identified in 13 existing gene essentiality datasets. We examine how genome-scale metabolic models have been applied to design a minimal metabolism for growth in simple and complex media. Additionally, we survey the progress of efforts to construct a minimal organism, either through implementation of combinatorial deletions in Bacillus subtilis and Escherichia coli or through the synthesis and implantation of synthetic genomes.     

The nucleotide‐bound/substrate‐bound conformation of the Mycoplasma genitalium DnaK chaperone

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains—nucleotide binding domain (NBD) and substrate binding domain (SBD)—connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide‐bound/substrate‐bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDβ‐SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.