Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.     

Characterization of the agent of "high plains disease": mass spectrometry determines the sequence of the disease-specific protein

The "32-kDa" protein specifically associated with high plains disease was characterized by time-of-flight mass spectrometry, after the agent had been isolated in pure culture by "vascular puncture inoculation," a novel mechanical means of transmission. Two isolates from different geographic locations each consisted of a mixture of subpopulations that were highly homologous to an amino acid sequence derived from a nucleotide sequence (U60141) deposited in GenBank trade mark by the Nebraska group as "the probable N-protein of high plains virus." However, the U60141 sequence was found to be incomplete; de novo sequencing of peptides produced by proteolytic digestions of the 32-kDa band from an SDS-PAGE separation showed that an additional 18 amino acid residues were present at the N terminus. BLAST (basic local alignment search tool) examination of the sequence showed no significant homology with any protein in the databases, indicating that the infectious agent of high plains disease is likely a member of a hitherto unclassified virus group.     

新生儿病房铜绿假单胞菌感染状况及耐药性的5年监测

目的监测2005年至2009年间铜绿假单胞菌在新生儿病房的分离情况及其耐药趋势,为临床感染的预防和治疗提供实验室依据。方法回顾分析2005年至2009年间新生儿病房所有标本中铜绿假单胞菌的分离率,菌株在标本中的分布以及对常见的13种抗菌药物的耐药性。结果铜绿假单胞菌的分离率有逐年升高的趋势,从2005年的1.19%增至2009年的5.16%;其在呼吸道标本中的分离率最高(86.3%),在其他标本中的检出率都相对较低。铜绿假单胞菌的耐药株也有逐年增多的趋势,特别是对复方新诺明的耐药性已发展成为全部耐药。结论铜绿假单胞菌在新生儿病房的分离率在不断地增加,其对多种抗菌药物有较高的耐药性,加之新生儿免疫系统发育不完善,应引起临床医师的高度重视。     

Iron activates NF-kappaB in Kupffer cells

Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappaB-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappaB and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (approximately 5-50 microM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappaBalpha, a concomitant increase in nuclear p65 protein, and increased DNA binding of p50/p50 and p65/p50 without affecting activator protein-1 binding. Addition of Fe2+ to the cells resulted in an increase in electron paramagnetic resonance-detectable.OH peaking at 15 min, preceding activation of NF-kappaB but coinciding with activation of inhibitor kappaB kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappaB, and TNF-alpha promoter activity and to induce the release of TNF-alpha protein by cultured Kupffer cells in a redox status-dependent manner. We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-alpha-dependent liver injury.     

Identification of cross-linked peptides from complex samples

We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein-interaction studies.     

Phenotypic features of Ferroplasma acidiphilum strains Yt and Y-2

Earlier, we described a new family of mesophilic, strictly autotrophic Fe(2+)-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and species F. acidiphilum (strain YT) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain YT and a new strain, F. acidiphilum Y-2, isolated from dense pulps produced during oxidation of arsenogold concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk Krai) ore deposits, respectively. The G + C content of DNA from strains YT and Y-2 comprised 35.1 and 35.2 mol%, respectively; the level of DNA-DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous oxide and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. Strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains YT and Y-2 were 34-36 and 40-42 degrees C, respectively. Comparative analysis of total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of total lipids: lipid I, beta-D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2:1. Thus, it was established that the intraspecific phylogenetic divergence within F. acidiphilum is manifested in two the strains by different temperature optima against the background of similarity in other phenotypic properties.     

Effects of chicken intestinal antimicrobial peptides on humoral immunity of chickens and antibody titres after vaccination with infectious bursal disease virus vaccine in chicken

Sixty chickens were randomly divided into two groups (30 chickens in each group) to determine the effect of oral administration of chicken intestinal antimicrobial peptides (CIAMP) on the humoral immune response. Chickens of both groups were fed the same diet. In the treatment group chickens received drinking water supplemented with CIAMP (1 microg/ml) right after hatching. Samples of blood, bursa of Fabricus, spleen and intestine were taken at day 1, 4, 7, 10 and 17 of experiment. CIAMP supplementation enhanced the content of IgG and IgM in serum from day 4-10 and day 10-17, respectively, (p < 0.05), IgM-forming cells in bursa of Fabricus and spleen at the age of 7 days (p < 0.05) and IgG-forming cells in bursa of Fabricus at the age of 4 days (p < 0.05). In addition, CIAMP enhanced the IgA-forming cells in caecal tonsils diffuse area at day 4 (p < 0.05). Furthermore, CIAMP enhanced the antibody response to infectious bursal disease virus vaccine (IBDV) in chickens 21 days following IBDV vaccine administration (p < 0.05). These results suggested that CIAMP could modulate the humoral immune response of chickens and increased the antibody titres of infectious bursal disease virus vaccine.     

Formaldehyde induces toxic effects and regulates the expression of damage response genes in BM-MSCs

In this study, we assessed the toxic effects of formaldehyde （FA） on mouse bone marrow mesenchymai stem cells （BM- MSCs）. Cytotoxicity was measured by using MTT assay. DNA strand breakage was detected by standard alkaline comet assay and comet assay modified with proteinase K （PK）. DNA-protein crosslinks （DPCs） were detected by KCI-SDS precipitation assay. We found that FA at a con- centration from 75 to 200 μM inhibited cell survival and induced DPCs over 125 μM. The PK-modified comet assay showed that FA-induced DNA strand breakage was increased in a dose-dependent manner from 75 to 200 μM. On the other hand, standard alkaline comet assay showed that DNA strand breakage was decreased with FA concen- tration over 125 μM. We confirmed by using Pearson cor- relation that there was a negative linear correlation between DPCs and survival rate （r = -0.987, P 〈 0.01） and positive linear relationships between DPCs and （i） sister chromatid exchange and （ii） micronucleus （r = 0.995, P〈 0.01; r = 0.968, P〈 0.01）. DNA damage RTz profiler polymerase chain reaction array was used to investigate the changes in the expression of damage response genes. Xpa and Xpc of the nucleotide excision repair pathway and Brca2, Rad51, and Xrcc2 of the homologous recombination pathway were all up-regulated in both 75 and 125 μM FA. However, the same genes were down-regulated with 175 μM FA. The expressions of Chekl and Husl, which are involved in cell cycle regulation, were altered in the same manner with 75, 125, and 175 μM FA. These results indicated that Xpa, Xpc, Brca2, Rad51, Xrcc2, Chekl, and Husl were essential for the BM-MSCs to counteract the effects of FA.