The role of the omega subunit of RNA polymerase in expression of the relA gene in Escherichia coli

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.     

Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.     

Functional domain analysis of human HP1 isoforms in Drosophila

Three subtypes of HP1, a conserved non-histone chromosomal protein enriched in heterochromatin, have been identified in humans, HP1alpha, beta and gamma. In the present study, we utilized a Drosophila system to characterize human HP1 functions. Over-expression of HP1beta in eye imaginal discs caused abnormally patterned eyes, with reduced numbers of ommatidia, and over-expression of HP1gamma in wing imaginal discs caused abnormal wings, in which L4 veins were gapped. These phenotypes were specific to the HP1 subtypes and appear to reflect suppressed gene expression. To determine the molecular domains of HP1 required for each specific phenotype, we constructed a series of chimeric molecules with HP1beta and HP1gamma. Our data show that the C-terminal chromo shadow domain (CSD) of HP1gamma is necessary for HP1gamma-type phenotype, whereas for the HP1beta-type phenotype both the chromo domain and the CSD are required. These results suggest human HP1 subtypes use different domains to suppress gene expression in Drosophila cells.     

Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif

Under ambient air conditions, NO inhibits NMDAR activity by reacting with the NR2A subunit C399 along with two additional cysteine pairs if their disulfide bonds are reduced to free thiol groups [NR1(C744,C798); NR2(C87,C320)]. Here we demonstrate that relative hypoxia enhances S-nitrosylation of NMDARs by a unique mechanism involving an "NO-reactive oxygen sensor motif" whose determinants include C744 and C798 of the NR1 subunit. Redox reactions involving these two thiol groups sensitize other NMDAR sites to S-nitrosylation and consequent receptor inhibition, while their own nitrosylation has little effect on NMDAR activity. The crystal structure of the ligand-binding domain of NR1 reveals a flexible disulfide bond (C744-C798), which may account for its susceptibility to reduction and subsequent reaction with NO that is observed with biochemical techniques. These thiols may be nitrosylated preferentially during increasing hypoxia or stroke conditions, thus preventing excessive activity associated with cytotoxicity while avoiding blockade of physiologically active NMDARs.     

Plant-type N-glycans containing fucose and xylose in Bryophyta (mosses) and Tracheophyta (ferns)

The presence of typical plant-type N-glycans (eg, M3FX, Gn2M3FX, and Le(a)2M3FX) in mosses, ferns, and other organisms was examined to determine which plant initially acquired glycosyltransferases to produce plant-type N-glycans during organic evolution. No M3FX-type N-glycan was detected in lichens (Cladonia humilis) or in any one of the three preland plants Enteromorpha prolifera, Ulva pertusa Kjellman, and Chara braunii Gmelin. In Bryophyta, M3FX-type N-glycan was detected at trace amounts in Anthocerotopsida (hornworts) and at certain amounts in Bryopsida (mosses), but not in Hepaticopsida (liverworts). Le(a)2M3FX was detected in some Bryopsida of relatively high M3FX content. Most Tracheophyta (ferns and higher plants) contained the three typical M3FX-type glycans as the main N-glycans in different ratios. These results suggest that organisms acquired xylosyltransferase and fucosyltransferase during the development of mosses from liverworts, and that later all plants retained both enzymes. Bryopsida have also obtained galactosyltransferase and fucosyltransferase to synthesize the Le(a) antigen.     

Association between prostaglandin E2 receptor gene and essential hypertension

BACKGROUND: Essential hypertension (EH) is a complex multifactorial polygenic disorder that is thought to result from an interaction between an individual's genetic makeup and various environmental factors. In the kidney, prostaglandins (PGs) are important mediators of vascular tone and salt and water homeostasis, and are involved in the mediation and/or modulation of hormonal action. In previous studies, mice deficient in the prostaglandin E2 (PGE(2)) EP2 receptor had resting systolic blood pressure (BP) that was significantly lower than that of wild-type controls. The BP of those mice increased when they were put on a high-salt diet, suggesting that the EP2 receptor is involved in sodium handling by the kidney. In the present study, we investigated the association between EH and nucleotide polymorphisms in the gene encoding the prostaglandin E2 receptor subtype EP2 (PTGER2). METHODS: We selected three single-nucleotide polymorphisms (SNP) in the human PTGER2 gene (rs1254601, rs2075797, and rs17197), and we performed a genetic association study of 266 EH patients and 253 age-matched normotensive (NT) controls. RESULTS: There was no significant difference in overall distribution of genotypes or alleles of any of the SNP between the EH and NT groups. However, among men, the A/A type of the SNP rs17197 (rs17197, A/G in 3'UTR) was significantly more frequent in EH subjects than in NT subjects (P=0.041). CONCLUSION: The present findings suggest that rs17197 is useful as a genetic marker of EH in men.     

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.     

Phenylimidazole derivatives as new inhibitors of bacterial enoyl-ACP reductase FabK

Novel FabK inhibitors with antibacterial activity against Streptococcus pneumoniae were synthesized and evaluated. Through SAR studies of our initial hit compound 2-(1H-benz[d]imidazol-2-ylthio)-N-(6-methoxycarbonylbenzo[d]thiazol-2-yl)acetamide, a series of novel phenylimidazole derivatives were discovered as potent FabK inhibitors.