The structure of a eukaryotic nicotinic acid phosphoribosyltransferase reveals structural heterogeneity among type II PRTases

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and can act as an important substrate in numerous biological processes. As a result, nature has evolved multiple biosynthetic pathways to meet this high chemical demand. In Saccharomyces cerevisiae, the NAD salvage pathway relies on the activity of nicotinic acid phosphoribosyltransferase (NAPRTase), a member of the phosphoribosyltransferase (PRTase) superfamily. Here, we report the structure of a eukaryotic (yeast) NAPRTase at 1.75 A resolution (locus name: YOR209C, gene name: NPT1). The structure reveals a two-domain fold that resembles the architecture of quinolinic acid phosphoribosyltransferases (QAPRTases), but with completely different dispositions that provide evidence for structural heterogeneity among the Type II PRTases. The identification of a third domain in NAPRTases provides a structural basis and possible mechanism for the functional modulation of this family of enzymes by ATP.     

Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.     

HMGB1 contributes to the development of acute lung injury after hemorrhage

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.     

Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Fibronectin-mediated adhesion rescues cell cycle arrest induced by fibroblast growth factor-1 by decreased expression of P21cip/waf in human chondrocytes

Summary In chondrocytes, fibroblast growth factors (FGFs) inhibit chondrocytes proliferation by upregulation of the cell cycle inhibitor p21^cip/waf. In this report, we first investigated the roles of fibronectin (FN)-mediated cell adhesion in the modulation of FGF-1's antiproliferative function in chondrocytes. In this study, we found that FN-mediated signaling could rescue cell cycle arrest induced by FGF-1 in primary human chondrocytes. This prevention of cell cycle arrest induced by FGF-1 was due to the suppression of the cell cycle inhibitor p21^cip/waf expression on adhesion to FN and its downstream activation of signaling pathways. Finally, we showed that this rescue induced by FN-mediated adhesion is dependent on the extracellular regulated kinase (ERK) signaling pathway. Taken together, these studies support that, despite FGF-FGF receptor's growth-inhibitory function, the FN-mediated signaling can collaborate to compensate for its negative effect on chondrocytes proliferation, providing evidence for cross talk between signals emerging from these cell surface molecules in chondrocyte.     

Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea)

To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.