The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoSdependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases, including life-threatening septicemia and possibly gastroenteritis, in individuals with underlying predisposed conditions such as liver damage, excess levels of iron, and immunocompromised conditions. Wound infections also result from exposure to seawater or from the handling of shellfish contaminated with V. vulnificus. The mortality from septicemia is very high (Ͼ50%), and death can occur within 1-2 days after the first signs of illness. Several potential virulence factors, including an endotoxin, polysaccharide capsule, iron-sequestering systems, cytolytic hemolysin, elastase, phospholipase A 2 , and other exotoxins, have been identified in V. vulnificus (for recent reviews, see Refs. 1 and 2).The elastase activity is from a neutral metalloprotease and represents the major proteolytic activity of V. vulnificus (3, 4). Our previous study revealed the existence of at least two proteases that are produced by V. vulnificus (5). Therefore, vvpE was designed to differentiate the elastase gene from other genes encoding other potential proteases of V. vulnificus (5). As such, a gene encoding the V. vulnificus elastase was recently cloned and sequenced by us (5) and by others (6). The deduced gene product was predicted to be a 609-amino acid polypeptide, and the mature elastase is a 45-kDa protein consisting of 413 amino acids generated by the deletion of the N-termi...

The interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd less than 2 microM) than for GDP (Kd = 15 microM) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the alpha- and beta-31P of IDP to Mn2+ are identical (3.5 A). The Mn2+ distance to the beta- and gamma-31P of ITP is also identical (3.7 A) and is 0.2 A further from the alpha-phosphorus. In the presence of P-enolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a second sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the phosphorus atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 X 10(4) s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

Zeolite-intercalated semiconductor quantum dots (QDs) have long been proposed to give very high third-order nonlinear optical (3NLO) responses. However, measurements of their 3NLO responses have not been possible due to the lack of methods to prepare optically transparent QD-incorporating zeolite films supported on optically transparent substrates and to confine QDs only within zeolite interiors. We found that the zeolite-Y films grown on indium-tin-oxide-coated glass plates (Ygs) remain firmly bonded to the substrates during ion exchange with Pb2+ ions, drying, and formation of PbS QDs by treating Pb2+ ions with H2S. A series of Ygs encapsulating different numbers (n = 0, 8, 14, 23, and 33) of PbS in a unit cell [(PbS)n-Yg] were prepared. The PbS QDs were expelled by adsorbed moisture to the external surfaces, and the expelled QDs formed large QDs. Coating of the (PbS)n-Ygs with octadecyltrimethoxysilane results in effective confinement of the QDs within the internal pores. The zeolite-encapsulated PbS QDs showed remarkably high 3NLO activities at 532 and 1064 nm which are unparalleled by other PbS QDs dispersed in other matrixes.

Background Progressive hepatic fibrosis with development of cirrhosis is a feature of almost all chronic liver diseases. Aims We assessed the performance of Fibroscan in patients with chronic viral hepatitis, and in comparison with and combined with several surrogate serum markers for predicting cirrhosis. Methods In this prospective multicenter cohort study, a novel panel of serum markers was constructed and serum levels of surrogate markers of liver fibrosis and Fibroscan were compared with the stage of fibrosis in the liver biopsy specimens obtained from 121 subjects with chronic viral hepatitis. Another 159 patients were enrolled to validate the diagnostic accuracy of this novel panel.Results Multivariate analysis identified platelet count and procollagen III N-terminal peptide (PIIINP) as independent predictors of liver cirrhosis. The PP score (combining of platelet count and PIIINP) showed significantly better diagnostic accuracy (areas under the receiver operating characteristic curves, AUROC: 0.885) than that of previously reported serologic tests, including APRI, Fibroscan and PP score, and previously reported serologic tests in the estimation group. Conclusions Fibroscan and surrogate serum markers had similar accuracy for predicting cirrhosis, and combining Fibroscan and serum markers did not improve the accuracy.