Rapid detection of quinolone-resistant Salmonella by real time SNP genotyping

A total of 63 isolates were screened for the gyrA mutation (87Asp-Tyr) in Salmonella enterica serovars using real time PCR. All of the isolates were successfully identified as resistant or susceptible, consistent with the MIC result of the agar dilution method and gyrA sequencing.     

Orally active anti-proliferation agents: novel diphenylamine derivatives as FGF-R2 autophosphorylation inhibitors

(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also performed metabolic stability studies showing that substitutions at the 7-position of quinoline affect its biological stability. In this study, we achieved a remarkable improvement in the solubility and metabolic stability of the diphenylamine derivative. The most promising compound 15e showed a significant decrease in tumor volume when orally administered.     

A simple titration method for determining Chlamydia infectivity using an image analyzing system

In the biological study of Chlamydia, it is very important to determine the infectivity titer of the organism. For many years researchers used the serial dilution method to determine this titer. This method consists of diluting the material to be examined, inoculating suitable dilutions into susceptible cell cultures and cultivating them. The number of inclusions formed in host cells can be calculated with the naked eye under a microscope. The precision and accuracy of this method, however, depend on the number of inclusions per field and the number of fields counted. In this report, we present a simple and rapid method for counting a large number of inclusions using an image analysis system and an appropriate number of samples, and propose a sampling method based on a statistical analysis of the data obtained with 84 microscopic fields.     

Role of phosphatidylinositol 3-kinase in the binding of Bordetella pertussis to human monocytes

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes by means of filamentous haemagglutinin (FHA), a bacterial surface protein that is recognized by complement receptor type 3 (CR3, alphaMbeta2 integrin). Previous work has shown that an FHA Arg-Gly-Asp (RGD, residues 1097-1099) site interacts with a complex composed of leucocyte response integrin (LRI, alphavbeta3 integrin) and integrin-associated protein (IAP, CD47) on human monocytes, resulting in enhancement of CR3-mediated bacterial binding. However, the pathway that mediates alphavbeta3-alphaMbeta2 integrin signalling remains to be characterized. Here we describe the involvement of phosphatidylinositol 3-kinase (PI3-K) in this pathway. Wortmannin and LY294002, inhibitors of PI3-K, reduced alphavbeta3/IAP-upregulated, CR3-associated bacterial binding to human monocytes. B. pertussis infection of human monocytes resulted in a marked recruitment of cellular PI3-K to the sites of B. pertussis contact. In contrast, cells infected with an isogenic strain carrying a G1098A mutation at the FHA RGD site did not show any recruitment of PI3-K. We found that ligation of FHA by alphavbeta3/IAP induced RGD-dependent tyrosine phosphorylation of a 60 kDa protein, which associated with IAP and PI3-K in human monocytes. These results suggest that PI3-K and a tyrosine phosphorylated 60 kDa protein may be involved in this biologically important integrin signalling pathway.     

Upward shift of the pH optimum of Acremonium ascorbate oxidase

A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5-7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.     

EmPLiCS: an empirical approach for structure-based design of natural peptide drugs

The computer implementation of a peptide drug-design strategy has been developed. The system is named EmPLiCS (Empirical Peptide Ligand Construction System) according to the strategy of the system, which searches for peptide-ligand structures by referring to empirical rules that are derived from known protein 3D structures. The system was tested on several known peptide-protein complexes. The results demonstrated the ability of this system to detect key residues of peptides that are crucial for interaction with their specific proteins. The system also showed the ability to detect the main chain trace of these peptides. Some of the main chain atoms were detected even though the complete primary structures were not reproduced, suggesting that main chain structure is important in peptide-protein recognition. The results of the present study demonstrated that the empirical rules-based system can generate significant information for use in the design of natural peptide drugs.     

Role of pressor mechanisms from the NTS and CVLM in control of arterial pressure

In the present study, we investigated the effects of inhibition of the caudal ventrolateral medulla (CVLM) with the GABA(A) agonist muscimol combined with the blockade of glutamatergic mechanism in the nucleus of the solitary tract (NTS) with kynurenic acid (kyn) on mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances. In male Holtzman rats anesthetized intravenously with urethane/chloralose, bilateral injections of muscimol (120 pmol) into the CVLM or bilateral injections of kyn (2.7 nmol) into the NTS alone increased MAP to 186 +/- 11 and to 142 +/- 6 mmHg, respectively, vs. control: 105 +/- 4 mmHg; HR to 407 +/- 15 and to 412 +/- 18 beats per minute (bpm), respectively, vs. control: 352 +/- 12 bpm; and renal, mesenteric and hindquarter vascular resistances. However, in rats with the CVLM bilaterally blocked by muscimol, additional injections of kyn into the NTS reduced MAP to 88 +/- 5 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Moreover, in rats with the glutamatergic mechanisms of the NTS blocked by bilateral injections of kyn, additional injections of muscimol into the CVLM also reduced MAP to 92 +/- 2 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Simultaneous blockade of NTS and CVLM did not modify the increase in HR but also abolished the increase in renal vascular resistance produced by each treatment alone. The results suggest that important pressor mechanisms arise from the NTS and CVLM to control vascular resistance and arterial pressure under the conditions of the present study.     

An Assay of Sterigmatocystin and Related Metabolites of Aspergillus versicolor by High Speed Liquid Chromatography

Among several type cultures that assimilated 1-hexadecene, Corynebacterium equi IFO 3730 was found to best accumulate 1, 2-epoxyhexadecane. The purified product exhibited +9.64 (c = 3.71, n-hexane) and was confirmed to have the (R) absolute configuration by correlating to known analogous compounds. The optical purity was determined to be 100% by PMR measurement of 1-methoxy-2-hexadecanol which was derived stereospecifically from the epoxide. The highest yield (41 % based on consumed 1-hexadecene) was achieved when 2.0% of octane and 0.1 % of Tween 80 were added to the medium containing 0.5 % of the olefin. C. equi also assimilated terminal olefins other than 1-hexadecene and produced the corresponding epoxides from substrates which have carbon chains longer than fourteen.     

Iterative ACORN as a high throughput tool in structural genomics

High throughput macromolecular structure determination is very essential in structural genomics as the available number of sequence information far exceeds the number of available 3D structures. ACORN, a freely available resource in the CCP4 suite of programs is a comprehensive and efficient program for phasing in the determination of protein structures, when atomic resolution data are available. ACORN with the automatic model-building program ARP/wARP and refinement program REFMAC is a suitable combination for the high throughput structural genomics. ACORN can also be run with secondary structural elements like helices and sheets as inputs with high resolution data. In situations, where ACORN phasing is not sufficient for building the protein model, the fragments (incomplete model/dummy atoms) can again be used as a starting input. Iterative ACORN is proved to work efficiently in the subsequent model building stages in congerin (PDB-ID: lis3) and catalase (PDB-ID: 1gwe) for which models are available.