Hwa-Ja Ryu

The pyrH gene of Vibrio vulnificus is an essential in vivo survival factor.

ABSTRACT We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

Abstract The 3C-like protease (3CLpro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09kcalmol−1 were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CLpro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CLpro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC50 ranged from 38.57±2.41 to 101.38±3.27μM. Two strong 3CLpro inhibitors were further identified as competitive inhibitors of 3CLpro with K i values of 9.11±1.6 and 9.93±0.44μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CLpro were also identified.

Cloning of a dextransucrase gene (fmcmds) from a constitutive dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM developed using VUV.

Dextransucrase (FMCMDS) from Leuconostoc mesenteroides B-512FMCM, a dextransucrase constitutive and hyper-producing strain, catalyzes the synthesis of dextran from sucrose. The coding region for fmcmds was isolated and sequenced. It consisted of an open reading frame (ORF) of 4699 bp, coding for a 1527 amino acid protein with a molecular mass of 170 kDa. However, it showed a dextransucrase activity band at 180 kDa in SDS-PAGE. Only one nucleotide changed in the promoter site and two amino acid residues were changed in the structural gene from that of the parent L. mesenteroides NRRL B-512F dsrS; an inducible dextransucrase gene of low productivity.

Inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition

Human intestinal maltase (HMA) is an α-glucosidase responsible for the hydrolysis of α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with IC50 of 20 ± 1.0 and 31.5 ± 1.0 μM, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants (Ki) calculated from a Dixon plot were 5.93 ± 0.26 and 7.88 ± 0.57 μM, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.