In vitro cleavage of hepatitis B virus C mRNA by 10-23 DNA enzyme

BACKGROUND: 10-23 DNA enzyme is one kind of de-oxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2. 2. 15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A1816UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. RESULTS: Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km,Kcat and Kcat/ Km for Drz-HBV-C-9, were 1.4 ×10-9 mol, 1.6 min-1 and 1.1 × 109 mol-1 · min-1, respectively. CONCLUSIONS: 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.

Antibody-Like Phosphorylation Sites in Focus of Statistically Based Bilingual Approach

In accordance with previous reports, the sequences related to phosporylated protein segments occur in conserved variable domains of immunoglobulins including first of all certain N-terminally located segments. Consequently, we look here for the sequences 1) composing human and mouse proteins different from antigen receptors, 2) identical with or highly similar to nucleotide sequence representatives of conserved variable immunoglobulin segments and 3) identical with or closely related to phosphorylation sites. More precisely, we searched for the corresponding actual pairs of DNA and protein sequence segments using five-step bilingual approach employing among others a) different types of BLAST searches, b) two in-principle-different machine-learning methods predicting phosphorylated sites and c) two large databases recording existing phosphorylation sites. The approach identified seven existing phosphorylation sites and thirty-seven related human and mouse segments achieving limits for several predictions or phylogenic parameters. Mostly serines phosporylated with ataxia-telangiectasia-related kinase (involved in regulation of DNA-double-strand-break repair) were indicated or predicted in this study. Hypermutation motifs, located in effective positions of the selected sequence segments, occurred significantly less frequently in transcribed than non-transcribed DNA strands suggesting thus the incidence of mutation events. In addition, marked differences between the numbers and proportions of human and mouse cancer-related sequence items were found in different steps of selection process. The possible role of hypermutation changes within the selected segments and the observed structural relationships are discussed here with respect to DNA damage, carcinogenesis, cancer vaccination, ageing and evolution. Taken together, our data represent additional and sometimes perhaps complementary information to the existing databases of empirically proven phosphorylation sites or pathogenically important spots.

Relationship between antimicrobial resistance and aminoglycoside-modifying enzyme gene expressions in Acinetobacter baumannii

Background Acinetobacter baumannii is one of the main gramnegative bacilli in clinical practice Nosocomial infections caused by multidrug resistance Acinetobacter baumannii is very difficult to treat This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycosidemodifying enzymes including Nacetyltransferases and Ophosphotransferases in Acinetobacter baumannii Methods Bacterial identification and antimicrobial susceptibility test were performed by PhoenixTM system in 247 strains of Acinetobacter baumannii Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multidrug resistant Acinetobacter baumannii were detected by agar dilution Four aminoglycosidemodifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencerResults The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50% Imipenem and meropenem showed high antibacterial activities with resistance rates of 32% and 41% MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multidrug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 933%, respectively But their resistance rates to tobramycin, netilmicin and neomycin were 867%, 933% and 467%, respectively Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected Their positive rates were 933%, 200% and 200%, respectively These three genes existed simultaneously in No19 strain Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 979% and 997% identities with GenBank genes (AY307113, S68058 and AY307114)Conclusion Multidrug resistant Acinetobacter baumannii strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycosidemodifying enzyme gene expressions