Alternative Expression of a Chitosanase Gene Produces Two Different Proteins in Cells Infected withChlorellaVirus CVK2

SeveralChlorellavirus CVK2 proteins had chitosanase and/or chitinase activities. A gene coding for an ORF of 328 amino acids (aa) with a predicted molecular mass of 36,769 Da was cloned from the viral genome. The predicted amino acid sequence of an N′-portion (174 aa) of this gene product (vChta-1) showed 22 to 25% identity with various bacterial chitosanases. A glutathioneS-transferase (GST)–vChta-1 fusion protein had strong chitosanase activity. Western blot analysis with antisera raised against the vChta-1 protein identified two proteins of 37 and 65 kDa in virus-infectedChlorellacells beginning at 240 min postinfection and continuing until cell lysis. The larger protein was packaged in the virion, while the smaller one remained in the cell lysate. Both chitosanase proteins were produced from the single gene,vChta-1,by a mechanism of alternative gene expression.     

Labdane‐type diterpenes from Hedychium gardnerianum with potent cytotoxicity against human small cell lung cancer cells

Seven labdane‐type diterpenes, coronarin E, coronarin A, yunnancoronarin A, yunnancoronarin B, hedyforrestin B, villosin, and hedyforrestin C were isolated from the rhizome of Hedychium gardnerianum and evaluated for cytotoxic activity against human small cell lung cancer (NCI‐H187) and non‐cancerous Vero cells. The results showed that villosin exhibited potent cytotoxic activity with IC₅₀ of 0.40 μM, which was higher than that of the drug ellipticine (IC₅₀ 1.79 μM). Moreover, ellipticine was very toxic to Vero cells (IC₅₀ 7.47 μM) whereas the toxicity of villosin was undetectable at concentration lower than 166.42 μM. The results have indicated that the lactone ring is essential for high cytotoxic activity and that the presence of a hydroxyl group at the 6 or 7 position causes decrease in activity. The very high cytotoxicity against the NCI‐H187 cells and the exceptionally high selectivity index (>416) of villosin suggested that this compound may be used as a potential lead molecule for antitumor therapeutic development. Copyright © 2009 John Wiley & Sons, Ltd.     

CMY-2, CMY-8b,and DHA-1 plasmid-mediated AmpC β-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae from a university hospital,Thailand

Between February 2005 and January 2006 in Srinagarind Hospital, Thailand, 44 from 1730 isolates (2.5%) of Escherichia coli and 8 from 982 isolates (0.8%) of Klebsiella pneumoniae were found to produce plasmid-mediated AmpC β-lactamases (pAmpCs) as detected by a cefoxitin–Hodge test followed by a multiplex polymerase chain reaction (PCR) technique. Fifteen of the 52 pAmpC-producing isolates also produced extended-spectrum β-lactamases. The ampC genes found in both organisms were bla_CMY-2 (46 isolates), bla_CMY-8b (4 isolates), and bla_DHA-1 (2 isolates). These genes were present on plasmids. Twenty-five of the 46 CMY-2–producing isolates could transfer cefoxitin resistance to E. coli UB1637 by conjugation. More than 90% of the pAmpC-producing isolates were resistant to cefoxitin, but 80% to 90% of them were susceptible or intermediately susceptible to ceftazidime or cefotaxime. Enterobacterial repetitive intergenic consensus PCR analysis revealed that most isolates were of different strains, indicating the ease of transmission of these resistance determinants. This is the first report of CMY-2, CMY-8b, and DHA-1 β-lactamases in Thai isolates.     

Use of latex agglutination test to determine rabies antibodies in production of rabies antisera in horses

A therapeutic anti-rabies immunoglobulin for human use has been produced mainly in horses. The presently available seroneutralization test, the rapid fluorescent focus inhibition test (RFFIT), is laborious and rather difficult to carry out in horse farms. This study was undertaken to develop a simple latex agglutination test (LAT) for determining rabies antibodies in horse sera. LAT was validated by testing a total of 468 horse serum samples characterized by RFFIT. Of these, 253 of 260 samples with antibody titers of less than 100 IU/ml had agglutination score of 1+, whereas 174 of 208 samples with antibody titers equal to or greater than 100 IU/ml had agglutination scores of 2-4+. Results of LAT correlated with those of RFFIT (r = 0.87, p < 0.0001). LAT has the advantages of being rapid, simple to perform, easy to interpret, and applicable as an on-site testing tool for the estimation of rabies antibodies in horses.     

Real-time PCR analysis of dog cerebrospinal fluid and saliva samples for ante-mortem diagnosis of rabies

The use of a 10-day observation to determine whether a dog is rabid is standard practice. This study was conducted in order to look for evidence of rabies vius in saliva and cerebrospinal fluid (CSF) of suspected live rabid dogs at the time of quarantine by using a SYBR Green real-time RT-PCR based assay for the detection of rabies virus RNA. Saliva and CSF of dogs were collected once on the day of admission for the 10-day quarantine. All test dogs were or became ill and died of rabies within the observation period. Thirteen of 15 dogs (87%) had saliva samples that were positive for rabies RNA. Two dogs with furious rabies had negative saliva samples. Positive CSF samples were found in 4 of 15 dogs (27%) whose saliva samples were positive. The time from sample collection to result was less than 5 hours. Because virus may be absent or present at very low level in both clinical fluids, samples taken for ante-mortem diagnosis cannot definitively rule out rabies.