Morphology and infectivity of virus that persistently caused infection in an AGS cell line.

A recent report has indicated that proteins and genes of simian virus 5 (SV5) are detected in a human gastric adenocarcinoma (AGS) cell line, which is widely provided for oncology, immunology, and microbiology research. However, the production of infective virions has not been determined in this cell line. In this study, the morphology and infectivity of the virus particles of the AGS cell line were studied by light and electron microscopy and virus transmission assay. The virus particles were approximately 176.0 ± 41.1 nm in diameter. The particles possessed projections 8-12 nm long on the surface and contained a nucleocapsid determined to be 13-18 nm in width and less than 1,000 nm in length. The virus was transmissible to the Vero cell line, induced multinuclear giant cell formation, and reproduced the same shape of antigenic virions. In this study, the persistently infected virus in the AGS cell line was determined to be infective and form reproducible virions, and a new morphological feature of SV5 was determined.

Intrabacterial proton-dependent CagA transport system in Helicobacter pylori.

Helicobacter pylori CagA modifies the signalling of host cells and causes gastric diseases. Although CagA is injected into gastric epithelial cells through the type IV secretion machinery, it remains unclear how CagA is transported towards the machinery in the bacterial cytoplasm. In this study, it was determined that the proton-dependent intracytoplasmic transport system correlates with the priming of CagA secretion from H. pylori. The cytotoxicity of neutral-pH- and acidic-pH-treated H. pylori was examined in the AGS cell line. The amount of phosphorylated CagA in AGS cells incubated with acidic-pH- and neutral-pH-treated H. pylori was determined by enzyme immunoassay and Western blot. The production of CagA and adherence of the treated bacteria were examined by enzyme immunoassay and light microscopy, respectively. To clarify how CagA is transported towards the inner membrane of the treated bacteria, the localization of CagA was analysed by immunoelectron microscopy. The proportion of hummingbird cells in the AGS cell line rapidly increased following the inoculation of acidic-pH-treated H. pylori but increased more slowly with neutral-pH-treated H. pylori, and the phenomenon correlated with the amount of phosphorylated CagA in AGS cells. CagA was densely localized near the inner membrane in the acidic-pH-treated bacterial cytoplasm, but this localization was not observed in the neutral-pH-treated bacterial cytoplasm, suggesting that CagA shifts from the centre to the peripheral portion of the cytoplasm as a result of an extracellular decrease in pH. This phenomenon depended on the presence of UreI, a proton-dependent urea channel, but not on the presence of urea. The pH treatments did not enhance CagA production or the adherence of the bacterium to AGS cells. The authors propose that H. pylori possesses a proton-dependent intracytoplasmic transport system that probably accelerates priming for CagA injection.

An improved method for detecting faecal Vibrio cholerae by PCR of the toxin A gene.

A method for removing inhibitor(s) of the PCR assay for the direct detection of cholera toxin A gene (ctxA) in human faeces is described. Inhibitors of the PCR were removed by centrifugation and the activity of the remaining inhibitors by dilution. Based on these data, a protocol was developed for pre-treatment of stool specimens for PCR assay, and a simple and rapid protocol was constructed for the diagnostic detection of the ctxA genes in stool specimens in combination with single band detection on gel electrophoresis, dot-blot hybridisation and enrichment culture. This protocol was applied to clinical specimens and showed that the PCR method gave 100% agreement with established culture methods for the detection of cholera toxin-producing Vibrio cholerae O1. This protocol was considered to be useful because of its simplicity and the rapidity of diagnosis.

A new improved method for the concentration of HIV-1 infective particles.

Improvement of the sensitivity of detection systems for human immunodeficiency virus-1 (HIV-1) has been carried out. One approach to improve the sensitivity is purification and/or concentration of the virus from a specimen. In this study, a method for concentrating HIV-1 using polyethylene glycol (PEG) has been re-evaluated and the optimal protocol for concentrating the virus from low-titer specimens was determined. That is, to obtain a virus pellet, a mixture of equal volumes of a specimen and 20% PEG 20,000 solution in saline is incubated at 4 degrees C for 16 h and then centrifuged at 17860 x g in a microcentrifuge for 20 min. HIV-1 in the pellet could be detectable by HIV-1 p24 antigen capture assay for viral protein, reverse transcriptase (RT) assay for viral enzyme, reverse transcriptase polymerase chain reaction (RT-PCR) assay for viral RNA and a virus infectivity assay.

Bactericidal activity of electrolyzed acid water from solution containing sodium chloride at low concentration, in comparison with that at high concentration.

Electrolyzed strong acid water (ESW) containing free chlorine at various concentrations is becoming to be available in clinical settings as a disinfectant. ESW is prepared by electrolysis of a NaCl solution, and has a corrosive activity against medical instruments. Although lower concentrations of NaCl and free chlorine are desired to eliminate corrosion, the germicidal effect of ESW with low NaCl and free-chlorine concentrations (ESW-L) has not been fully clarified. In this study, we demonstrated that ESW-L possesses bactericidal activity against Mycobacteria and spores of Bacillus subtilis. The effect was slightly weaker than that of ESW containing higher NaCl and free-chlorine concentrations (ESW-H), but acceptable as a disinfectant. To clarify the mechanism of the bactericidal activity, we investigated ESW-L-treated Pseudomonas aeruginosa by transmission electron microscopy, a bacterial enzyme assay and restriction fragment length polymorphism pattern (RFLP) assay. Since the bacterium, whose growth was completely inhibited by ESW-L, revealed the inactivation of cytoplasmic enzyme, blebs and breaks in its outer membrane and remained complete RFLP of DNA, damage of the outer membrane and inactivation of cytoplasmic enzyme are the important determinants of the bactericidal activity.