Shinichi Morimatsu

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.

Disinfection potential of electrolyzed solutions containing sodium chloride at low concentrations

Electrolyzed products of sodium chloride solution were examined for their disinfection potential against hepatitis B virus (HBV) and human immunodeficiency virus (HIV) in vitro. Electrolysis of 0.05% NaCl in tap water was carried out for 45 min at room temperature using a 3 A electric current in separate wells installed with positive and negative electrodes. The electrolyzed products were obtained from the positive well. The oxidation reduction potential (ORP), pH and free chlorine content of the product were 1053 mV, pH 2.34 and 4.20 ppm, respectively. The products modified the antigenicity of the surface protein of HBV as well as the infectivity of HIV in time- and concentration-dependent manner. Although the inactivating potential was decreased by the addition of contaminating protein, recycling of the product or continuous addition of fresh product may restore the complete disinfection against bloodborne pathogens.

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.

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.

Contrast-enhancement for the image of human immunodeficiency virus from ultrathin section by immuno electron microscopy

A simple contrast-enhancement method is described for electron microscopic imaging of the human immunodeficiency virus (HIV) from a sample embedded in Lowicryl K4M resin, by immuneelectron microscopy. Ultrathin sections were treated with a mixture of ruthenium red dye (RR) and osmium tetroxide (OSO4). This treatment provided good contrast enhancement of the entire ultrastructural image of virus particles without the loss of immunolabelling. RR/OsO4 solution is simple to prepare and provides a better contrast than that which is achieved during conventional post-embedding immunoelectron microscopy. Treatment of ultrathin sections from low temperature-embedded samples with RR/OsO4 solution is recommended.

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.

Targets of a protease inhibitor, KNI-272, in HIV-1-infected cells

The targets of a protease inhibitor, KNI‐272, in the HIV‐1 life cycle were investigated in this study. Neither expression of HIV‐1 Gag proteins nor production of virus particles was detected in cells infected acutely with HIV‐1 cultured in the presence of KNI‐272. Although HIV‐1 proviral DNA was detected in the cells by PCR, the inhibitor depressed the amount of the proviral DNA in a concentration dependent manner. These results indicate that one of the targets of KNI‐272 occurs in the stage before the expression of viral structural proteins. No direct inhibition of reverse transcription was found with the inhibitor. To confirm the inhibition of viral protease, persistently HIV‐1‐infected cells were cultured in the presence of the inhibitor and examined by electron microscopy for the morphology of HIV‐1 particles. Doughnut‐shaped immature particles were observed in the extracellular space of the cells, and disrupted semicircular shaped particles were also seen at the higher concentration of KNI‐272. A bioassay for infectivity showed that the virus particles were not infectious, and immunofluorescent assay using anti‐p17 antibody, that does not react with the precursor of Gag protein, revealed that Gag precursor p55 protein in the cells was not processed. Thus, KNI‐272 blocked the maturation of viral particles. Consequently, KNI‐272 has at least two inhibition targets in the stages of the HIV‐1 life cycle. J. Med. Virol. 63:203–209, 2001.

[Detection of HTLV-I/ATLV by using the culture of lymph node lymphocytes with adult T-cell leukemia/lymphoma].

We cultured the cervical lymph node lymphocytes of a patient suffering from cutaneous T-cell lymphoma. His anti-ATLV antibody was positive by indirect immunofluorescent method (IF). ATLV was detected on these cultured cells by IF. Type C particles were observed in the cultured cells by electron microscopy. These particles were measured to be 60 to 120 nm in diameter with electron dense core, and were considered as ATLV. This case showed a possibility of detecting ATLV by culture of lymph node lymphocytes from such a patient.

We Detected HTLV-I/ATLV by Means of Culture of Lymphonodal Lymphocytes with Adult T-Cell Leukemia/Lymphoma

We cultured the cervical lymph node lymphocytes of a patient suffering from cutaneous T-cell lymphoma. His anti-ATLV antibody was positive by indirect immunofluorescent method (IF). ATLV was detected on these cultured cells by IF. Type C particles were observed in the cultured cells by electron microscopy. These particles were measured to be 60 to 120 nm in diameter with electron dense core, and were considered as ATLV. This case showed a possibility of detecting ATLV by culture of lymph node lymphocytes from such a patient.