Enterovirus isolation from children with acute respiratory infections and presumptive identification by a modified microplate method

AbstractObjective: To evaluate a modified microplate method, utilizing HEF, HEp-2, Vero, MDCK and newly introduced RD-18S and GMK cell lines, for virus isolation.Methods: From June to October 2001, 723 throat swab specimens taken from children with acute respiratory infections (ARIs) were inoculated onto these cells. To analyze cell sensitivity, we also inoculated 20 serotypes of stocked enteroviruses.Results: During the period, we isolated 40 Coxsackie A2 (CoxA2), 13 CoxA4, 16 CoxA16, 1 CoxB2, 11 CoxB3, 2 CoxB5, 54 echo16, 2 entero7l and 1 polio2. By observing a cell sensitivity pattern with HEF, HEp-2, Vero, RD-18S, and GMK, we could finally differentiate five enterovirus groups: CoxA except for CoxA16, CoxA16/entero7l, CoxB, echovirus, and poliovirus.Conclusions: With this system, the RD-18S cell line enabled us to isolate CoxA virus, except for CoxA16, for the first time. Differentiation of five enterovirus groups by cell sensitivity simplified the specific identification by neutralization test as a presumptive identification. A modified microplate method may be an appropriate cell combination for virus isolation, especially for enteroviruses, and is expected to be used routinely for virologic diagnosis and to clarify the epidemiology of ARI in children

Physiological and Ultrastructural Studies on the Origin of Activator Calcium in Body Wall Muscles of Spoon Worms

To examine the origin of activator Ca and its translocation during contraction in body wall muscles (BWM) of spoon worms, Urechis unicinctus , physiological and ultrastructural studies, including cytochemistry, were performed. The potassium (K-) contracture tension was significantly reduced by the removal of external Ca, and by the application of Mn, La and verapamil. On the other hand, caffeine induced a prolonged contraction. The removal of Ca and Mg from the external solution, and the rapid cooling caused an irregular or oscillatory contraction. These results suggested that, in BWM fibers, the activator Ca is supplied partially from both external solution and intracellular Ca-accumulating structures. Ultrastructural observations revealed that the muscle fibers contain a relatively large amount of sarcoplasmic reticulum (SR). The fractional volume of the SR relative to the fiber volume was 2~5% in all fibers of three muscle layers. To demonstrate the Ca localization, the muscle fibers were fixed by pyroantimonate (PA) methods at resting and contracting states. In the resting fibers, the PA precipitates were exclusively localized in the SR and the inner surface of plasma membrane. On the other hand, in the contracting fibers, they were diffusely distributed in the central regions of myoplasm, and had disappeared from the SR and plasma membrane. X-ray microanalysis revealed that the PA precipitates contain Ca. With the results of physiological experiments, these results indicate that the activator Ca originates not only from the external solution, but also from the intracellular Ca-accumulating structures, the SR and the inner surface of plasma membrane.Full-Length Pape

LET dependence of oxidative DNA base damage in 2-deoxyguanine aqueous solution irradiated with heavy ions

To evaluate the contribution of indirect action mediated by OH radicals in biological effects of high LET radiations, we examined the production of 8-hydroxy-2-deoxyguanosine (8-OHdG) in 2-deoxyguanosine aqueous solution using various ion species with an LET range from 20 to 300 keV/mm. The 8-OHdG yield decreased with increasing LET. In the hypoxic irradiation condition, the yield showed constant or rather increasing tendency above about 100 keV/mm, which is consistent with an oxygen-in-the-track hypothesis to explain the diminishment of oxygen effect.The 2nd Asia-Pacific Symposium on Radiation Chemistry (APSRC-2008

LET dependence of the formation of oxidative damage 8-hydroxy-2\u27-deoxyguanosine(8-OHdG)in 2\u27-deoxyguanosine aqueous solution irradiated with heavy ions

To evaluate the contribution of indirect action mediated by OH radicals in biological effects of high LET radiations, we examined the production of 8-hydroxy-20-deoxyguanosine (8-OHdG) in 20-deoxyguanosine aqueous solution using various ion species with an LET range from 20 to 300 keV/mm. The 8-OHdG yield decreased with increasing LET. In the hypoxic irradiation condition, the yield showed constant or rather increasing tendency above about 100 keV/mm, which is consistent with an oxygen-in-the-track hypothesis to explain the diminishment of oxygen effect

Protective effects of amino acids on plasmid DNA damage induced by therapeutic carbon Ions

Radioprotectors with few side effects are useful for carbon ion therapy, which directly induces clustering damage in DNA. With the aim of finding the most effective radioprotector, the authors have investigated the effects of selected amino acids which might have chemical DNA-repair functions against therapeutic carbon ions. In the present study, we employed five amino acids: tryptophan (Trp), cysteine (Cys), methionine (Met), valine (Val), and alanine (Ala). Samples of supercoiled pBR322 plasmid DNA with a 17 milli molar (mM) amino acid were prepared in TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 7.5). Phosphate-buffered saline (PBS) was also used in assays of the 0.17 mM amino acid. The samples were irradiated with carbon ion beams (290 MeV/u) on 6 cm spread-out Bragg peak at the National Institute of Radiological Sciences-Heavy Ion Medial Accelerator in Chiba, Japan. Breaks in the DNA were detected as changes in the plasmids and quantified by subsequent electrophoresis on agarose gels. DNA damage yields and protection factors for each amino acid were calculated as ratios relative to reagent-free controls. Trp and Cys showed radioprotective effects against plasmid DNA damage induced by a carbon ion beams, both in PBS and TE buffer, comparable to those of Met. The double-strand break (DSB) yields and protective effects of Trp were comparable to those of Cys. The yields of both single-strand breaks and DSBs correlated with the scavenging capacity of hydroxyl radicals (rate constant for scavenging hydroxyl radicals multiplied by the amino acid concentration) in bulk solution. These data indicated that the radioprotective effects of amino acids against plasmid DNA damage induced by carbon ion irradiation could be mainly explained by the scavenging capacity of hydroxyl radicals. These findings imply that some amino acids such as Trp, Cys and Met have good potentials as radioprotectors preventing DNA damages in normal tissues in carbon ion therapy