Chemical composition and immunological characterization of polysaccharide antigen from Eubacterium saburreum T18.

Two types of polysaccharide were obtained from the oral microorganism Eubacterium saburreum T18 by formamide extraction and subsequent gel filtration and ion-exchange chromatography. One polysaccharide, which was composed of D-glycero-D-galacto-heptose, had antigenic activity in an immunoprecipitation reaction with rabbit anti-T18 serum due to immunoglobulin M antibodies. The second polysaccharide was composed of D-glycero-D-manno-heptose and L-rhamnose, but it did not have immunoprecipitation activity. These polysaccharide antigens were not alkali labile and differed from E. saburreum L44 and T27 antigens, which were composed of D-glycero-D-galacto-heptose.     

Development of a particle agglutination assay system for detecting Japanese encephalitis virus-specific human IgM,using hydroxyapatite-coated nylon beads

Japanese encephalitis virus-specific IgM is a reliable indicator for serodiagnosis of Japanese encephalitis. A particle agglutination (PA) assay system was developed to detect anti-Japanese encephalitis virus IgM in human serum samples. The newly developed PA assay consisted of hydroxyapatite-coated nylon beads and V-bottom 96-well microplates. Hydroxyapatite-coated nylon beads were coated with Japanese encephalitis virus antigens. Japanese encephalitis virus antigen-coated, hydroxyapatite-coated nylon beads agglutinated in the IgM-captured wells when anti-Japanese encephalitis virus IgM-positive serum samples were used. A button pattern was formed at the bottom of the wells when anti-Japanese encephalitis virus IgM-negative serum samples were used. Thirty anti-Japanese encephalitis virus IgM-positive serum samples from Japanese encephalitis-confirmed cases were tested by the PA assay. All these serum samples were determined to be Japanese encephalitis virus IgM-positive. IgM titers determined by the PA assay corresponded to those determined by enzyme-linked immunosorbent assay. The titers were consistent in two independent PA assays. These results indicate that the newly developed PA assay is a reliable method for detecting anti-Japanese encephalitis virus IgM in human serum samples and that this assay will be a suitable diagnostic system especially in rural areas of Asia.     

High-performance liquid chromatography with peroxyoxalate chemiluminescence detection of bisphenol A migrated from polycarbonate baby bottles using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a label

A highly sensitive and selective high-performance liquid chromatographic method with peroxyoxalate chemiluminescence detection for the determination of bisphenol A at sub-ppb levels is described. Bisphenol A was derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride and the excess unreacted reagent was removed by a simple solid-phase extraction procedure with recoveries of approximately 60%. The separation was carried out isocratically on an ODS column and the derivatized bisphenol A was detected by peroxyoxalate chemiluminescence. A mixture of bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate (0.6 mM) and hydrogen peroxide (25.0 mM) dissolved in acetonitrile was used as a chemiluminescence reagent solution with a mixture of imidazole-HNO3 buffer (40.0 mM, pH 7.0): acetonitrile (17:83, v/v) as a mobile phase. The linear standard curve was obtained over the range from 0.57 (2.5) to 22.8 (100) ppb (nM) (r=0.996) with a detection limit of 0.38 ppb (2.8 fmol on column) at a signal-to-noise ratio of 3. The method was successfully applied to the determination of bisphenol A in hot water in contact with commercially available baby bottle samples.     

Immunohistochemical study of copper-zinc and manganese superoxide dismutases in the lungs of human fetuses and newborn infants: Developmental profile and alterations in hyaline membrane disease and bronchopulmonary dysplasia

To determine the late gestational development of copper-zinc (CnZn) and manganese (Mn) superoxide dismutases (SOD) in human lung, immunohistochemical localization was performed for each SOD. The lung samples were taken from five aborted fetuses, four fetuses in which intrauterine death occurred, one full-term neonate, two premature infants with hyaline membrane disease and one premature infant with bronchopulmonary dysplasia (BPD). Morphometry was performed, and the percent area of positive staining was computed. The bronchial epithelium was intensely stained from the early stages of gestation (i.e. 17 weeks), while the staining intensity for both CuZnSOD and MnSOD in the peripheral airways increased gradually during lung development. The mean percent area of the staining for CuZnSOD and MnSOD from 16 to 38 weeks was increased 30-fold and 8-fold, respectively, and further increases were observed postnatally. CuZnSOD staining was markedly decreased in lungs with respiratory disorders. However, proliferating type II pneumocytes were intensely stained for MnSOD in the BPD lungs, making the staining area 3-fold larger than that in the control lungs. These results clearly depict age-related increases in staining for both CuZnSOD and MnSOD and an alteration in SOD distribution associated with neonatal respiratory disorders.     

Effects of ATP antagonists on purinoceptor-operated inward currents in rat phaeochromocytoma cells

The effects of suramin, reactive blue 2 (RB2) and d-tubocurarine (d-TC) were investigated electrophysiologically to elucidate the mechanisms underlying their antagonism of P₂ purinoceptor-mediated responses. All three compounds inhibited an adenosine triphosphate (ATP)-activated inward current in rat phaeochromocytoma PC12 cells in a concentration-dependent manner. The order of potency was RB2 > suramin > d-TC. The inhibition induced by suramin or RB2 was reversible, whereas that induced by d-TC was not reversed after a 5-min rinse. The inactivation of the ATP-activated current was accelerated by d-TC but not by suramin or RB2. RB2 administered simultaneously with ATP exerted much weaker inhibition compared to that induced by prior administration, suggesting that RB2 is a slowly acting antagonist. This was not observed for suramin or d-TC. Suramin and RB2 caused a parallel shift in the concentration/response curve for the ATP-activated current. With d-TC the maximal response of ATP was decreased but the concentration producing half-maximal response was unchanged. The voltage dependency of the ATP-activated current showed less inward rectification in the presence of d-TC. Suramin or RB2 did not affect the voltage dependency. These results suggest that suramin and RB2 reversibly block binding of ATP to receptors, whereas d-TC blocks ion permeability through the ATP-activated channel.