Gas chromatographic—mass spectrometric determination of tiopronin in human blood using acrylic acid esters as a derivatization reagent for the thiol group

A gas chromatographic—mass spectrometric method for determining tiopronin, which has a thiol group, in human blood has been described. To prevent the oxidative degradation of tiopronin in the blood, its thiol group was immediately protected by treatment with isobutyl acrylate, which reacted readily with tiopronin in a 0.1 M Na₂HPO₄ solution (pH 9.1). The reaction was quantitative within 30 min. The blood sample was deproteinized and purified by a combination of liquid—liquid extraction and solid-phase extraction. Finally, the carboxyl moiety of the ester adduct was derivatized to the pentafluorobenzyl ester. The derivatives of tiopronin and the internal standard were analysed with gas chromatography—mass spectrometry. The precision of the method was satisfactory, and the calibration curve had good linearity in the concentration range investigated. The limit of determination of tiopronin in blood was estimated to be ca. 1 ng/ml.     

Bird carcass detection from integrated trials at multiple wind farms

It is often necessary to estimate the number of wind turbine collision fatalities to assess impacts to birds following construction of wind farms. Detection of bird carcasses at wind turbines in the field is affected by carcass persistence and searcher detection rate. Integrated detection trials, which integrate carcass persistence and searcher detection trials into the periodic fatality search, have been proposed as an effective method for estimating these parameters. The purpose of our study was to test whether and how environmental factors affect integrated detection trial outcomes at multiple wind farms. We conducted this study at 10 wind farms in various environments of Japan. Binary data on trial outcomes in open versus forested areas served as our response variable in a generalized additive mixed model informed by days into trial, carcass body mass, season, whether snow covered the ground, and precipitation. For both ground cover types, days into trial and body mass were included in all the top models, suggesting that these factors most influenced bird carcass detection probability in integrated trials. The best model in open areas included days into trial, body mass, snow, and precipitation, and the best model in forested areas included days into trial, body mass, snow, precipitation, and season. Values of area under the curve indicated high accuracy of the best model for both ground cover types. The survey design needs to be appropriate to the size of the target species and to the environment in which the impacts will occur, such as the site's seasonality, its ground cover, and whether snow will cover the ground. Frequency of post-construction fatality monitoring should also be set cautiously, especially at wind farms located on small-bird migration routes, at wind farms in open areas, in areas with snow-covered ground in winter, or in forested areas during spring and summer because detection probabilities decline fastest under such conditions.     

Purification and characterization of pig lung carbonyl reductase.

A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.     

Changes in food intake and growth rate in mice under hypergravity.

Mice exposed to hypergravity, especially soon after start of exposure, diminished the body weight and the food intake. The amount of food intake was kept less than that of the ground control during hypergravity breeding for 2 weeks. Furthermore, the weight of testis relative to the body weight increased compared to that of 1 G control although the relative weights of liver and kidney were not changed. The purpose of this study was whether the low growth rate of the body weight and the increase of the relative testis weight were induced by the decrease of the food intake under hypergravity. We divided 3 weeks old male mice to 3 groups; the 1 G (ground control), the food restricted (FR) under 1 G, and the 3 G group. The 3 G group bred for 2 weeks under the centrifuge at 3 G. The FR group was given the same amount of food as the group ate. The changes in the body weight and the relative weights of testis, spleen and seminal vesicle of the FR group were similar to those of the 3 G group. The hunger test revealed that only the FR group was hunger. Our results suggested that the decrease of the food intake both in response to hypergravity and the food restriction induced the decrease of the body weight but the increase of the relative testis weight.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

FAB CID-MS/MS characterization of tetrasaccharide tri- and tetrasulfate derived from the antigenic determinant recognized by the anti-chondroitin sulfate monoclonal antibody MO-225

The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MS/MS mass spectrometry/mass spectrometry - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose     

Immunohistochemistry of Grandry corpuscles in the oral mucosa of the duck bill: a light- and electron-microscopic study

Grandry corpuscles in the oral mucosa of the upper bill of the duck were immunohistochemically studied using antisera against calcitonin gene-related peptide (CGRP), galanin, methionine-enkephalin, neuropeptide Y (NPY), somatostatin, substance P (SP) and vasoactive intestinal peptide (VIP). Grandry corpuscles in the lamina propria selectively showed only SP-like immunoreactivity. Herbst corpuscles distributed near Grandry corpuscles were negative to all antisera applied. Although immunoreactive products in the Grandry corpuscles were found as granules in the peripheral cytoplasm of the Grandry cell, the axon terminals and satellite cells exhibited no reactivity. In pre-embedding electron-microscopic sections, SP-like immunoreactive products visualized with 3,3-diaminobezidine were localized in the granules of Grandry cells, but no labeling was observed in the cytoplasmic matrix or cell organelles. Electron-immunocytochemical labeling with colloidal gold by the post-embedding method clearly demonstrated that the SP antigen was localized only in the granules. It is presumed that Grandry cells have a secretory function. However, the function and the method of release of the SP contained in the observed granules remains obscure. Some CGRP-, NPY-, SP- and VIP-like-immunoreactive nerve fibers with varicosities associated with blood vessels and nerve fiber bundles of various sizes were observed in the lamina propria, but no such fibers penetrated into the intraepitherial layer. Nerve fibers positive for SP and VIP were also found in the interlobular connective tissue of the palatine glands. Some SP-positive neurons were detected in the vicinity of the palatine glands.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.