Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose

The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.     

Phenotypic profile and functions of T cell receptor-gamma delta-bearing cells from patients with primary immunodeficiency syndrome

TCR-gamma delta-bearing T cells have been reported to be increased in several immunodeficient patients. However, their functional role and phenotypic characterization have not yet been well documented. In this study we examined the surface phenotypes and functional properties of TCR-gamma delta+ cells from several patients with primary immunodeficiency syndrome. It was demonstrated that TCR-gamma delta+ cells detected by TCR-delta 1 mAb were increased in some of the patients, particularly in patients with Wiskott-Aldrich syndrome and severe combined immune deficiency. The TCR-gamma delta+ cells showed such a unique profile that more than 60% of the cells expressed delta-TCS1, which is normally present in a lesser amount, and that most of the cells lacked CD5 T lineage marker. TCR-gamma delta+ cells from the patients with primary immunodeficiency syndrome served as NK cells as observed in normal individuals, while displaying weak LAK and allogeneic cell-specific killer activities. The TCR-gamma delta+ cells were classified into several subpopulations according to their antigenic phenotype, then their NK activity of normal individuals and patients, lymphokine-activated killer and allo-specific killer activities of normal individuals were compared among the subpopulations. Delta-TCS1+ cells mediated almost the same killer activities as total TCR-gamma delta+ cells, whereas CD8+ TCR-gamma delta+ cells displayed stronger cytotoxic activities in both normal subjects and the patients with primary immunodeficiency syndrome.     

Throughfall,stemflow and interception of rainwater in an evergreen broadleaved forest

In order to analyze the dyanmics of heavy metals in a forest ecosystem, throughfall and stemflow were collected for individual rain showers in an evergreen broad-leaved forest dominated byCastanopsis cuspidata. The relation between throughfall (or stemflow) (Px) and gross rainfall (P) was approximated by a linear regression equationPx=a(P-b). The values of coefficient “a” were 0.32–2.02 for throughfall at each sampling point and the mean values for 1976 and 1977–1978 were 0.682 and 0.767, respectively. The stemflow volume differed widely among individual trees, depending mainly on the tree form of each species. In particular, the tree form ofPasania edulis was found to be especially suited to collecting stemflow. OneP. edulis tree collected 64% of the rainwater that fell onto its crown as stemflow. The ratio of stemflow to gross rainfall decreased in summer resulting from an increase in leaf biomass and an increase in air temperature. In fact, the values of coefficient “a” for mean stemflow per unit area were 0.180 for summer and 0.229 for other seasons in 1976, and 0.145 for summer and 0.155 for other seasons in 1977–1978 for different sampled trees.     

Observation of water and Na+ in tissues of theBruguiera gymnorrhiza by1H-and23Na-NMR imaging

Distributions of free water, which is called water in this investigation, in mangrove (Bruguiera gymnorrhiza (L.) Lam.) tissues were examined by using¹H-NMR imaging, and accumulation of Na⁺ in hypocotyls was examined by using high resolution²³Na-NMR and²³Na-NMR imaging in relation to their morphology. Water located preferentially in the epidermis and the outer layer of cortex adjacent to the epidermis, and around vascular bundles of a root, a branch stem, and hypocotyls. Amount of water detected in the middle parts of cortex and pith was small unlikeAucuba japonica branch tissue. On the other hand, relatively high concentration of Na⁺ was detected in the pith besides the epidermis and the outer layer of the cortex adjacent to the epidermis, and around vascular bundles of the hypocotyl. The localization of Na⁺ did not correspond to that of water. Concentrations of Na⁺ accumulated (up to 22mM) in the hypocotyl were approximately 10 times higher than those observed in tissues of ordinary plants. The characteristic water distribution and accumulation of Na⁺ in the mangrove are considered to relate to their ecological nature for the adaptation to saline environments.     

Differences in body sizes and physiological conditions among castes in the ponerine ant Cryptopone sauteri (Hymenoptera: Formicidae)

In several poneroid ant species, mated workers alone undertake reproduction. The reproductive systems of such species have been examined extensively. However, few studies have investigated species with alate queens, which reproduce after shedding their wings. We compared body sizes and the numbers of ovarioles between queens and workers in the ant Cryptopone sauteri with alate queens. We also compared ovariole development between the castes to evaluate their reproductive systems. Approximately 60% of the nests collected were queenless. We often detected unmated queens in the nests throughout the year, but did not obtain strong evidence for their reproduction. Although significant differences were observed in the number of ovarioles and body characteristics between the queens and workers, the differences were not as prominent as those observed in Formicinae and Myrmicinae. We propose two alternative hypotheses, failure of nuptial flight or postponement of reproduction, to explain the presence of unmated queens in the nests.     

Screening of plants for phytoremediation of oil-contaminated soil

Several species of ornamental flowering plants were evaluated regarding their phytoremediation ability for the cleanup of oil-contaminated soil in Japanese environmental conditions. Thirty-three species of plants were grown in oil-contaminated soil, and Mimosa, Zinnia, Gazania, and cypress vine were selected for further assessment on the basis of their favorable initial growth. No significant difference was observed in the above-ground and under-ground dry matter weight of Gazania 180 days after sowing between contaminated and non-contaminated plots. However, the other 3 species of plants died by the 180^th day, indicating that Gazania has an especially strong tolerance for oil-contaminated soil. The total petroleum hydrocarbon concentration of the soils in which the 4 species of plants were grown decreased by 45–49% by the 180^th day. Compared to an irrigated plot, the dehydrogenase activity of the contaminated soil also increased significantly, indicating a phytoremediation effect by the 4 tested plants. Mimosa, Zinnia, and cypress vine all died by the 180^th day after seeding, but the roots themselves became a source of nutrients for the soil microorganisms, which led to a phytoremediation effect by increase in the oil degradation activity. It has been indicated that Gazania is most appropriate for phytoremediation of oil-contaminated soil.     

Order of binding of substrate to valyl-tRNA synthetase from Bacillus stearothermophilus in amino acid activation reaction

Amino acid activation reaction with valyl-tRNA synthetase (EC 6.1.1.9) from Bacillus stearothermophilus was studied kinetically by measuring ATP-PPi exchange to find the order of the binding of substrate to the enzyme. The effects of the concentration of the substrates (L-valine and ATP) and two dead-end inhibitors (L-valinol and adenosine) on the reaction rate were analyzed. The results indicate that L-valine and ATP are bound to the enzyme in a random sequence. This conclusion is consistent with the one previously suggested by static binding experiments.     

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of l-cysteine

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing l-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC–mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and ¹H and ¹³C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and l-cysteine in foods, and might act as a mutagen.