Fast product formation and slow product release are important features in a hysteretic reaction mechanism of glutathione transferase T2-2.

The reaction mechanism of rat glutathione transferase T2-2 has been studied using pre-steady-state and steady-state kinetics. Several parts of the catalytic cycle including binding of substrates, product formation, and product release were investigated. Under saturating conditions, a two-step product release was found to be rate limiting in the enzyme-catalyzed reactions between the nucleophilic substrate glutathione and either of the two electrophilic substrates 1-menaphthyl sulfate and 4-nitrobenzyl chloride. The rate constant for pre-steady-state product formation on rat glutathione transferase T2-2 has an observed pK(a) value of 5.7 apparently due to ionization of the sulfhydryl group of glutathione. This rate constant is approximately 2 orders of magnitude higher than k(cat) at pH values of >6. It can be predicted from the pH dependence that product formation would be the sole rate-limiting step at pH values of <3. A hysteretic mechanism of rGST T2-2 is proposed based on a slow conformational transition detected in pre-steady-state displacement experiments.     

A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince,pear and olive genotypes under iron deficiency stress

Quince (Cydonia oblonga Mill.), pear (Pyrus communis L.) and olive (Olea europaea L.) genotypes were evaluated for their tolerance to iron deficiency stress by growing young plants in three types of aerated nutrient solutions: (1) with iron, (2) without iron or (3) low in iron and with 10 mM bicarbonate. Plants were obtained either from rooted softwood cuttings or from germination of seeds. The degree of tolerance was evaluated with several indices: (1) the chlorophyll content, (2) the root Fe³⁺ reducing capacity and (3) the whole plant relative growth. Fifteen hours before Fe³⁺ reducing capacity determination, iron was applied to the roots of plants with iron-stress, since this method resulted in increasing the reductase activity. All quince and pear genotypes increased the root Fe³⁺ reducing capacity when grown in the treatments for iron-stress, in relation to control plants of the same genotypes. In olive cultivars, the Fe³⁺ reducing capacity was lower in the iron-stress treatments than in the control one. Studying the relationship between relative growth and chlorophyll content for each genotype under iron-stress, in relation to both indices in control plants, a classification of species and genotypes was established. According to that, most olive cultivars and some pear rootstocks and cultivars appear more iron-efficient than quince rootstocks. Our study shows that in some woody species, determining root Fe³⁺ reducing capacity is not the best method to establish tolerance to iron deficiency stress.     

Ethological study of early language

Sign language studies of cross-fostered chimpanzees measure the effect of special rearing conditions on the development of very young chimpanzees. Cross-fostered chimpanzees, like human children, develop gradually in a process that takes many years. Here we discuss details of the procedure, the overlap between human and chimpanzee infants in the contents of the first 50-item vocabularies, and the ways in which the signs of the chimpanzees exhibit the fuzziness of natural language categories. We also compare the cross-fostering approach with more traditional modular approaches to the study of language-like behavior in nonhuman animals. Project Washoe was originally supported by grants MH-12154 from the National Institute of Mental Health and GB-7432 from the National Science Foundation. We gratefully acknowledge this support and the support that later sign language studies of chimpanzees have received since then from NIH, NSF, the National Geographic Society, the Grant Foundation, the Spencer Foundation, the University of Nevada, and the UNR Foundation.     

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.     

Effect of training on the rating of perceived exertion at the ventilatory threshold

The purpose of this study was to determine the effect of training on the rating of perceived exertion (RPE) at the ventilatory threshold. College students were assigned to either training (n = 17) or control (n = 10) groups. Trainers completed 18 interval training sessions (five X 5 min cycling at 90-100% VO2max) and 8 continuous training sessions (40 min running or cycling) in 6 weeks. Pre- and post-training, cardiorespiratory, metabolic, and perceptual variables were measured at the ventilatory threshold during graded exercise tests on a cycle ergometer. Ventilatory threshold was that point above which VE X VO2-1 increased abruptly relative to work rate. Post-training means of trained and control subjects were compared using analysis of covariance, with pre-training values as covariates. Following training, the adjusted means for the trained subjects were significantly greater (p less than 0.05) than for controls for VO2max (6%), and for work rate (20%), VO2 (23%), and %VO2max (13%) at the ventilatory threshold. However, adjusted means for RPE at the ventilatory threshold were not significantly different (2%). Both before and after training, exercise at the ventilatory threshold was perceived as 'somewhat hard' to 'hard' (RPE = 13-15) by both groups. The relationship between RPE and %VO2max was altered by training, with trained subjects having a lower RPE at a given %VO2max. It is concluded that RPE at the ventilatory threshold is not affected by training, despite that after training the ventilatory threshold occurs at a higher work rate and is associated with higher absolute and relative metabolic and cardiorespiratory demands.     

Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells

Methylisocyanate (MIC) induced mutagenic responses in the absence of exogenous activation in the mouse lymphoma cell forward mutation assay at concentrations as low as 8-24 microM. MIC produced predominantly small mutant colonies, suggesting the possibility of clastogenic activity. The intermediate hydrolysis product, methylamine, was also mutagenic without exogenous activation but required several hundred-fold higher concentrations (ca. 3 mM). N,N'-Dimethylurea, the final product in the reaction of methylisocyanate and water, was totally refractory in either the presence or absence of S9 for concentrations up to 57 mM (5 mg/ml). The ethyl ester of N-methylcarbamic acid was also tested since it was the only available analogue to the highly reactive N-methylcarbamic acid intermediate. This compound was mutagenic only in the presence of S9 at doses exceeding 5-40 microM, which suggested the possibility that the free acid, produced by enzymatic hydrolysis, is also mutagenic. The mutagenic activity of the ester resulted solely in the production of small mutant colonies.     

The effect of phenformin and other adenosine triphosphate (ATP)-lowering agents on insulin binding to IM-9 human cultured lymphocytes

In the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM-9 human cultured lymphocytes. After a 24-hr preincubation, phenformin induced a twofold increase in specific 125I-insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I-insulin binding and energy metabolism in IM-9 cells was investigated. The phenformin-induced increase in insulin binding to IM-9 cells was related to a time- and dose-dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2-deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM-9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.     

Deanol Affects Choline Metabolism in Peripheral Tissues of Mice

Abstract: The enzymatic hydrolysis of UDP-galactose in rat and calf brain was studied. The hydrolysis occurs in two steps: The first is the conversion of UDP-galactose to galactose-1-phosphate catalyzed by nucleotide pyrophosphatase (EC 3.6.1.9), and the second is the conversion of the latter to free galactose by alkaline phosphatase (EC 3.1.3.1). The overall conversion has a pH optimum of 9.0, but there is considerable activity at pH 7.4, which is the optimum for UDP-galactose:ceramide galactosyltransferase in the synthesis of cerebrosides. Preparations from cytosol from calf brain cerebellum or stem that were enriched in UDP-galactose hydrolytic activity inhibit cerebroside synthesis under conditions optimal for the synthesis. Microsome-rich and nuclear debris fractions contain the highest apparent specific activity among the subcellular fractions studied. Hydrolysis of UDP-galactose occurs in all areas of brain, brainstem having the highest activity. The apparent specific activity in jimpy mouse brain homogenate is nearly twice as high as in the control brain homogenate.