Group-selective reagent modification of the sodium- and chloride-coupled glycine transporter under native and reconstituted conditions.

Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the Vmax for glycine transport and to a lesser extent act on the apparent Km. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.     

Purification and properties of a coagulant proteinase isolated from bushmaster (Lachesis muta) venom (Serpentes: Viperidae)

The venom of Lachesis muta is a rich source of a thrombin-like enzyme. Its coagulant proteinase was purified by DEAE -Sephadex A -50 followed by agmatine CH -Sepharose and gel filtration on Sephadex G-100. On polyacrylamide gel electrophoresis at pH 8.4 a single band was observed. Its molecular weight by gel filtration was 49,000. The coagulant and esterolytic activities toward human fibrinogen and Tame of the inudasa were 662 NIH units/mg of protein and 4.37 delta OD225/min x 10(-3)/micrograms/ml, respectively. These values represent 23 and 5.7 fold increase over the crude venom. The enzyme mudasa, was evaluated with serum from human patients at Hospital Nacional de Ni?os Dr. Carlos Sáenz Herrera and found to be a valuable reagent for the quantification of fibrinogen on heparinized plasma.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

Glyceraldehyde-3-phosphate dehydrogenase activity studied under physiological conditions with a linear assay.

An assay method for glyceraldehyde-3-phosphate dehydrogenase in which none of the primary products accumulate and which gives linear kinetics under physiological conditions has been developed. It is based on the use of the 1,3-diphosphoglycerate produced by the enzyme for the formation of NADPH, while the NADH produced is recycled with an auxiliary system. Revised Km values at pH 7.4 for the muscle (rabbit and rat) enzyme are: glyceraldehyde-3-P, 50 μM; NAD, 100 μM; Pi, 10 mM. The rat erythrocyte enzyme gave similar values except for glyceraldehyde-3-P which was 300 μM. Cooperativity for NAD⁺ tends to be positive but is a variable parameter.     

Can we disentangle predator–prey interactions from species distributions at a macro‐scale? A case study with a raptor species

There is a debate on whether an influence of biotic interactions on species distributions can be reflected at macro‐scale levels. Whereas the influence of biotic interactions on spatial arrangements is beginning to be studied at local scales, similar studies at macro‐scale levels are scarce. There is no example disentangling, from other similarities with related species, the influence of predator–prey interactions on species distributions at macro‐scale levels. In this study we aimed to disentangle predator–prey interactions from species distribution data following an experimental approach including a factorial design. As a case of study we selected the short‐toed eagle because of its known specialization on certain prey reptiles. We used presence–absence data at a 100 km2 spatial resolution to extract the explanatory capacity of different environmental predictors (five abiotic and two biotic predictors) on the short‐toed eagle species distribution in peninsular Spain. Abiotic predictors were relevant climatic and topographic variables, and relevant biotic predictors were prey richness and forest density. In addition to the short‐toed eagle, we also obtained the predictor's explanatory capacities for 1) species of the same family Accipitridae (as a reference), 2) for other birds of different families (as controls) and 3) artificial species with randomly selected presences (as null models). We run 650 models to test for similarities of the short‐toed eagle, controls and null models with reference species, assessed by regressions of explanatory capacities. We found higher similarities between the short‐toed eagle and other species of the family Accipitridae than for the other two groups. Once corrected by the family effect, our analyses revealed a signal of predator–prey interaction embedded in species distribution data. This result was corroborated with additional analyses testing for differences in the concordance between the distributions of different bird categories and the distributions of either prey or non‐prey species of the short‐toed eagle. Our analyses were useful to disentangle a signal of predator–prey interactions from species distribution data at a macro‐scale. This study highlights the importance of disentangling specific features from the variation shared with a given taxonomic level.     

Biochemical and physiological changes produced by Azotobacter chroococcum (INIFAT5 strain) on pineapple in vitro-plantlets during acclimatization

This study highlights some of the effects of the application of Azotobacter chroococcum (INIFAT5 strain) on in vitro-pineapple plantlets during acclimatization. The bacteria were sprayed immediately after transplanting to the ex vitro environment; the plants were then sprayed every 4 week. Subsequently (4 months) the evaluated variables included plantlet fresh and dry weights, leaf and root lengths, and composition of minerals, amino-acids, carbohydrates and proteins. Photosynthesis indicators were also evaluated. Significant effects of the application of Azotobacter over pineapple plantlets during acclimatization were observed in the mineral, amino-acid, carbohydrate and protein levels, as well as, in the photosynthesis indicators. Contrastingly, plant growth parameters showed modest increases caused by the bacteria, although they were statistically significant. Looking into specific minerals, the following significant effects of Azotobacter should be highlighted: increased levels of nitrogen, phosphorous, potassium, magnesium, copper and zinc. Moreover, contents of all amino-acids recorded showed significant increases in their levels in sprayed plantlets. Carbohydrates were also increased in leaves of plantlets bio-fertilized with the bacteria, mainly sucrose and fructose. Chlorophyll b levels were also significantly increased by Azotobacter. The biofertilizer did not modify levels of calcium, iron or manganese.