Crystal structure of the GpIbalpha-thrombin complex essential for platelet aggregation

Direct interaction between platelet receptor glycoprotein Ibalpha (GpIbalpha) and thrombin is required for platelet aggregation and activation at sites of vascular injury. Abnormal GpIbalpha-thrombin binding is associated with many pathological conditions,including occlusive arterial thrombosis and bleeding disorders. The crystal structure of the GpIbalpha-thrombin complex at 2.6 angstrom resolution reveals simultaneous interactions of GpIbalpha with exosite I of one thrombin molecule,and with exosite II of a second thrombin molecule. In the crystal lattice,the periodic arrangement of GpIbalpha-thrombin complexes mirrors a scaffold that could serve as a driving force for tight platelet adhesion. The details of these interactions reconcile GpIbalpha-thrombin binding modes that are presently controversial,highlighting two distinct interfaces that are potential targets for development of novel antithrombotic drugs.     

Toxicokinetics of Malathion in Larval Stages of the Toad Bufo arenarum (Hensel): Effect of Exogenous Spermidine

The aim of this work was to study the absorption, biotransformation, and excretion of malathion (¹⁴C-methoxy) and its metabolites in larval stages of the toad Bufo arenarum (Hensel). Also, changes in malathion metabolization by the action of the exogenous polyamine spermidine were studied. Malathion clearance from the media was uniexponential, and spermidine reduced the uptake in the larvae, causing an increase in the apparent half-life of the toxicant. Concomitant with this effect, spermidine increased the level of induction of mixed-function oxidases due to malathion and caused a progressively higher malaoxon/malathion ratio. As a consequence of the higher conversion to the active metabolite malaoxon, spermidine also provoked a significant enhancement in the inhibitory effect of Malathion on acetylcholinesterase activity. [methoxy¹⁴C]malathion metabolites, such as carboxylesterase and glutathione S-transferase products, were detected in the toad larvae and in the media. The excreted products of carboxylesterase activity were about 70% of the total radioactivity, and the glutathione S-transferase products (methyl glutathione) were 20–30% of the total radioactivity. No significant variations in the levels of excreted products due to the action of exogenous spermidine were detected. Malathion inhibited carboxylesterase activity, independent of the presence of spermidine in the media. In turn, glutathione S-transferase activity was induced by spermidine, but was not affected by the exposure to low concentrations of malathion for 48 h. We conclude that the presence of spermidine in the medium modifies malathion toxicokinetics, increasing its toxicity in B. arenarum larvae.     

Histochemical study of the intestinal absorption,liver and lens effect with zinc‐supplemented diets for gilthead seabream

To describe the effect and the distribution of zinc in different tissues of a representative marine fish species, gilthead seabreams (Sparus aurata Linnaeus, 1758) were fed different diets containing zinc in different sources (organic, inorganic, encapsulated, blood‐rich diet or fishmeal). The effect was monitored by histology in the lens, liver and intestine. In addition, the anterior and posterior intestine was studied by means of autometallography, a histochemical silver‐based staining method to determine the zinc flow and distribution. The histology of lens did not improve by dietary zinc as there was no occurrence of cataracts. In the liver, the experimental diets were correlated with different changes in the tissue architecture. In the intestine, no histological changes were found using haematoxylin and eosin stain. However, with the use of autometallography stain, it was possible to visualize and describe the route that zinc follows through the enterocytes and the lamina propria. Organic and inorganic zinc diet produced the richest silver deposition in the anterior intestine.     

Antimicrobial activity in bacteria isolated from Senegalese sole, Solea senegalensis, fed with natural prey

Intestinal bacteria in marine fish may produce antimicrobial substances which inhibit pathogenic bacteria. The aim of this study was to determine the influence of a change of fish diet on the antimicrobial activity of the culturable aerobic gut microflora of Senegalese sole, Solea senegalensis. Pre‐adult 15‐month‐old fish previously fed on an artificial diet, were fed polychaetes (Hediste diversicolor), which form part of the natural diet of Senegalese sole. Samples were taken0, 3 and 6 weeks after start of the experiment from the stomach, small and large intestine of the fish. The bacterial strains isolated from these samples were sub‐cultured to pure cultures and stored at ?80°C. Several biochemical tests were run to obtain some basic phenotypic characteristics of the isolated strains. Amplification and sequencing of 16S rDNA fragments were used to identify the majority of the bacterial strains isolated. The identification by use of this molecular approach gave results in agreement to the phenotypic characterization. Feeding with polychaetes significantly increased (P<0.05) the numbers of presumptive Vibrio isolates in the gut. The number of bacterial strains with antimicrobial activity, as determined by two in vitro approaches, was significantly (P<0.05) increased by feeding with polychaetes.     

Characterization of volatile compounds of Mezcal, an ethnic alcoholic beverage obtained from Agave salmiana

Commercial mezcals (white, white with worm, rested, rested with worm, and aged) produced from Agave salmiana were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). Thirty-seven compounds were identified, and nine of them were classified as major compounds of mezcal (MCM). Saturated alcohols, ethyl acetate, ethyl 2-hydroxypropanoate, and acetic acid form the MCM group. Minor compounds of mezcal group include other alcohols, aldehydes, ketones, large chain ethyl esters, organic acids, furans, terpenes, alkenes, and alkynes. Most of the compounds found in mezcals in this study are similar to those present in tequilas and other alcoholic beverages. However, mezcals contain unique compounds such as limonene and pentyl butanoate, which can be used as markers for the authenticity of mezcal produced from A. salmiana.     

Effect of organogenesis on steroidal saponin biosynthesis in calli cultures of Ruscus aculeatus

Calli cultures derived from Ruscus aculeatus rhizomes were investigated for their potential to biosynthesize saponins. The capacity of undifferentiated tissues to form steroidal saponins is very limited, but when the calli developed organogenesis, mainly aerial shoots and roots, the saponin production increased significantly. Plantlets regenerated from aerial shoots of Ruscus calli showed a saponin pattern similar to that of the callus cultures but the levels of saponins found in the aerial part and roots were significantly greater.     

Carbohydrate use and assimilation by litter and soil fungi assessed by carbon isotopes and BIOLOG assays

Soil fungi are integral to decomposition in forests and other habitats, yet identifying probable functional roles of different taxa is problematic. Here, we compared carbohydrate assimilation patterns derived from stable isotope analyses on cultures with patterns of metabolic activity measured on Biolog^® SF-P plates for 12 taxa of soil- and litter-inhabiting saprotrophic fungi isolated from Douglas-fir (Pseudotsuga menziesii) ecosystems. To determine the relative assimilation of carbon from malt extract versus sucrose by ¹³C stable isotope analyses, we cultured fungi with malt extract (consisting primarily of glucose and maltose) plus either C₃- or C₄-derived sucrose as carbon sources. Rhodotorula graminis and F. oxysporum assimilated the highest proportion of sucrose, a Mortierella isolate and an unidentified sterile isolate (FPC 341) assimilated the lowest proportion of sucrose, and remaining cultures assimilated similar and intermediate proportions of sucrose. On Biolog plates, low metabolic activity of Mortierella and FPC 341 on sucrose and R. graminis and F. oxysporum on maltose were qualitatively consistent with isotopic results. Assimilation of sucrose calculated isotopically was correlated with the ratio of sucrose: maltose activity calculated from Biolog assays (r²=0.45, P=0.0145, n=12). Metabolic activity on Biolog plates for six other common soil carbohydrates were also determined: glucose, fructose, galactose, cellobiose, lactose, and glycogen. Metabolic activity was greatest overall on maltose and glucose and lowest on fructose. Two of the isolates (Aspergillus flavus and F. oxysporum) had higher metabolic activity on the glucose-containing disaccharide cellobiose than on glucose, strongly suggesting preferential uptake of cellobiose compared to glucose and suggesting the potential ability to use cellulose. The high metabolic activity of these cultures on galactose, a primary constituent of hemicellulose, also suggested cellulolytic capabilities. Our results indicated that stable isotope studies and Biolog assays may provide complementary information to characterize metabolic potential of fungi in forest litter and soil.