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.     

Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems

The structure of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems was studied both in situ and in isolated milled "wood" lignins by several analytical methods. The presence of p-coumarate and ferulate in the cortex and pith, as well as in their isolated lignins, was revealed by pyrolysis in the presence of tetramethylammonium hydroxide, and by 2D NMR, and indicated that ferulate acylates the carbohydrates while p-coumarate acylates the lignin polymer. 2D NMR showed a predominance of alkyl aryl ether (β-O-4') linkages (82% of total interunit linkages), with low amounts of "condensed" substructures, such as resinols (β-β'), phenylcoumarans (β-5'), and spirodienones (β-1'). Moreover, the NMR also indicated that these lignins are extensively acylated at the γ-carbon of the side chain. DFRC analyses confirmed that p-coumarate groups acylate the γ-OHs of these lignins, and predominantly on syringyl units.     

Comparison of respiratory function during TIVA (romifidine,ketamine, midazolam) and isoflurane anaesthesia in spontaneously breathing ponies Part I: blood gas analysis and cardiorespiratory variables

ObjectiveTo compare pulmonary function and gas exchange in ponies during maintenance of anaesthesia with isoflurane or by a total intravenous anaesthesia (TIVA) technique.Study designExperimental, cross–over study.AnimalsSix healthy ponies weighing mean 286 (range 233–388) ± SD 61 kg, age 13 (9-16) ± 3 years.MethodsThe ponies were anaesthetized twice, a minimum of two weeks apart. Following sedation with romifidine [80 μg kg^?1 intravenously (IV)], anaesthesia was induced IV with midazolam (0.06 mg kg^?1) and ketamine (2.5 mg kg^?1), then maintained either with inhaled isoflurane (Fe’Iso = 1.1 vol%) (T-ISO) or an IV infusion of romifidine (120 μg kg^?1 hour^?1), midazolam (0.09 mg kg^?1 hour^?1 IV) and ketamine (3.3 mg kg^?1 hour^?1) (T-TIVA). Ponies were placed in lateral recumbency. Breathing was spontaneous and Fi’O₂ 60%. After an instrumentation/stabilisation period of 30 minutes, arterial and mixed venous blood samples were taken simultaneously every 10 minutes for 60 minutes and analysed immediately. Oxygen extraction ratio (O₂ER) and venous admixture were calculated. Tidal volume (TV), minute volume (MV), respiratory rate (f_R), packed cell volume (PCV), arterial blood pressure and heart rate (HR) were measured and recorded. Data were analysed with mixed model anova (a = 0.05). Treatments were compared overall and at two selected time points (T30 and T60) using Bonferroni correction.ResultsArterial and mixed venous partial pressures of O₂ and CO₂, and TV were significantly lower and MV and f_R were higher in T-TIVA compared to T-ISO. Venous admixture did not differ between treatments. O₂ER was significantly higher in T-TIVA. Mean arterial pressure was higher and HR was lower in T-TIVA compared to T-ISO.Conclusions and clinical relevanceWhilst arterial CO₂ was within an acceptable range during both protocols, the impairment of oxygenation was more pronounced with the T-TIVA evidenced by lower arterial and venous oxygen partial pressures.     

Phytoremediation of soil contaminated with low concentrations of radionuclides

Primary sludge, secondary sludge, and wood ash from a pulp and paper mill were combined with sand to create a synthetic topsoil (C:N ratio of 18:1) to restore an abandoned gravel pit. Synthetic topsoil was applied to field microcosms at rates equivalent to 0, 2170, 4341, or 6511 kg N/ha; each was seeded with grass. Fifteen chemical constituents in leachate were measured during two field seasons. Cadmium, Ni and Zn were mobilized rapidly by soil disturbance. Chloride and SO₄-S eluted rapidly from the sludge along with Na. Nitrate leached with Ca late in each field season when sludge N-mineralization and nitrification exceeded plant uptake and microbial immobilization. Ammonium elution was negligible. Dissolved organic carbon (DOC) was mobilized by decomposition of organic matter in the sludge, as were Mg and K. Copper eluted with DOC, probably as an organic ligand. Lead and ortho-P were below our detection limits. We concluded that a synthetic topsoil with a 30:1 C:N ratio applied at a rate of 2100-4300 kg N/ha should provide adequate plant nutrition while minimizing water quality hazards.     

Ecological Risk Assessment of Alfalfa Medicago Varia L.) Genetically Engineered to Express a Human Metallothionein (hMT) Gene

The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO₄ by addition of CuSO₄ solutions to soil and (2) to identify potential unintended effects of the genetic engineering on root and shoot biomass, shoot nutrient content, arbuscular mycorrhizal infection and on the metabolic functions of microbial communities in the rhizosphere. In the absence of exogenous CuSO₄ additions to soil shoot biomass and the macronutrient (C, P, K, Ca, Mg and N) content of plants expressing hMT were not significantly different from the parental control. In the 0.5 mM and 1.0 mM CuSO₄ treatments transgenic plants expressing the commonly used transgenic β-glucuronidase (GUS) marker had significantly higher Fe content than the parental genotype. Significant differences were observed in the carbon substrate utilization patterns of rhizosphere microbial communities among the transgenic plants; no significant differences were observed in the percent mycorrhizal infection of parental and transgenic plants. Shoot biomass increased significantly in all genotypes treated with 0.5 mM CuSO₄ and decreased in all genotypes at CuSO₄ concentrations of 1.5 mM and 2.0 mM. Root dry weights decreased significantly in all genotypes at concentrations of 1.0 mM, 1.5 mM and 2.0 mM CuSO₄. The largest decreases in root dry weight were observed in hMT genotypes grown in soil treated with 1.5 and 2.0 mM CuSO₄. In plants treated with 1.5 mM CuSO₄, shoots of transgenic plants expressing the hMT gene accumulated nominally, but not statistically significantly higher levels of Cu in shoot tissue. Our results were surprising with regard to lack of sufficient efficacy of the current hMT constructs for significant accumulation of Cu from soil treated with CuSO₄. However, our results suggest the utility of applying adverse levels of CuSO₄ or other environmental stressors to identify potential unintended effects of genetic engineering that may not be apparent under typically more optimal plant growth test conditions.     

<Superscript>15</Superscript>N uptake by mycorrhizal native and invasive plants from a N-eutrophied shrubland: a greenhouse experiment

In the coastal sage scrub (CSS) community of southern California, Artemisia californica and other native shrubs are gradually being replaced by invasive annual grasses, especially Bromus madritensis ssp. rubens. This decline may be attributed, in part, to local atmospheric N deposition, which causes elevated soil NO₃ ^–. Unaffected soils have low N with a relatively higher concentration of NH₄ ⁺ than NO₃ ^–. The objectives in this study were: (1) to compare the short-term uptake of ¹⁵NO₃ ^– and ¹⁵NH₄ ⁺ by B. madritensis and A. californica and (2) to discern whether ¹⁵NO₃ ^– or ¹⁵NH₄ ⁺ uptake is influenced by the mycorrhizal status of either plant species. Analysis of ¹⁵N concentrations indicated that both A. californica and B. madritensis took up more ¹⁵NO₃ ^– than ¹⁵NH₄ ⁺, but overall, B. madritensis took up 6–15 times more of both forms of ¹⁵N than A. californica. Mycorrhizal A. californica had an increased ¹⁵NH₄ ⁺ concentration in roots but not shoots. In B. madritensis, the only mycorrhizal response was a reallocation of ¹⁵NO₃ ^–, with mycorrhizal plants retaining a higher proportion of ¹⁵NO₃ ^– in roots. Overall, arbuscular mycorrhizae had a small effect on ¹⁵N uptake in this short-term study. However, the ability of B. madritensis to take up so much ¹⁵N may explain in part why it has been so successful in replacing A. californica in CSS.