Vascular smooth muscle relaxation by a lectin from Pisum arvense: evidences of endothelial NOS pathway

The vasorelaxant effect of the lectin of Pisum arvense (PAL) seeds was investigated in rat aorta. PAL (10-100 μg/ml) was applied on aorta rings, with or without endothelium, pre-contracted with phenylephrine (Phe; 0.1 μM). Participation of endothelium derived relaxant factors was evaluated incubating the tissue with indomethacin (10 μM), L-nitro arginine methyl ester (L-NAME, 100 μM) and tetraethylammonium (TEA, 5 mM) before addition of PAL. The role of the lectin domain was investigated by addition of PAL into tissue in presence of glucose (3x 10?? M), or N-acetyl Dglucosamine (GlcNAc; 3 x 10?? M). The importance of extracellular calcium (Ca2?e) or interaction with muscarinic receptors in the relaxant effect was evaluated by addition of PAL into aorta rings containing calcium free solution (OCa) and atropine (1 μ M), respectively. PAL induced concentration-dependent relaxation in endothelized aorta (IC50 =58.38 ± 1.87 μg/ml), which was reversed by L-NAME and glucose. The lectin effect was totally inhibited when the preparation was inserted in OCa, but not in presence of atropine. Summarizing, our data showed a relaxant effect of PAL in isolated rat aorta rings in presence of endothelium, suggestive of interaction between the lectin carbohydrate binding sites with specific receptors located in vascular endothelial cells leading to nitric oxide synthase activation. This effect seems to require Ca2?e but is independent on muscarinic receptors interaction.     

Throwing the baby out with the bathwater: does laurel forest restoration remove a critical winter food supply for the critically endangered Azores bullfinch?

The invasive Clethra arborea has a dual-role in the diet of the Azores bullfinch, a critically endangered bird species endemic to the island of São Miguel (Azores, Portugal). This is a crucial winter food resource but it lowers the availability of native laurel forest species that compose most of the bird’s diet throughout the year. The removal of this and other invasive alien species is part of current laurel forest habitat restoration programmes, disregarding the impact on the Azores bullfinch population. In order to evaluate the first responses of the Azores bullfinch to habitat restoration, we studied bird diet, foraging behaviour, food availability and habitat occupancy in managed (without C. arborea) and control areas. Significant increases in the availability of native food resources in managed areas were noticeable in the diet, particularly the intake of Ilex perado ssp. azorica and Prunus lusitanica ssp. azorica flower buds. In most of the studied months birds heavily used and foraged in managed over control areas. The one exception was in December, when a resource-gap occurred in managed areas, which may be overcome in the short-term due to re-establishment of native plants following removal of invasive aliens.     

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Maximization of fructose esters synthesis by response surface methodology

Enzymatic synthesis of fructose fatty acid ester was performed in organic solvent media, using a purified lipase from Candida antartica B immobilized in acrylic resin. Response surface methodology with a central composite rotatable design based on five levels was implemented to optimize three experimental operating conditions (temperature, agitation and reaction time). A statistical significant cubic model was established. Temperature and reaction time were found to be the most significant parameters. The optimum operational conditions for maximizing the synthesis of fructose esters were 57.1°C, 100 rpm and 37.8 h. The model was validated in the identified optimal conditions to check its adequacy and accuracy, and an experimental esterification percentage of 88.4% (±0.3%) was obtained. These results showed that an improvement of the enzymatic synthesis of fructose esters was obtained under the optimized conditions.     

Immunohistochemical analysis of smooth muscle cells and volumetric density of the elastic system fibers of wild boar (Sus scrofa) penis

The purpose of the present study was to verify the smooth muscle cell distribution and elastic system fibers volumetric density (Vv) in the corpus spongiosum and corpus cavernosum of the wild boar penis. Adult wild boars (n=13) were used. The penile mid shaft fragments were fixed with 4% phosphate buffered formalin solution and/or Bouin's liquid during 24-48 h, and processed using standard histological techniques. The sections were stained with Weigert's Resorcin-Fucsin with previous oxidation. The elastic system fibers Vv was determined in 25 random fields of each fragment using M42 test system. For immunohistochemical analysis, monoclonal anti-alpha actin smooth muscle was used. The histochemical methods detected elastic system fibers in both corpus spongiosum and corpus cavernosum of all animals. The elastic fibers Vv average was 36.6%+/-0.9 for corpus spongiosum and 11.7%+/-0.5 for corpus cavernosum. Through immunocitochemistry, a small quantity of smooth muscle cells was visualized in intimate relationship with blood vessels wall. The great amount of elastic fibers and the smooth muscle cell distribution beneath the endothelium suggest that these fibers may have an important role in penile erection process in the penis of wild boars.     

Occurrence and Diversity of Yeasts in the Mid-Atlantic Ridge Hydrothermal Fields Near the Azores Archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.     

Influence of high-intensity exercise training and anabolic androgenic steroid treatment on rat tissue glycogen content

To increase tissue glycogen content many athletes use anabolic androgenic steroids (AAS). However, the literature concerning the effects of androgens on glycogen metabolism is conflicting. This study aimed to determine the influence of training and AAS on body weight (bw), triglycerides, glucose, tissue glycogen and transaminases levels. Male Wistar rats, randomized into four groups (sedentary vehicle (SV), sedentary AAS (SA), trained vehicle (TV) and trained AAS (TA)), were treated with nadrolone (5 mg/Kg, 2x/week, i.m.) or vehicle. Trained rats performed jumps into water (4 sets, 10 repetitions, 30 sec rest) carrying a 50-70% body wt-load strapped to the chest (5 days/week,6 weeks). Two days after the last session, the animals were killed (bifatorial ANOVA+Tukey test; P < 0.05). Trained animals presented lower bw (TV:345+/-7 vs. SV:380+/-7 and TA:328+/-4 vs SA:370+/-11 g) and triglycerides levels (TV:77+/-3 vs. SV:98+/-4 and TA:79+/-3 vs. SA:98+/-8 mg/dL) and higher glycogen content in liver (TV:5.3+/-0.2 vs. SV:3.9+/-0.1 and TA:5.3+/-0.3 vs. SA:4.6+/-0,2 mg/100 mg) and in gastrocnemious (TV:0.70+/-0.02 vs. SV:0.49+/-0.01 and TA:0.73+/-0.03 vs. SA:0.57+/-0.02 mg/100 mg) than sedentary ones. In the cardiac muscle, the association between training and AAS increased glycogen content (TA:0.19+/-0.01 > SV:0.13+/-0.01=TV:0.13+/-0.01=SA:0.14+/-0.01 mg/100 mg). In the soleus AAS increased glycogen (SA:0.53+/-0.03 vs. SV:0.43+/-0.01 and TA:0.58+/-0.02 vs. TV:0.48+/-0.01 mg/100 mg). Exercise training and AAS had no effect on blood glucose and transaminases levels. Training and AAS effects on glycogen supercompensation are tissue-dependent and the effects of association between them were only observed in the cardiac muscle. These data emphasize the necessity of more studies to confirm greater effects of AAS than those promoted by physical exercise.     

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.     

Effects of voltage-gated Na+ channel toxins from Tityus serrulatus venom on rat arterial blood pressure and plasma catecholamines

Scorpion toxins interact with ionic channels of excitable cells, leading to a massive release of neurotransmitters. Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: alpha- and beta-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, we compared the effects of these toxins on mean arterial pressure (MAP) and catecholamines release in rats. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). All experiments were performed on conscious unrestrained rats previously catheterised. The toxins (15 and 30 microg/kg) and TsV (50 and 100 microg/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5-3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity.     

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.