Proteomic View of the Venom from the Fire Ant Solenopsis invicta Buren

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytochemical studies of the nuclei of the venom glands' cells of Apis mellifera (Hymenoptera, Apidae)

Cytochemistry studies of the nuclei of the venom glands' cells of worker bees of Apis mellifera indicated that there is a higher activity in the young workers while there is a predominance of degenerative characteristics in the older workers. In addition, we demonstrated that there is an occurrence of differential nuclear synthetic activities between the cells of the distal and the proximal regions of the secretory filament and of the venom reservoir. Signs of a higher nuclear activity were evidenced at the distal regions of this gland in 14-day old workers, while at the more proximal regions of the venom gland of 40-day old workers we identified the most obvious signs of degeneration. Therefore, it was evident that the process of glandular degeneration begins at the distal region of the venom gland instead of beginning at the proximal region as had been established previously. In addition, characteristics of nuclear synthetic activities were noted in the cells of the proximal region of the reservoir; these cells were thought to be non-secretory.

How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH2), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO-) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO-, a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH2 and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO- leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH2 exists mainly as a rigid structure, EMP-AF-COO- presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

Effects of Spider Venom Toxin PWTX-I (6-Hydroxytrypargine) on the Central Nervous System of Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aerobic metabolism during predation by a boid snake

We quantified the oxygen uptake rates ((V) over dot O-2) and time spent, during the constriction, inspection, and ingestion of prey of different relative sizes, by the prey-constricting boid snake Boa constrictor amarali. Time spent in prey constriction varied from 7.6 to 16.3 min, and (V) over dot O-2 during prey constriction increased 6.8-fold above resting values. This was the most energy expensive predation phase but neither time spent nor metabolic rate during this phase were correlated with prey size. Similarly, prey size did not affect the (V) over dot O-2 or duration of prey inspection. Prey ingestion time, on the other hand, increased linearly with prey size although (V) over dot O-2 during this phase, which increased 4.9-fold above resting levels, was not affected by prey size. The increase in mechanical difficulty of ingesting larger prey, therefore, was associated with longer ingestion times rather than proportional increases in the level of metabolic effort. The data indicate that prey constriction and ingestion are largely sustained by glycolysis and the intervening phase of prey inspection may allow recovery between these two predatory phases with high metabolic demands. The total amount of energy spent by B. c. amarali to constrict, inspect, and ingest prey of sizes varying from 5 to 40% of snake body mass varied inversely from 0.21 to 0.11% of the energy assimilated from the prey, respectively. Thus, prey size was not limited by the energetic cost of predation. on the contrary, snakes feeding on larger prey were rewarded with larger energetic returns, in accordance with explanations of the evolution of snake feeding specializations. (C) 2002 Elsevier B.V. All rights reserved.

Autonomic control of heart rate during forced activity and digestion in the snake Boa constrictor

Reptiles, particularly snakes, exhibit large and quantitatively similar increments in metabolic rate during muscular exercise and following a meal, when they are apparently inactive. The cardiovascular responses are similar during these two states, but the underlying autonomic control of the heart remains unknown. We describe both adrenergic and cholinergic tonus on the heart during rest, during enforced activity and during digestion (24-36h after ingestion of 30% of their body mass) in the snake Boa constrictor. The snakes were equipped with an arterial catheter for measurements of blood pressure and heart rate, and autonomic tonus was determined following infusion of the beta -adrenergic antagonist propranolol (3mg kg(-1)) and the muscarinic cholinoceptor antagonist atropine (3 mg kg-1).The mean heart rate of fasting animals at rest was 26.4 +/- 1.4 min(-1), and this increased to 36.1 +/- 1.4 min(-1) (means +/- S.E.M.; N=8) following double autonomic block (atropine and propranolol). The calculated cholinergic and adrenergic tones were 60.1 +/- 0.3% and 19.8 +/- 2.2%, respectively. Heart rate increased to 61.4 +/- 1.5 min(-1) during enforced activity, and this response was significantly reduced by propranolol (maximum values of 35.8 +/-1.6 min(-1)), but unaffected by atropine. The cholinergic and adrenergic tones were 2.6 +/- 2.2 and 41.3 +/- 1.9 % during activity, respectively. Double autonomic block virtually abolished tachycardia associated with enforced activity (heart rate increased significantly from 36.1 +/- 1.4 to 37.6 +/- 1.3 min(-1)), indicating that non-adrenergic, non-cholinergic effectors are not involved in regulating heart rate during activity. Blood pressure also increased during activity.Digestion was accompanied by an increase in heart rate from 25.6 +/- 1.3 to 47.7 +/- 2.2 min(-1) (N=8). In these animals, heart rate decreased to 44.2 +/- 2.7 min-1 following propranolol infusion and increased to 53.9 +/- 1.8 min-1 after infusion of atropine, resulting in small cholinergic and adrenergic tones (6.0 +/- 3.5 and 11.1 +/- 1.1 %, respectively). The heart rate of digesting snakes was 47.0 +/- 1.0 min(-1) after double autonomic blockade, which is significantly higher than the value of 36.1 1.4 min-1 in double-blocked fasting animals at rest. Therefore, it appears that some other factor exerts a positive chronotropic effect during digestion, and we propose that this factor may be a circulating regulatory peptide, possibly liberated from the gastrointestinal system in response to the presence of food.

Ventilatory compensation of the alkaline tide during digestion in the snake Boa constrictor

The increased metabolic rate during digestion is associated with changes in arterial acid-base parameters that are caused by gastric acid secretion (the 'alkaline tide'). Net transfer of HCl to the stomach lumen causes an increase in plasma HCO3- levels, but arterial pH does not change because of a ventilatory compensation that counters the metabolic alkalosis. It seems, therefore, that ventilation is controlled to preserve pH and not P-CO2, during the postprandial period. To investigate this possibility, we determined arterial acid-base parameters and the metabolic response to digestion in the snake Boa constrictor, where gastric acid secretion was inhibited pharmacologically by oral administration of omeprazole. The increase in oxygen consumption of omeprazole-treated snakes after ingestion of 30% of their own body mass was quantitatively similar to the response in untreated snakes, although the peak of the metabolic response occurred later (36 h versus 24 h). Untreated control animals exhibited a large increase in arterial plasma HCO3- concentration of approximately 12 mmol 1(-1), but arterial pH only increased by 0.12 pH units because of a simultaneous increase in arterial P-CO2 by about 10 mmHg. Omeprazole virtually abolished the changes in arterial pH and plasma HCO3- concentration during digestion and there was no increase in arterial P-CO2. The increased arterial P-CO2 during digestion is not caused, therefore, by the increased metabolism during digestion or a lower ventilatory responsiveness to ventilatory stimuli during a presumably relaxed state in digestion. Furthermore, the constant arterial P-CO2, in the absence of an alkaline tide, of omeprazole-treated snakes strongly suggests that pH rather than P-CO2 normally affects chemoreceptor activity and ventilatory drive.

Diet of the colubrid snake, Thamnodynastes strigatus (Günther, 1858) from Paraná State, Brazil, with field notes on anuran predation

São apresentados dados da dieta de Thamnodynastes strigatus (Serpentes: Colubridae), a partir da dissecção de 44 espécimes. No tubo digestivo dos exemplares examinados foram encontrados anfíbios anuros (71,4% da amostra), roedores (14,3%), peixes (10,7%) e lagartos (3,6%). A maioria das espécies de anuros (Bufo sp., Leptodactylus sp., Physalaemus cuvieri e Scinax fuscovarius) encontradas no exame de conteúdo estomacal de T. strigatus, utiliza o solo ou o nível d'água como sítio de vocalização. Também são apresentados dados sobre a observação de eventos de predação na natureza.

When Basking Is Not an Option: Thermoregulation of a Viperid Snake Endemic to a Small Island in the South Atlantic of Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Localized matrix metalloproteinase (MMP)-2 and MMP-9 activity in the rat ventral prostate during the first week of postnatal development

The initial events in prostatic morphogenesis involve cell proliferation, epithelial canalization and outgrowth toward the stroma. We have hypothesized that stromal rearrangement takes place at the sites of epithelial growth and branching and that this rearrangement involves the action of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9. Thus, the purpose of the present study was to characterize structural aspects of epithelial growth during the first week of postnatal development of the rat ventral prostate and to investigate the expression, localization and activity of MMP-2 and MMP-9 during this period by histological, ultrastructural and immunocytochemical analysis, in addition to gel zymography, in situ zymography and Western blotting. An increasing complexity of prostatic architeture was observed within the first postnatal week. Concurrently, the stroma became more organized and some cells differentiated into smooth muscle cells. Reticulin fibers appeared in a basket-like arrangement around both growing tips and epithelial sprouts, associated with a fainter staining for laminin. MMP-2 and MMP-9 activities were detected. MMP-2/MMP-9 expression decreased during the first week. Developing epithelial cords showed strong and difuse gelatinolytic activity. This activity coincided with the distribution of MMP-2 as determined by immunocytochemistry. on the other hand, MMP-9 was rather concentrated at the epithelial tips. These results suggest that gelatinolytic activity (with contribution of both MMP-2 and MMP-9) in the epithelium and at the epithelium-stroma interface are at least in part responsible for the tissue remodeling that allows epithelial growth and its projection into the surrounding stroma.

Matrix metalloproteinase (MMP)-2 and MMP-9 activity and localization during ventral prostate atrophy and regrowth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

Initial structural analysis of an alpha(4)beta(4) C-type lectin from the venom of Crotalus durissus terrificus

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 Angstrom, and diffraction data were collected to 2.7 Angstrom. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.