Produção de lipases de Aspergillus niger e Aspergillus fumigatus através de fermentação em estado sólido, avaliação da especificidade do substrato e seu uso em reações de esterificação e alcoólise: evaluation of substrate specificity and use in esterification and alcoholysis reactions

Filamentous fungi were cultured under solid state fermentation of soybean residues to produce lipases. Enzymes produced by Aspergillus niger esterified oleic and butyric acids in the presence of ethanol, while enzymes produced by Aspergillus fumigatus demonstrated no esterification activity toward lauric acid. In case of A. niger, direct lyophilization of fermented bran led to higher esterification activity. The esterification of oleic acid by enzymes of A. fumigatus was neither influenced by pH adjustment nor by the extraction process. Conversions to ethyl esters were higher after pH adjustment with lyophilized liquid extract of A. niger.

Contexts, non-specificity, and minimalism

Atlas (2007) argues that semantic minimalism (as defended by Cappelen & Lepore 2005) fails because it cannot deal with semantic non-specificity. I argue that thereis a plausible version of minimalism-viz., situated minimalism-which doesn't succumb to the non-specificity charge insofar as non-specificity can be dealt with at a postsemantic level. Thus, pragmatics plays no rolewhen it comes to determining the (minimal) proposition expressed. Instead, pragmatic and other extra-semantic considerations enter the scene in characterizing the situation vis-à-vis which the proposition is evaluated. For this reason a plausible form of minimalism must embrace a form of truth-relativism: a proposition is not universally true/false, but true/false only relative to a situation. I show how the position defended is not only (i) more cognitively plausible than either (semantic) minimalism as proposed by Cappelen & Lepore or the positions appealing to pragmaticintrusion into the proposition expressed, but is also (ii) in accordance with ordinary people's intuitions.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

Distribution of 131I-labeled Bothrops erythromelas venom in mice

Bothrops erythromelas is responsible for many snake bites in northeastern Brazil. In the present study we determined the in vivo distribution of the venom following its subcutaneous injection into mice. B. erythromelas venom and albumin were labeled individually with 131I by the chloramine T method, and separated in a Sephacryl® S-200 column. The efficiency of labeling was 68%. Male Swiss mice (40-45 g), which had been provided with drinking water containing 0.05% KI over a period of 10 days prior to the experiment, were inoculated dorsally (sc) with 0.3 ml (2.35 x 105 cpm/mouse) of 131I-venom (N = 42), 131I-albumin or 131I (controls, N = 28 each). Thirty minutes and 1, 3, 6, 12, 18 and 24 h after inoculation, the animals were perfused with 0.85% NaCl and skin and various organs were collected in order to determine radioactivity content. There was a high rate of venom absorption in the skin (51%) within the first 30 min compared to albumin (20.1%) and free iodine (8.2%). Up to the third hour after injection there was a tendency for venom and albumin to concentrate in the stomach (3rd h), small intestine (3rd h) and large intestine (6th h). Both control groups had more radioactivity in the digestive tract, especially in the stomach, but these levels decreased essentially to baseline by 12-18 h postinjection. In the kidneys, the distribution profiles of venom, albumin and iodine were similar. Counts at 30 min postinjection were low in all three groups (1.37, 1.86 and 0.77, respectively), and diminished to essentially 0% by 12-18 h. Albumin tended to concentrate in muscle until the 3rd h postinjection (1.98%). There was a low binding of labeled venom in the liver (<0.54%), thyroid (<0.11%) and lungs (<0.08%), and no iodinated venom was detected in brain, heart, diaphragm, spleen or bladder. The low venom binding observed in most internal organs, comparable to that of albumin, suggests that B. erythromelas venom does not specifically target most internal organs. That is, the systemic effects of envenomation are mainly due to an indirect action

Snake venom metalloproteinases and disintegrins: interactions with cells

Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.

Effect of toxin-g from Tityus serrulatus scorpion venom on gastric emptying in rats

The effect of toxin-g from Tityus serrulatus scorpion venom on the gastric emptying of liquids was studied in 176 young adult male Wistar rats (2-3 months of age) divided into subgroups of 8 animals each. Toxin-g was injected iv at doses of 25, 37.5, 50 or 100 µg/kg and the effect on gastric emptying was assessed 30 min and 8 h later. A time-course study was also performed by injecting 50 µg of toxin-g /kg and measuring the effect on gastric emptying at times 0.25, 0.5, 1, 2, 4, 8, 24 and 48 h post-venom. Each envenomed animal was paired with its saline control and all received a saline test meal solution containing phenol red (60 µg/ml) as a marker. Ten minutes after administering the test meal by gavage the animals were sacrificed and gastric retention was determined by measuring the residual marker concentration of the test meal. A significant delay in gastric emptying, at 30 min and 8 h post-venom, was observed only after 50 and 100 µg of toxin-g /kg compared to control values. The responses to these two doses were significantly different after 8 h post-venom. Toxin-g (50 µg/kg) significantly delayed the gastric emptying of liquids at all times studied, with a peak response at 4 h after toxin administration compared to control values. These results indicate that the iv injection of toxin-g may induce a rapid, intense and sustained inhibition of gastric emptying 0.25 to 48 h after envenomation.

Collagenase specificity in chondrosarcoma metastasis

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

Extracellular matrix molecules as targets for brown spider venom toxins

Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues.

Identification of hemolytic and neuroactive fractions in the venom of the sea anemone Bunodosoma cangicum

Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC50 = 14 µg/ml and a maximum dose of 22 µg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K+ channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na+ channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously.