Nerve growth factor-induced migration of endothelial cells.
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Nerve growth factor (NGF) is a well known neurotropic and neurotrophic agonist in the nervous system, which recently was shown to also induce angiogenic effects in endothelial cells (ECs). To measure NGF effects on the migration of cultured ECs, an important step in neoangiogenesis, we optimized an omnidirectional migration assay using human aortic endothelial cells (HAECs) and validated the assay with human recombinant basic fibroblast growth factor (rhbFGF) and human recombinant vascular endothelial growth factor (rhVEGF). The potencies of nerve growth factor purified from various species (viper, mouse, and recombinant human) to stimulate HAEC migration was similar to that of VEGF and basic fibroblast growth factor (bFGF) (EC50 of approximately 0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by approximately 30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(/)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11 -epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist SU-5416 [3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system. (+info)
Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis.
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Lebestatin, a new member of the lysine-threonine-serine (KTS)-disintegrin family, was purified to homogeneity from Tunisian snake (Macrovipera lebetina) venom. It is a single-chain polypeptide composed of 41 amino acids. The amino-acid sequence of lebestatin shows that it displays a pattern of cysteines similar to other short disintegrins, but contains the sequence KTS rather than RGD in its integrin-binding loop. Lebestatin presents a high homology with obtustatin and viperistatin. Lebestatin interacts specifically with the alpha1beta1 integrin. It was thus able to inhibit both adhesion and migration of PC12 and alpha1beta1 integrin-expressing CHO cells (CHO-alpha1) to type I and IV collagens. This disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect in vivo when using the 8-day-old embryo chick chorioallantoic membrane model. (+info)
Viper bites: treat or ignore? Review of a series of 99 patients bitten by Vipera aspis in an alpine Swiss area.
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In a well defined alpine area of Switzerland (Valais, about 300000 inhabitants, tourists included) we studied retrospectively over 32 years, 99 patients bitten by vipers (Vipera aspis, the likely unique species in this area). The annual incidence was estimated at 3/100,000, as in other European countries. The mortality was 0% for the adults. The patients, 72 adults and 17 children (13 years and less), were classified in four groups: grade 0 no envenomation (8%), grade 1 minimal (42%), grade 2 moderate (40%), and grade 3 severe envenomation (10%). The 10 patients of grade 3 showed impressive clinical signs and blood abnormalities, as exemplified by our three most severe cases. Only patients of grade 3 must be treated with antivenom and other intensive treatments, but all patients, even grade 1, especially small children, must be observed for several hours. (+info)
Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system.
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We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors. (+info)
Thrombin-like enzymes from venom gland of Deinagkistrodon acutus: cDNA cloning, mechanism of diversity and phylogenetic tree construction.
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AIM: To clone cDNAs of thrombin-like enzymes (TLEs) from venom gland of Deinagkistrodon acutus and analyze the mechanisms by which their structural diversity arose. METHODS: Reverse transcription-polymerase chain reaction and gene cloning techniques were used, and the cloned sequences were analyzed by using bioinformatics tools. RESULTS: Novel cDNAs of snake venom TLEs were cloned. The possibilities of post-transcriptional recombination and horizontal gene transfer are discussed. A phylogenetic tree was constructed. CONCLUSION: The cDNAs of snake venom TLEs exhibit great diversification. There are several types of structural variations. These variations may be attributable to certain mechanisms including recombination. (+info)
Isolation and preliminary crystallographic studies of two new phospholipases A2 from Vipera nikolskii venom.
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Snake-venom phospholipases A2 (PLA2s) represent a good model for studies of structure-function relationships, mainly because of their small size and diverse pharmacological and toxicological activities. To obtain new members of the abundant PLA2 family, the venom of the viper Vipera nikolskii was fractionated for the first time and two new proteins, VN5-3 and VN4-3, were isolated. Both proteins show phospholipase A2 activity and may possess neurotoxic activity. Based on the determined partial amino-acid sequences, the new proteins can be classified as basic Asp49 phospholipases A2. They were crystallized using the hanging-drop vapour-diffusion method and crystals of both proteins belong to space group R32, with similar unit-cell parameters: a = b = 76.29, c = 303.35 A for protein VN5-3 and a = b = 76.28, c = 304.39 A for protein VN4-3. Diffraction data sets to 3.0 and 2.2 A resolution were collected and processed for the VN5-3 and VN4-3 crystals, respectively. Preliminary analysis indicates that there are two molecules in the asymmetric unit for both crystals. Further crystallographic studies will help in understanding the structural basis for the multiple functions of snake-venom PLA2s. (+info)
Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution.
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Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts. (+info)
Evidence for a Mullerian mimetic radiation in Asian pitvipers.
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Mullerian mimicry, in which toxic species gain mutual protection from shared warning signals, is poorly understood in vertebrates, reflecting a paucity of examples. Indirect evidence for mimicry is found if monophyletic species or clades show parallel geographic variation in warning patterns. Here, we evaluate a hypothesis of Mullerian mimicry for the pitvipers in Southeast Asia using a phylogeny derived from DNA sequences from four combined mitochondrial regions. Mantel matrix correlation tests show that conspicuous red colour pattern elements are significantly associated with sympatric and parapatric populations in four genera. To our knowledge, this represents the first evidence of a Mullerian mimetic radiation in vipers. The putative mimetic patterns are rarely found in females. This appears paradoxical in light of the Mullerian prediction of monomorphism, but may be explained by divergent selection pressures on the sexes, which have different behaviours. We suggest that biased predation on active males causes selection for protective warning coloration, whereas crypsis is favoured in relatively sedentary females. (+info)