PLA2

^aMagro AJ,^aTakeda AA,^bSoares AM,^aFontes MR

The crystal structure of an acidic phospholipase A₂ isolated from Bothrops jararacussu venom (BthA-I) chemically modified with ρ-bromophenacyl bromide (BPB) has been determined at 1.85 Å resolution. The catalytic, plateletaggregation inhibition, anticoagulant and hypotensive activities of BthA-I are abolished by ligand binding. Electron-density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate-binding cleft. The BthA-I–BPB complex contains three structural regions that are modified after inhibitor binding: the Ca²⁺- binding loop, β-wing and C-terminal regions. Comparison of BthA-I–BPB with two other BPB-inhibited PLA₂ structures suggests that in the absence of Na⁺ ions at the Ca2⁺-binding loop, this loop and other regions of the PLA²s undergo structural changes. The BthA-I–BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the ‘pancreatic’ loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA-I–BPB, leading to a new hypothesis regarding the abolition of this activity by BPB.

^aMaiorano VA , ^b,cMarcussi S, ^aDaher MA, ^a,cOliveira CZ, ^dCouto LB, ^dGomes OA, ^aFranca SC, ^cSoares AM, ^aPereira PS.

Antiophidian properties of the aqueous extract of Mikania glomerata

Aqueous extracts, prepared from dried or fresh roots, stems or leaves of Mikania glomerata, a plant found in Mata Atlantica in Southeastern Brazil, were able to efficiently neutralize different toxic, pharmacological, and enzymatic effects induced by venoms from Bothrops and Crotalus snakes. Phospholipase A₂ activity and the edema induced by Crotalus durissus terrificus venom were inhibited around 100 and approximately 40%, respectively, although this inhibition was only partial for Bothrops venoms. The hemorrhagic activity of Bothrops venoms (Bothrops altenatus, Bothrops moojeni, Bothrops neuwiedi, and Bothrops jararacussu) was significantly inhibited by this vegetal species, while the clotting activity of Crotalus durissus terrificus, Bothrops jararacussu, and Bothrops neuwiedi venoms was totally inhibited. Although, the mechanism of action of Mikania glomerata extract is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. Since the extract of Mikania glomerata significantly inhibited the studied snake venoms, it may be used as an alternative treatment to serumtherapy and, in addition, as a rich source of potential inhibitors of PLA₂s, metalloproteases and serineproteases, enzymes involved in several physiopathological human and animal diseases.

XII Congresso de Iniciação Científica da Universidade Federal de São Carlos - UFSCar

Biondo R, Soares AM, Bertoni BW, Franca SC, Pereira AM.

Direct organogenesis of Mandevilla illustris (Vell) Woodson and effects of its aqueous extract on the enzymatic and toxic activities of Crotalus durissus terrificus snake venom.

In order to produce explants of Mandevilla illustris (Vell) Woodson for the "Cerrado in vitro", the Germplasm Bank of UNAERP, we carried out a micropropagation protocol using MS or MS/3 medium supplemented with different concentrations of 6-benzyladeninepurine (BA), Zeatin or 2-isopentenyladenine for nodal segment growth, and alpha-naphthaleneacetic acid, indole-3-butyric acid (IBA) or 1,4 dithiothreitol for rooting. For nodal segments, all the cytokinins tested yielded similar results. However, 2.22 micro M BA is more economical to use. MS/3 medium supplemented with 0.49 micro M IBA was the most appropriate medium for rooting, resulting in 29% rooted explants. The crude aqueous extract from the subterranean system (SS) of M. illustris was assayed for its inhibitory action on the enzymatic activity of Crotalus durissus terrificus snake venom, isolated basic phospholipase A2 (CB) and crotoxin. It totally inhibited the phospholipase activity of crude Cdt venom and CB toxin and inhibited the phospholipase activity of crotoxin by 49%. The toxic action of both the crude venom and crotoxin was partially inhibited-there was a prolonged survival time and a 40.0% decrease in lethality.

Otero R^a, Nunez V^a, Jimenez SL^a, Fonnegra Ra, Osorio RG^a, Garcia ME^a, Diaz A^a

Twelve of 74 ethanolic extracts of plants used by traditional healers for snakebites in the northwest region of Colombia, were active against lethal effect of Bothrops atrox venom when they were i.p. injected into mice (18–20 g). After preincubation of sublethal doses of every extract (0.5–4.0 mg/mouse) with 1.5 i.p. lethal dose 50% (LD₅₀) (99.3 μg) of venom, seven of them demonstrated 100% neutralizing capacity within 48 h. These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia ( Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae). The other five extracts showing partial neutralization (45–80%; 10–30% survival rate in the control group receiving the venom alone; P<0.05) were: leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae). When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 μg=1.5 i.m. LD₅₀), C. limon, T. elegans, B. orellana and T. rosea extracts had partial and significant neutralizing capacity against B. atrox venom lethal effect. C. limon extract was also partially effective when it was administered either i.v. 15 min before or i.p. 5 min after an i.m. venom injection. Three of the 12 extracts with anti-lethal effect (C. limon, D. croatii and S. acuta) were devoid of antiphospholipase A₂ activity, when they were tested against one minimum indirect hemolytic dose of B. atrox venom (2 μg) in agarose-erythrocyte-egg yolk gels.