Viper metalloproteinase (Agkistrodon halys pallas) with antimicrobial activity against multi-drug resistant human pathogens

Metalloproteinases are abundant enzymes in crotalidae and viperidae snake venoms. Snake venom metalloproteinases (SVMPs) comprise a family of zinc-dependent enzymes, which display many different biological activities. A 23.1 kDa protein was isolated from Agkistrodon halys (pallas, Chinese viper) snake venom. The toxin is a single chain polypeptide with a molecular weight of 23146.61 and an N-terminal sequence (MIQVLLVTICLAVFPYQGSSIILES) relatively similar to that of other metalloprotein-like proteases isolated from the snake venoms of the Viperidae family. The antibacterial effect of Agkistrodon halys metalloproteinase (AHM) on Burkholderia pseudomallei (strains TES and KHW), Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacterium) was studied at a concentration 120 microM. Interestingly, we found that the metalloproteinase exhibited antibacterial properties and was more active against S. aureus, P. vulgaris, P. mirabilis and multi-drug resistant B. pseudomallei (strain KHW) bacteria. AHM variants with high bacteriostatic activity (MIC 1.875-60 microM) also tended to be less cytotoxic against U-937 human monocytic cells up to 1 mM concentrations. These results suggest that this metalloprotein exerts its antimicrobial effect by altering membrane packing and inhibiting mechanosensitive targets.     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa)

Bothrops snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA(2) from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (pI 5.05 and 4.49) Asp49 PLA(2), with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA(2) previously isolated from this venom (PA2_BOTIN, SwissProt accession number ) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA(2) showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA(2) from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA(2) that consisted of three alpha-helices, a beta-wing, a short helix and a calcium-binding loop. BinTX-I (30 microg/paw) produced mouse hind paw edema that was maximal after 1h compared to after 3h with venom (10 and 100 microg/paw); in both cases, the edema decreased after 6h. BinTX-1 and venom (40 microg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Identification of beta-type phospholipase A(2) inhibitor in a nonvenomous snake,Elaphe quadrivirgata

A novel serum protein inhibiting specifically the enzymatic activity of the basic phospholipase A(2) (PLA(2)) from the venom of the Chinese mamushi snake (Agkistrodon blomhoffii siniticus) was purified from a nonvenomous Colubridae snake, Elaphe quadrivirgata. The purified inhibitor was a 150-kDa glycoprotein having a trimeric structure, composed of two homologous 50-kDa subunits. Their amino acid sequences, containing leucine-rich repeats, were typical of the beta-type PLA(2) inhibitor (PLIbeta), previously identified from the serum of A. blomhoffii siniticus. The inhibitor inhibited exclusively group II basic PLA(2)s and did not inhibit other kinds of PLA(2)s. This is the first paper reporting the existence of PLIbeta in a nonvenomous snake. The existence of PLIbeta in the nonvenomous snake reflects that PLIbetas are widely distributed over the snake species and participate commonly in regulating the physiological activities of the unidentified target PLA(2)s.     

Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A(2) homologue from Bothrops pirajai venom

The complete amino acid sequence of the 121 amino acid residues of piratoxin II, a phospholipase A(2) like myotoxin from Bothrops pirajai venom, is reported. PrTX-II is a basic protein with a molecular mass of 13740 Da, a calculated pI of 9.03, but an experimental pI of 8.4 +/- 0.2, showing sequence similarity with other bothropic (90-99%) or non-bothropic ( approximately 80%) Lys49 PLA(2)-like myotoxins. This similarity falls to approximately 70% when this sequence is aligned with that of Asp49 PLA(2). Due to the substitution of Asp49 by Lys49 and alterations in the calcium binding loop structure, as the replacement of Gly32 by Leu32, piratoxin-II shows no PLA(2) activity when assayed on egg yolk. Piratoxin-II showed the same primary structure as piratoxin-I, except that it has Lys116 for Leu116. Despite this slightly higher basicity at the C-terminal region, piratoxin-II was shown to be less myotoxic than piratoxin-I. The change Leu --> Lys induced an alteration of the molecule surface shape and probably of the environment charge high enough to slightly decrease the myotoxic activity. When aligned with B. jararacussu bothropstoxin-I and with B. asper Basp-II, piratoxin-II revealed a single (position 132) and a quintuple (positions 17, 90, 111, 120 and 132) amino acid substitution, respectively, suggesting a common evolutionary origin for these three myotoxins.     

Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.     

Structures of cadmium-binding acidic phospholipase A2 from the venom of Agkistrodon halys Pallas at 1.9A resolution

Phospholipase A(2) coordinates Ca(2+) ion through three carbonyl oxygen atoms of residues 28, 30, and 32, two carboxyl oxygen atoms of residue Asp49, and two (or one) water molecules, forming seven (or six) coordinate geometry of Ca(2+) ligands. Two crystal structures of cadmium-binding acidic phospholipase A(2) from the venom of Agkistrodon halys Pallas (i.e., Agkistrodon blomhoffii brevicaudus) at different pH values (5.9 and 7.4) were determined to 1.9A resolution by the isomorphous difference Fourier method. The well-refined structures revealed that a Cd(2+) ion occupied the position expected for a Ca(2+) ion, and that the substitution of Cd(2+) for Ca(2+) resulted in detectable changes in the metal-binding region: one of the carboxyl oxygen atoms from residue Asp49 was farther from the metal ion while the other one was closer and there were no water molecules coordinating to the metal ion. Thus the Cd(2+)-binding region appears to have four coordinating oxygen ligands. The cadmium binding to the enzyme induced no other significant conformational change in the enzyme molecule elsewhere. The mechanism for divalent cadmium cation to support substrate binding but not catalysis is discussed.     

Structural and serological studies of the O-antigen of Proteus mirabilis O-9

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with chemical methods: [chemical structure: see text] where the degree of O-acetylation is approximately 70%. Immunochemical studies using rabbit polyclonal anti-Proteus mirabilis O-9 serum showed the importance of the O-acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti-P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

Identification of vascular endothelial growth factor receptor-binding protein in the venom of eastern cottonmouth. A new role of snake venom myotoxic Lys49-phospholipase A2

Vascular endothelial growth factor (VEGF165) and its receptor KDR (kinase insert domain-containing receptor) are central regulators of blood vessel formation. We herein report a KDR-binding protein we have isolated in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49-phosholipase A2 (Lys49PLA2), an inactive PLA2 homologue with strong myotoxicity, in which Lys49 substitutes Asp49, a key residue for binding the essential cofactor Ca2+. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. KDR-bp also binds to a lesser extent with Flt-1 and IgG but not to other receptors with similar immunoglobulin-like domain structures such as platelet-derived growth factor receptor alpha. The interaction between KDR-bp and KDR was blocked by VEGF165, and KDR-bp specifically inhibited VEGF165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. Lys49PLA2s from another snake venom were found to exhibit similar receptor binding properties to KDR-bp. This is the first report to demonstrate that an exogenous factor antagonizes VEGF and its receptor system. Our observation offers further insight into the as yet unknown molecular mechanism of myotoxic activity of snake venom Lys49PLA2s. Furthermore, KDR-bp would make a valuable tool for studying the structure and function of KDR, such as that expressed on skeletal muscle cells.     

Crystal structure of a myotoxic Asp49-phospholipase A2 with low catalytic activity: Insights into Ca2+-independent catalytic mechanism

A myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2S. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu) and other Asp49-PLA2S. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA2S, making a hydrogen bond with the atom O delta 2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA2S which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA2S indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA2S.     

Cloning and cDNA sequence analysis of Lys(49) and Asp(49) basic phospholipase A(2) myotoxin isoforms from Bothrops asper

Snake venom myotoxic phospholipases A(2) contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA(2)s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys(49) and one Asp(49) basic PLA(2) myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val-->Leu(18) and Ala-->Val(23) substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant (>75% sequence identity) identities with other crotalid venom Lys(49) PLA(2)s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

A novel autotransporter of uropathogenic Proteus mirabilis is both a cytotoxin and an agglutinin

One of the six predicted Proteus mirabilis autotransporters (ATs), ORF c2341, is predicted to contain a serine protease motif and was earlier identified as an immunogenic outer membrane protein in P. mirabilis. The 3.2 kb gene encodes a 117 kDa protein with a 58-amino-acid-long signal peptide, a 75-kDa-long N-terminal passenger domain and a 30-kDa-long C-terminal translocator. Affinity-purified 110 kDa AT exhibited chymotrypsin-like activity and hydrolysed N-Suc-Ala-Ala-Pro-Phe-pNa and N-Suc-Ala-Ala-Pro-Leu-pNa with a K(M) of 22 muM and 31 muM, respectively, under optimal pH of 8.5-9.0 in a Ca(2+)-dependent manner. Activity was inhibited by subtilase-specific inhibitors leupeptin and chymostatin. Both the cell-associated and purified form elicited cytopathic effects on cultured kidney and bladder epithelial cells. Substrate hydrolysis as well as cytotoxicity was associated with the passenger domain and was compromised upon mutation of any of the catalytic residues (Ser366, His147 and Asp533). At alkaline pH and optimal cell density, the AT also promoted autoaggregation of P. mirabilis and this function was independent of its protease activity. Cytotoxicity, autoaggregation and virulence were significantly reduced in an isogenic pta mutant of P. mirabilis. Proteus toxic agglutinin (Pta) represents a novel autotransported cytotoxin with no bacterial homologues that works optimally in the alkalinized urinary tract, a characteristic of urease-mediated urea hydrolysis during P. mirabilis infection.     

Crystal structure of Bn IV in complex with myristic acid: a Lys49 myotoxic phospholipase A₂ from Bothrops neuwiedi venom

The LYS49-PLA₂s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA₂ is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA₂ was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH₃(CH₂)₁₂COOH) and its overall structure was refined at 2.2 Å resolution. The Bn IV crystals belong to monoclinic space group P2₁ and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 Å. The biological assembly is a “conventional dimer” and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes.     

Pharmacological Study of Edema and Myonecrosis in Mice Induced by Venom of the Bushmaster Snake (<Emphasis Type="Italic">Lachesis muta muta</Emphasis>) and Its Basic Asp49 Phospholipase A<Subscript>2</Subscript> (LmTX-I)

Previous in vitro studies show that Lachesis muta venom and its purified Asp49 phospholipase A₂, named as LmTX-I, display potent neurotoxic and myotoxic activities. Here, an in vivo study was conducted to investigate some pharmacological effects of the venom or its LmTX-I toxin, after intra-muscular injection in tibialis anterior (TA) and following subplantar injection in hind paws of mice. Findings showed that LmTX-I increased plasma creatine kinase activity and produced strong myonecrosis and inflammatory reactions in TA muscle. In addition to these effects, the venom also induced intense local hemorrhage. Pre-treatment of the venom with EDTA (5 mM) significantly inhibited the edema and hemorrhage. Histological examination showed that L. muta venom caused inner dermal layer thickening in the pad hind paw. In addition, there was marked inflammatory cell infiltration, particularly of neutrophils, and hemorrhage. LmTX-I also demonstrated edema-forming activity, which was inhibited by pretreatment with indomethacin.     

Asp49 phospholipase A(2)-elaidoylamide complex: a new mode of inhibition

The inhibition of phospholipase A(2)s (PLA(2)s) is of pharmacological and therapeutic interest because these enzymes are involved in several inflammatory diseases. Elaidoylamide is a powerful inhibitor of a neurotoxic PLA(2) from the Vipera ammodytes meridionalis venom. The X-ray structure of the enzyme-inhibitor complex reveals a new mode of Asp49 PLA(2) inhibition by a fatty acid hydrocarbon chain. The structure contains two identical homodimers in the asymmetric unit. In each dimer one subunit is rotated by 180 degrees with respect to the other and the two molecules are oriented head-to-tail. One molecule of elaidoylamide is bound simultaneously to the substrate binding sites of two associated neurotoxic phospholipase A(2) molecules. The inhibitor binds symmetrically to the hydrophobic channels of the two monomers. The structure can be used to design anti-inflammatory drugs.     

Antibacterial activity of ethanolic and aqueous extracts of Acacia aroma Gill. ex Hook et Arn

The purpose of the present study was to investigate the antibacterial activity of seven ethanolic extracts and three aqueous extracts from various parts (leaves, stems and flowers) of A. aroma against 163 strains of antibiotic multi-resistant bacteria. The disc diffusion assay was performed to evaluate antibacterial activity of the A. aroma crude extracts, against several Gram-positive bacteria (E. faecalis, S. aureus, coagulase-negative stahylococci, S. pyogenes, S. agalactiae, S. aureus ATCC 29213, E. faecalis ATCC 29212) and Gram-negative bacteria (E. coli., K. pneumoniae, P. mirabilis, E. cloacae, S. marcescens, M morganii, A. baumannii, P. aeruginosa, S. maltophilia, E. coli ATCC 35218, P. aeruginosa ATCC 27853, E. coli ATCC 25922). All ethanolic extracts showed activity against gram-positive bacteria. Among all obtained extracts, only leaf and flower fluid extracts showed activity against Gram-negative bacteria. Based on this bioassay, leaf fluid extracts tended to be the most potent, followed by flower fluid extracts. Minimal inhibitory concentration (MIC) values of extracts and antibiotics were comparatively determined by agar and broth dilution methods. Both extracts were active against S. aureus, coagulase-negative stahylococci, E. faecalis and E. faecium and all tested Gram-negative bacteria with MIC values from 0.067 to 0.308 mg/ml. In this study the minimal bactericidal concentration (MBC) values were identical or twice as high than the corresponding MIC for leaf extracts and four or eight times higher than MIC values for flower extracts. This may indicate a bactericidal effect. Stored extracts have similar antibacterial activity as recently obtained extracts. The A. aroma extracts of leaves and flowers may be useful as antibacterial agents against Gram- negative and Gram-positive antibiotic multi-resistant microorganisms.     

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.