Direct Hemolytic Activity Research Articles

The Prediction of Milk Whey Extract (MWE) Bioactive Compounds Based on Proximate Analysis and its Effects on Blood Coagulation: A New Approach

The present work demonstrates the milk whey extraction, proximate analysis, and biochemical characterization of bioactive compounds in MWE. Effects of MWE on plasma coagulation and platelet aggregation. Proximate analysis of MWE was done according to AOAC. Whey protein banding was confirmed in 12%, 15%, and 18% SDS-PAGEs. A quantitative analysis of bioactive compounds was done. The anticoagulant effects of MWE were tested using plasma recalcification time in both PRP and PPP, further confirmed by PT and APTT assays and in tail bleeding assays at concentrations of 0-100 µg. The non-toxic property of MWE was screened by edema, hemorrhage, and direct hemolytic activities. MWE proximate analysis showed the presence of both micro- and macronutrients. Qualitative analysis confirmed the presence of proteins and carbohydrates. A similar protein-banding pattern was observed in 12%, 15%, and 18% SDS-PAGE’s in both reduced and non-reduced conditions. MWE prolonged the clotting time of human citrated plasma, both PRP and PPP, against the control 210 sec to 770 sec, suggesting its anticoagulant property. MWE delayed the clot progress of only the APTT ratio (8.99 ± 0.09), and PT was not altered, suggesting its role in an intrinsic pathway of blood coagulation. MWE exhibited antiplatelet activity in PRP against ADP and epinephrine-induced platelet aggregation. The percentage of inhibition was 74% and 100% for ADP and epinephrine, respectively. Interestingly, MWE exhibits nontoxic properties, it does not cause hemolysis, hemorrhage, or edema. Milk whey extraction and studies confirm bioactive compounds, and these exhibit antithrombotic, antiplatelet, and non-toxic properties. Hence, MWE not only acts as a good nutritive source but also may prove tobe a therapeutic bioactive compound in the management of cardiovascular disease.

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra.

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra.Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed.Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice.Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.

Molecular profiling and bioactive potential of an endophytic fungus Aspergillus sulphureus isolated from Sida acuta: a medicinal plant

Context: Sida acuta Burm.f. (Malvaceae) extracts are reported to have applications against malaria, diuretic, antipyretic, nervous and urinary diseases. No fungal endophytes of S. acuta are reported. Objective: Isolation, identification and evaluation of antibacterial, antioxidant, anticancer and haemolytic potential of fungal endophytes from the ethnomedcinal plant S. acuta. Materials and methods: Sida acuta stem segments were placed on PDA medium to isolate endophytic fungi. The fungus was identified by genomic DNA analysis and phylogenetic tree was constructed using ITS sequences (GenBank) to confirm species. The antibacterial efficacy of Aspergillus sulphureus MME12 ethyl acetate extract was tested against Gram-positive and Gram-negative pathogenic bacteria. DPPH free radical scavenging activity, anticancer and DNA fragmentation against EAC cells, and direct haemolytic activity (100–500 μg/mL) using human erythrocytes were determined. Results and discussion: The ethyl acetate extract of A. sulphureus (Fresen.) Wehmer (Trichocomaceae) demonstrated significant antibacterial potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi compared to streptomycin. MIC against test pathogens was in the range of 15.6–62.5 μg/mL. The antioxidant results revealed significant RSA from 12.43% to 62.02% (IC50 = 350.4 μg/mL, p ≤ 0.05). MME12 offered considerable inhibition of EAC proliferation (23% to 84%, IC50 = 216.7 μg/mL, p ≤ 0.05) supported by DNA fragmentation studies. The extract also offered insignificant haemolysis (5.6%) compared to Triton X-100. Conclusions: A single endophytic fungus, A. sulphureus MME12 was isolated and identified using molecular profiling. The above-mentioned findings support the pharmacological application of A. sulphureus MME12 extract and demand for purification of the active principle(s).

Bungarus craeruleus (Indian common Krait) venom: An Attempt to observe the hemolytic property

Snakes are found in almost every part of the world. When the temperature of the air is fairly warm snakes may be seen at any hour of the day or night. Although they are found all over the world, but are not found in Antarctica as they do not like cold. In the present study the focus is on one of the venomous snake that is Indian common krait or Bungarus caeruleus. This snake’s venom is neurotoxic in nature. In author’s previous research paper the toxicity has been calculated as LD 50 and the venom was found most toxic through intracerebral route of inoculation. Hemotoxins are mostly found in viperids but here an attempt has been made to analyse the presence in bungarotoxin too. It has been shown by previous workers that the direct hemolytic activity is not present in the venom of Indian common Krait but to observe the indirectly hemolytic activity, an attempt has been made here. The study is also an attempt to see another character of venom of Bungarus caeruleus after toxicity.

Mechanism of in vivo anticoagulant and haemolytic activity by a neutral phospholipase A2 purified from Daboia russelii russelii venom: Correlation with clinical manifestations in Russell's Viper envenomed patients

A 13.0 kDa neutral phospholipase A2 (NEUPHOLIPASE) purified from venom of Daboia russelii russelii from eastern India was identified by peptide mass fingerprinting analysis. It exerted dose-dependent PLA2, anticoagulant and indirect haemolytic activities. NEUPHOLIPASE showed preferential binding followed by hydrolysis of phosphatidylserine > phosphatidylcholine >> phosphatidylethanolamine. Circular dichroism analysis of NEUPHOLIPASE showed a high content of alpha helix (54.6%) followed by beta-turn (29.7%) in its secondary structure. Gas-chromatographic analysis of plasma from PLA2-treated mice suggested preferential hydrolysis of pro-coagulant phospholipid PS was the primary mechanism to account for in vivo anticoagulant effect of NEUPHOLIPASE. The NEUPHOLIPASE-treated mice blood showed a significant decrease (p < 0.01) in WBC as well as RBC counts with a corresponding decline in Hb content due to indirect damage to erythrocyte membranes by plasma phospholipids hydrolysis products rather than the direct haemolytic activity of PLA2 under study. NEUPHOLIPASE was non-lethal to BALB/c mice, however; it was detrimental to liver of treated-mice. Pathological symptoms observed in NEUPHOLIPASE-treated mice were correlated with the actual clinical manifestations in Russell's Viper envenomed patients from eastern India indicating some contribution of NEUPHOLIPASE in Russell's Viper venom induced toxicity and pathogenesis.

Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera

Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits – basic and toxic phospholipase A2 (PLA2) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 109 pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones – D12, E3, F6, D10 and G5 exhihest binding affinity for the toxic component. Clones A1, D12 and C12 recognized preferentially vipoxin's acidic component. Clones E3, G5 and H4 inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E3, G5, H4, C12, D10 and A11) inhibited direct hemolytic activity of vipoxin and its pure PLA2 subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A2 activity for the vipoxin toxicity and its effective neutralization.

Enzymatic and structural characterization of a basic phospholipase A 2 from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A 2 (CgPLA 2) from the extract of the anemone Condylactis gigantea. CgPLA 2 was isolated with a high purity level through three chromatographic steps, showing p I ˜ 8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA 2 showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca 2+-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA 2 with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA 2 revealed 84%, 61%, 43% and 42% similarity to the PLA 2s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively.

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A 2 are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L. obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca 2+ and Mg 2+ ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L. obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L. obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation.

Toxic activities of Brazilian centipede venoms

Centipedes have a venom gland connected to a pair of forceps, which are used to arrest preys. Human victims bitten by centipedes usually manifest burning pain, paresthesia and edema, which may develop into superficial necrosis. The aim of this work was to characterize and compare toxic activities found in venoms of three species of Brazilian centipedes— Otostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDS–PAGE (4–20%), important differences were noticed among venoms (between 7 and 205 kDa). Few bands showed feeble caseinolytic, fibrinogenolytic and gelatinolytic activities by zymography, but strong hyaluronidase activity was observed in S. viridicornis and O. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, indicating that these enzymes are metalloproteinases. All venoms induced nociception, edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity. No coagulant activity was detected in centipede venoms. Low phospholipase A 2 activity was observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had intense direct hemolytic activity on human erythrocytes. Cross-reactivity among venoms was observed using species-specific sera raised in rabbits. Differences were noticed among centipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it could induce a more severe envenomation.

Cloning and characterization of Photobacterium damselae ssp. piscicida phospholipase: an enzyme that shows haemolytic activity

A phospholipase gene of Photobacterium damselae ssp. piscicida (ppp) was cloned from a genomic library and its nucleotide sequence was determined. The open reading frame consisted of 1218 bp encoding a protein of 405 amino acids with a predicted molecular mass of 46 kDa. The PPP had identities (53-55%) with phospholipase and haemolysin of Vibrio spp., while it showed low identities (23-26%) with glycerophospholipid cholesterol acyltransferase of Aeromonas spp. A recombinant PPP (rPPP) with a His tag at the C-terminus expressed in Escherichia coli and purified showed phospholipase activity. The rPPP also showed lecithin-dependent haemolytic activity against mammalian erythrocytes and direct haemolytic activity against fish erythrocytes. The culture supernatant of wild-type P. damselae ssp. piscicida showed phospholipase activity, while that of a PPP gene knockout mutant did not.

In vitro hemolysis and lipid peroxidation-inducing activity of the tentacle extract of the sea anemone (Paracondylactis indicus Dave) in rat erythrocytes

<b>Objective</b> : <i>In vitro </i>hemolytic activity of the tentacle extract of <i>Paracondylactis indicus</i> (Dave), a sea anemone found in the eastern coastal region of West Bengal (India), was determined in rat erythrocytes.<br><b> Materials and Methods</b> : Acute toxicity study was carried out with the tentacle extract followed by detailed biochemical studies to evaluate the direct and indirect hemolytic activities of the extract. The effects of pH, temperature, divalent cations and chelating agents on hemolysis were also evaluated. Efforts were also made to elucidate the mechanism of the hemolytic activity of the extract.<br><b> Results</b> : The tentacle extract of <i>P. indicus</i> (Dave) produced significant hemolysis (both direct and indirect) in washed rat erythrocytes. The direct hemolytic activity of the tentacle extract was found to be both temperature- and pH-dependent. Divalent cations (Ca²⁺ , Mg²⁺ ) produced significant enhancement of direct hemolytic activity. Hemolysis (both direct and indirect) was significantly diminished in the presence of Zn²⁺ , EDTA and <i>p</i>-bromophenacyl bromide. The tentacle extract was found to induce lipid peroxidation in washed erythrocytes, which was inhibited by chlorpromazine. The tentacle extract demonstrated significant proteolytic activity, which was found to decrease in the presence of protease inhibitors (EDTA, EGTA and PMSF).<br><b> Conclusion</b> : <i>P. indica</i> tentacle extract (and in particular, the 60% cut fraction) probably contains enzymatic components like phospholipase(s) along with proteases, which may be the contributing factors for its observed hemolytic activity.

Biological activities of a lectin from Bothrops jararacussu snake venom

Snake venoms contain saccharide-binding lectins. In this work, we examined the biological activities of a lectin (BjcuL) purified from Bothrops jararacussu snake venom by chromatography on non-derivatized Sepharose 4B and Sephacryl S-200 HR. The protein, a homodimer with subunits of 14.5 kDa, gave a single immunoprecipitin line in immunoelectrophoresis and cross-reacted in ELISA with antivenoms raised against Bothrops spp. (lanceheads), Micrurus spp. (coral snakes), Crotalus durissus terrificus (South American rattlesnake), and arthropod ( Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus) venoms. BjcuL agglutinated human formaldehyde-fixed erythrocytes at ≥100 ng/ml and was inhibited by lactose and EDTA (≥2 mM) and high concentrations (>100 mM) of glucose and sucrose, but not by N-acetylglucosamine. BjcuL had no direct hemolytic activity and was devoid of esterase, PLA 2 and proteolytic activities. The lectin (up to 200 μg/ml) did not aggregate human platelet-rich plasma (PRP) or washed platelets (WP), nor did it alter the aggregation induced by ADP in PRP or by thrombin in WP. When injected into mouse hind paws, BjcuL (10–100 μg/paw) caused edema and increased vascular permeability, with a maximum effect after 1 h that persisted for up to 6 h (edema) or gradually decreased after the peak interval (vascular permeability). No hemorrhage was observed in BjcuL-injected paws. In anesthetized rats, B. jararacussu venom (200 μg/kg, i.v.) produced sustained hypotension (maximum decrease of ∼60%) whereas a similar dose of BjcuL decreased the blood pressure by ∼15%, with a rapid return to the resting level.

TRICHOMONAS VAGINALIS: IDENTIFICATION OF SOLUBLE AND MEMBRANE-ASSOCIATED PHOSPHOLIPASE A1AND A2ACTIVITIES WITH DIRECT AND INDIRECT HEMOLYTIC EFFECTS

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.

Intravascular hemolysis induced by Lonomia obliqua caterpillar bristle extract: an experimental model of envenomation in rats

Hemostatic disturbances are frequent findings in human accidents caused by Lonomia obliqua caterpillars in the southern region of Brazil. In severe envenomation, patients may present life-threatening bleedings. Such disturbances may be mimicked in rats, which also develop intravascular hemolysis. The scope of this study was to investigate the time-course and intensity of intravascular hemolysis induced by i.d. injection of 750 μg/kg crude L. obliqua bristle extract in rats. Total blood cell count, reticulocyte count, plasma hemoglobin and haptoglobin assays were performed in control and envenomed rats at 1, 6, 24 and 48 h after envenomation. Rats presented a drastic drop of haptoglobin levels at 1 and 6 h with increased plasma hemoglobin levels, a decrease in packed cell volume values at 6, 24 and 48 h, and increased reticulocyte counts throughout after envenomation. Such observations indicated that intravascular hemolysis occurred as early as 1 h following envenomation, and lasted for more than 6 h. Intravascular hemolysis is probably induced by phospholipase A 2 and other proteins with direct hemolytic activity present in crude caterpillar bristle extract.

In vitro immunomodulatory activity of plants used by the Tacana ethnic group in Bolivia

One hundred and seventy-eight ethanolic plant extracts from the pharmacopoeia of the Tacana, an ethnic group from Bolivia, were screened for immunomodulatory activity using complement cascade inhibition and ADP-induced platelet aggregation inhibition assays. Six impaired both complement pathways (classical and alternative): stem bark from Astronium urundeuvea (Anacardiaceae), Cochlospermum vitifolium (Cochlospermaceae), Terminalia amazonica (Combretaceae), Triplaris americana (Polygonaceae), Uncaria tomentosa (Rubiaceae) and Euterpe precatoria (Arecaceae) roots. Inhibition of complement cascade was independent of essential ion complexation, and was not due to direct hemolytic activity on target red blood cells. For A. urundeuvea, C. vitifolium, and T. amazonica, anti-inflammatory activity relied on cyclo-oxygenase inhibition. Four of these species (A. urundeuva, T. americana, U. tomentosa and E. precatoria) are used traditionally to treat inflammatory processes.

In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes

Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes. Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane.

Micrurus spixii (PERUVIAN CORAL SNAKE) VENOM - PRELIMINARY BIOCHEMICAL AND ENZYMATIC CHARACTERIZATION

Micrurus spixii venom was studied after fractionation by Sephadex G-100 SF gel filtration chromatography. Several enzymatic activities and biological effects were investigated in whole venom and fractions. The venom was resolved in four peaks in a range of about 73.2-10.7 kDa molecular weight. Alkaline phosphatase and acetylcholinesterase activities were found in peak I, and procoagulant activity was seen in peak II. Phospholipase A2, hemorrhagic, and proteolytic activities were detected in peak III. A second procoagulant factor and proteinase were present in peak IV. Thrombin-like enzyme and direct hemolytic activities were not found in any assayed samples.

Characterization of PaxA and Its Operon: a Cohemolytic RTX Toxin Determinant from Pathogenic Pasteurella aerogenes

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

Tyr→Trp-substituted peptide 115-129 of a Lys49 phospholipase A 2 expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect

Myotoxin II is a group II Lys49 phospholipase A 2 (PLA 2) isolated from the venom of the snake Bothrops asper. Previous studies on a synthetic peptide derived from its heparin-binding, cationic/hydrophobic sequence 115–129 demonstrated a direct functional role of this particular region in the in vitro cytolytic and bactericidal actions of the protein. Nevertheless, no significant myonecrosis has been observed after local intramuscular injection of peptide 115-129 (p115-129) in mice. Since the membrane-damaging action of p115-129 was proposed to depend on its amphiphilic character, the present study examined the effects of substituting its cluster of three tyrosine residues by tryptophan residues, on its toxic/pharmacological activities in vitro and in vivo. This substitution resulted in a drastic enhancement of the membrane-damaging activities of the peptide (p115-W3), together with the clear expression of myotoxic activity in vivo. Both the heparin-binding and antigenic characteristics of p115-129 were essentially conserved in p115-W3, suggesting that the modification did not lead to radical structural alterations. In addition to myotoxicity, cytotoxicity, and bactericidal action, p115-W3 exerted edema-forming activity in the mouse footpad assay. Thus, the synthetic 13-mer p115-W3 reproduced all the known toxic effects of myotoxin II. In spite of its potent membrane-damaging actions, p115-W3 did not acquire direct hemolytic activity upon mouse erythrocytes, an effect which is not present in myotoxin II, but that has been ascribed to the presence of tryptophan in other cationic, membrane-damaging peptides such as mellitin from bee venom. The myotoxic activity of p115-W3 herein described constitutes the first example of a short, PLA 2-based linear synthetic peptide with the ability to reproduce this effect of a parent protein in vivo. This finding is in clear support of the proposed relevance of the C-terminal region 115–129 in all the membrane-damaging mechanisms exerted by myotoxin II, including the myotoxic mechanism.

Anticomplement activities of oleanolic acid monodesmosides and bisdesmosides isolated fromTiarella polyphylla

Seven known oleanolic acid glycosides (1-7) were isolated from the MeOH extract of Tiarella polyphylla. The structures were identified to be 3-O-(beta-D-glucopyranosyl) oleanolic acid (1), 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl] oleanolic acid (2), 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl] oleanolic acid (3), 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl] oleanolic acid 28-O-beta-D-glucopyranosyl ester (4), 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl] oleanolic acid 28-O-beta-D-glucopyranosyl ester (5), 3-O-[a-L-rhamnopyranosyl-(1-->3)-beta-D-glucuronopyranosyl] oleanolic acid (6), and 3-O-[alpha-L-rhamnopyranosyl-(1-->3)-beta-D-glucuronopyranosyl] oleanolic acid 28-O-beta-D-glucopyranosyl ester (7) on the basis of physicochemical and spectral data. These triterpene glycosides were tested for the anticomplement activity and hemolytic activity. Bisdesmosidic saponins, 4, 5, and 7, showed anticomplement activity; in contrast, monodesmosidic saponins, 1-3, and 6, showed direct hemolytic activity. Methyl esterified monodesmosidic saponins showed anticomplement activity at a low concentration and hemolytic activity at a high concentration.