Constituintes químicos e atividade antiedematogênica de Peltodon radicans (Lamiaceae)

Most of the snakebite incidents in the Amazon region involve Bothrops atrox, whose venom presents the most potent edematogenic and necrotic activities in the genus. This work describes the studies of isolation of the chemical constituents and antiedematogenic activity of the species Peltodon radicans (Lamiaceae), which is used in the treatment of snakebites and scorpion stings in the region. The extracts presented aliphatic hydrocarbons, 3beta-OH,beta-amirin (1), 3beta-OH,alpha-amirin (2), beta-sitosterol (3), stigmasterol (4), ursolic acid (5), 2alpha,3beta,19alpha-trihydroxy-urs-12-en-28-oic acid (tormentic acid, 6), methyl 3beta-hydroxy,28-methyl-ursolate (7), sitosterol-3-O-beta-D-glucopyranoside (8), and stigmasterol-3-O-beta-D-glucopyranoside (9). The flower extracts presented the higher antiedematogenic activity. This is the first report on the study of the flowers, stem, and roots of this plant.

Neutralizing capacity of a new monovalent anti-Bothrops atrox antivenom: comparison with two commercial antivenoms

Three horse-derived antivenoms were tested for their ability to neutralize lethal, hemorrhagic, edema-forming, defibrinating and myotoxic activities induced by the venom of Bothrops atrox from Antioquia and Chocó (Colombia). The following antivenoms were used: a) polyvalent (crotaline) antivenom produced by Instituto Clodomiro Picado (Costa Rica), b) monovalent antibothropic antivenom produced by Instituto Nacional de Salud-INS (Bogotá), and c) a new monovalent anti-B. atrox antivenom produced with the venom of B. atrox from Antioquia and Chocó. The three antivenoms neutralized all toxic activities tested albeit with different potencies. The new monovalent anti-B. atrox antivenom showed the highest neutralizing ability against edema-forming and defibrinating effects of B. atrox venom (41 ± 2 and 100 ± 32 µl antivenom/mg venom, respectively), suggesting that it should be useful in the treatment of B. atrox envenomation in Antioquia and Chocó

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

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.

Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation.

Missale ad usum ecclesiae Parisiensis.

Cf. notice du ms. par Leroquais, Sacramentaires, II, 180-183 n° 366. Les incipit des proses ont été relevés dans la table des incipit. F. 1-6v Calendrier de Paris : 3 janv., en rouge, «Genovefe virg. IX lc.» (1); 22 avr., «Inventio corporis s. Dyonisii sociorumque ejus. Oportune virg. semid.» (2v); 28 mai, en rouge, «Germani ep. Parisiensis semid. Carauni mart. memoria» (3); 25juin, en violet, «Translatio s. Eligii ep.» (3v); en violet, «In prima dominica hujus mensis [augusti] fit d. de cruce» [réception à Notre-Dame d'un fragment de la vraie Croix] (4v); 9 oct., en rouge, «Dyonisii sociorumque ejus duplex» (5v); 28 oct., «Germani ep. Translatio s. Genovefe virg. mart. memoria» (5v); 3 nov. «Marcelli ep. Parisiensis dupl.»; 13 nov., «Gendulphi ep. et conf. [Parisiensis] dupl.»; 26 nov., en violet, «Genovefe virg. de miraculo [ardentium] IX lc.» (6); 4 déc., «Susceptio capillorum b. Marie et capitis b. Dyonisii in ecclesia Parisiensi» (6v). Le calendrier ne contient pas la fête de s. Louis au 25 août (établie en 1298) ni au 17 mai la translation du chef de s. Louis à la Sainte-Chapelle (en 1306); — Ajout du XIVe s. au 30 avr., «Eutropii mart. et conf.» [culte établi à Paris en 1296] (2v); cf. Leroquais, Bréviaires, I, CXII-CXIII, Tableau chronologique des fêtes parisiennes. — Au début de chaque mois, vers sur les jours égyptiaques (éd. Hennig, Traditio, XI (1955), 84 III), sauf celui de janvier (1) qui correspond au premier vers de la série éd. par Riese (Anthol. lat., I2 (1906), 680a); — À la fin de février, vers sur l'année bissextile : «Byssextum sexte martis tenuere kalende...»; cf. ms. Latin 3162, f. 102 (1v); — À la fin de mars, vers sur la date de Pâques : «Post martis nonas ubi sit nova luna requiras...» (2); — Notations astrologiques et de comput, passim. F. 7-353v Temporal, avec parties chantées notées. Incomplet du début par la perte du premier f., il commence dans la prose du premier dimanche de l'Avent : «... [eterna indefici]ens mundi vita...» (7). À remarquer : «dominica IIIa [in Adventu] more ecclesie Parisiensis» (24v); — Cérémonie des Cendres avec rubriques liturgiques (62v-66v); — Dimanche des Rameaux, «congregatis processionibus conventualibus in ecclesia B. Marie, capiceriis portantibus capsam et tribus clericis in albis paratis tres textus, exitur de ecclesia nichil cantando et sic eundum est ad ecclesiam Sancte Genovefe de Monte...» (147); cf. Leroquais, 181; — Dans l'évangile des Rameaux, le Christ est désigné par la lettre L, le narrateur par la lettre C et les autres par la lettre S (153-158); dans les autres évangiles de la Passion (162-186 passim), ces lettres suscrites ont été effacées et remplacées à la mine de plomb de façon sporadique, le Christ étant alors désigné par une croix; — «Oremus pro papa nostro N...» (186v); «... pro christianissimo rege nostro N...» (187); — Parmi les litanies, «s.Stephane... s. Dyonisi...» (191v)..., « s. Lucane... s. Justine... s. Gendulphe... s. Germane...» (199)..., « s. Genovefa...» (201v). — «Incipit ordo misse. Sacerdos primo induat se rocheto dicens : Actiones nostras...» (203v-206); pour les rubriques liturgiques, cf. Leroquais, 181; — Préfaces notées : «Incipiunt prefationes...» (206-210); — Canon de la messe (210-213); pour les rubriques liturgiques, cf. Leroquais, 182; — «In die Re[sur]rectionis» (214); — «In die sancto [Pentecostes]» (262v); — [De sancta Trinitate] (280v); — «Dominica XXVa» (348v); — «In dedicatione ecclesie» (350-353v). La fête du Saint-Sacrement ne figure pas. F. 354-496v Sanctoral, avec parties chantées notées. À noter parmi les saints parisiens : 3 janv., «S. Genovefe virg.» (373v); 22 avr., «Inventio corporis s. Dyonisii, Rustici et Eleuterii» (398v); — 28 mai, «S. Germani ep. Parisiensis» (407v); — 10 juin, «S. Landerici Parisiensis ep.» (410); — 26 juill., «In translatione s. Marcelli» (432); — 11août, «De receptione s. corone...» (443v); —, 4 oct., «Auree virg. [abb. Parisiis]» (480); 9 oct., «In die [s. Dyonisii]», avec octave (481v); — 28oct., «In translatione s. Genovefe» (487); — 3nov., «S. Marcelli Parisiensis ep.», avec octave (489v); — 26 nov., «S. Genovefe de miraculo ardentium» (495); — «De s. Gendulpho. Prosa» (495). F. 497-541 Commun des saints, avec pièces chantées notées. F. 541-559 Messes votives, sans parties notées à l'exception de la messe des morts (551v-556). Aux ff. 546v-547, en marge de la messe «pro amico», deux additions d'une écriture cursive de la fin du XIVe s. précisent : «ducem nostrum et duxissem ejusque prolem». — Prières diverses (556-559). F. 559-563 Rituel de mariage. «Incipit ordo ad sponsam benedicendam. Cum venerint ante valvas ecclesie sponsus et sponsa, accinctus sacerdos alba et stola... auxilium et argentum super scutum positum benedicat dicens : Manda Deus... Tunc aspergatur aqua benedicta et thurificetur et sponsus et sponsa; quo facto sacerdos dicat : Bones genz nos avons faiz les bans III foiz de ces II genz et encore les faison nous, que se il i a nul ne nule qui sache enpeschement par quoi l'un ne puisse avoir l'autre par loi de mariage, si le die. Et re[spon]dent assistentes : Nous ni savons se bien non. Quo audito, accipiat sacerdos manum dexteram sponse et ponat in dextera manu sponsi et dicat... nominandos eos : Vos Marie et vos Jeham vous prometez, fianciez et jurez l'un à l'autre à garder la foi et la loiauté du mariage... Tunc sacerdos tradat anulum sponso et sponsus autem per manum sacerdotis primo in police sponse... dicens... : Marie de cest annel t'espous et de mon cors t'ennor et de douaire qui est devisiez entre mes amis et les tiens. In nomine... Secundo in indice dicens... Tercio in medio dicens : Et Spiritus sancti. Amen... Postea sacerdos extensa manu super illos dicat orationes... Tunc sacerdos centus [sic] sponsum per manum dexteram et sponsam et introducat eos in ecclesiam...» (559-559bis); — Messe de mariage, comportant deux préfaces notées : «Deinde celebratur missa...» (559bis-562v); pour les rubriques liturgiques, cf. Leroquais, 183. — «Missa celebrata, recedant sponsus et sponsa et, ipsis stantibus ante hostium domus illorum presentibus pane et vinum [sic], faciat sacerdos benedictionem super panem dicens : Benedic Domine creaturam... Tunc sponsus mordet in pane, postea sponsa. Item benedictio super vinum... Tunc sponsus bibat, postea sponsa. Quo facto introducit eos sacerdos per manum in domum... Item in sero benedictio thalami... Tunc turificet thalamum, postea sponsum et sponsam sedentes vel jacentes in lecto suo benedicat dicens : Benedic Domine adulescentulos...» (562v-563) (éd. avec variantes par E. Martène, De antiquis Ecclesiae ritibus, II, 374-376, d'après le ms. Latin 859A); même ordo dans le ms. NAL 2649, f. 333-336. F. 563-565v Exorcisme de l'eau et du sel (563-564v). — «Pro Terra sancta» (564v-565). — «Pro rege nostro» (565). — «Benedictio pere et baculi peregrinorum» (565-565v). — «Benedictio crucis... Benedictio novorum fructuum... Benedictio panis» (565v).

Studies on Venom of the fish Scatophagus Argus from Cochin Estuary-A Biochemical Approach

Studies reveal the presence of enzymes and different proteins in the venom of S.argus. The present study detected the presence of phosphodiesterase in S. argus venom. S. argus venom has displayed the presence of micromolar concentration of acetylcholine. Phospholipase activity in S. argus venom shows values below the detection threshold indicating that the venom does not possess this enzyme. The proteolylic activity of S. argus venom on casein and gelatin were assayed due to the probable involvement of proteases in causing the instability of biological activities of the fish venom. Caseinase and gelatinase enzymes were detected in S. argus venom. Though exact relationships of these enzymes and proteins in envenomation are not traced, the involvement of enzymes in envenomation cannot be ruled out. Further studies are required to find the mechanism of action of these enzymes and proteins present in S. argus venom. The present study opens new dimensions for isolation of the lethal compound present in S. argus venom. The preliminary study carried out here shows the presence of a lethal factor between 6.5 KDa - 68 KDa. Studies conclude that fish venom possesses many bioactive substances, especially peptides, proteases and enzymes that bind with high affinity to physiological targets and can be trapped for therapeutic purposes in the near future. Even though this study reveals the conundrums of S. argus venom, it opens new vistas of research on the venom components and the application and design of the venom as a drug.

L'oviparisme en la família Scorpaenidae (Pisces: Scorpaeniformes)

En aquest treball de tesi doctoral es descriu l'estructura gonadal, gametogènesi i cicle anual de Scorpaena porcus, S. scrofa i S. elongata per tal d'aprofundir en el coneixement dels diferents graus d'especialització dins l'oviparisme en peixos. Es pretén fer èmfasi en aquells trets inusuals que difereixen del què s'ha descrit fins ara per la majoria de les espècies ovípares de teleostis, i relacionar-los amb l'estratègia reproductiva de les espècies que els presenten El mostreig s'ha dut a terme de forma intensiva entre l'any 2000 i 2002, tot i que s'ha continuat recol·lectant mostres fins a l'agost de 2004, amb més o menys freqüència segons la necessitat. En total s'han obtingut 258 exemplars de S. porcus, 119 de S. scrofa i 46 de S. elongata. S'ha pogut veure com S. porcus, S. scrofa i S. elongata són espècies ovípares ovulípares especialitzades, ja que la seva estructura ovàrica presenta tot una sèrie de trets molt particulars i poc comuns a la resta de peixos ovípars estudiats fins ara: posició central del raquis musculo-connectiu que es troba envoltat completament pel lumen i la paret ovàrica; presència de peduncles en oòcits en fase de creixement secundari; escassetat i mida petita dels alvèols corticals; poc gruix de la zona radiata; i secreció d'una massa gelatinosa que engloba els ous en el moment de la posta. Totes aquestes característiques també s'han observat en Scorpaena notata, suggerint que l'especialització de l'oviparisme ovulípar es dóna en tot el gènere Scorpaena. L'estructura testicular i l'espermatogènesi d'aquestes espècies també és força particular. Per una banda els testicles són del tipus lobular no-restringit, tot i que presenten algunes característiques com la ordenació parcial dels cists segons l'estadi de maduració de les cèl·lules germinals que contenen i l'absència de lumen central, que actualment només s'ha observat en testicles del tipus lobular restringit de les espècies de la sèrie Aterinomorpha. Per l'altre banda, l'espermatogènesi és del tipus semicística, és a dir, els cists s'obren abans d'acabar tot el procés i les cèl·lules germinals s'acaben de desenvolupar a la llum del lòbul fins a espermatozoides. La posta d'aquestes espècies és múltipla i constisteix en una massa gelatinosa d'ous pelàgica, que en el cas de S. porcus s'alliberen entre els mesos de juny i agost, i en S. scrofa, a partir del mes de juliol. S'ha relacionat la presència d'aquesta massa amb un aparellament mascle-femella, de manera que el mascle alliberaria l'esperma directament sobre la massa d'ous, assegurant així la fartilització total dels mateixos.

Dynamics of QoI sensitivity in Mycosphaerella fijiensis in Costa Rica during 2000 to 2003

From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G 143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002,found no significant variation in frequency on spatial scales <10 in. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G 143A allele in bulk samples from I I farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.

Evolutionary analysis of novel serine proteases in the venom gland transcriptome of Bitis gabonica rhinoceros

Background: Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings: Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance: Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues.

The genus Ananteris Thorell, 1891 (Scorpiones, Buthidae) in southeast Brazil, with the description of three new species

A new synthesis of the geographical distribution of the genus Ananteris Thorell, 1891 (Scorpiones, Buthidae) in Brazil is proposed. Three new species, Ananteris chagasi sp. n., Ananteris kuryi sp. n., and Ananteris bernabei sp. n. are described from the States of Bahia, Espirito Santo and Minas Gerais. Only isolated records of Ananteris species where known up to now, for the States of Minas Gerais and Bahia. The record of an Ananteris species from the State of Espirito Santo is new. The total number of Ananteris species is now raised to 67 and from this, 16 species are found in Brazil. The geographical distribution of the genus is considerably enlarged in the country.

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. (C) 2010 Elsevier Ltd. All rights reserved.

Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation

Symptoms evoked by Thalassophryne nattereri fish envenomation include local oedema, severe pain and intense necrosis with strikingly inefficient healing, continuing for several weeks or months. Investigations carried out in our laboratory showed that, in the venom-induced acute inflammation, thrombosis in venules and constrictions in arterioles were highly visible, in contrast to a notable lack of inflammatory cell. Nevertheless, the reason that the venom toxins favour delayed local inflammatory response is poorly defined. In this study, we analysed the movement of leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice, the production of pro-inflammatory mediators and the venom potential to elicit matrix metalloproteinase production and extracellular matrix degradation. Total absence of mononuclear and neutrophil influx was observed until 14 days, but the venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases (MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue degradation of collagenous fibre. An influx of mononuclear cells was noted very late and at this time the levels of IL-6, IL-1 beta and MMP-2 remained high. Additionally, the action of venom on the cytoskeletal organization was assessed in vitro. Swift F-actin disruption and subsequent loss of focal adhesion was noted. Collectively these findings show that the altered specific interaction cell-matrix during the inflammatory process creates an inadequate environment for infiltration of inflammatory cells.