Rat snake, Taylor Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Rat snake, Taylor Slough

This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. This image is made available for non-commercial or educational use only.

Action of anti-bothropic factor isolated from Didelphis marsupialis on renal effects of Bothrops erythromelas venom

Acute renal failure is the most common complication in the lethal cases caused by snakebites in Brazil. Among the Brazilian venom snakes, Bothrops erythromelas is responsible for the majority of accidents in Northeastern Brazil. Didelphis marsupialis serum could inhibit myonecrotic, hemorrhagic, edematogenic hyperalgesic and lethal effects of envenomation determined by ophidian bites. In the present study, we evaluated the action of the anti-bothropic factor isolated from D. marsupialis on the renal effects promoted by B. erythromelas venom without systemic interference. Isolated kidneys from Wistar rats were perfused with Krebs-Henseleit solution containing 6% bovine serum albumin. We analyzed renal perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), and the percentages of sodium and potassium tubular transport (%TNa +, %TK +). The B. erythromelas venom (10 μg mL -1) decreased the PP (ct=108.71±5.09 mmHg; BE=65.21±5.6 mmHg*) and RVR (ct=5.76±0.65 mmHg mL -1 g -1 min -1; BE=3.10±0.45 mmHg mL -1 g -1 min -1*) . On the other hand, the GFR decreased at 60 min (ct 60=0.76±0. 07 mL g -1 min -1; BE 60=0.42±0.12 mL g -1 min -1*) and increased at 120 min (ct 120=0.72±0.01 mL g -1 min -1; BE 120=1.24±0.26 mL g -1 min -1*). The UF increased significantly when compared with the control group (ct=0.14±0.01 mL g -1 min -1; BE=0.47±0.08 mL g -1 min -1*). The venom reduced the %TNa + (ct 90=79.18±0.88%; BE 90=58.35±4.86%*) and %TK + (ct 90=67.20±4.04%; BE 90=57. 32±5.26%*) The anti-bothropic factor from D. marsupialis (10 μg mL -1) incubated with B. erythromelas venom (10 μg mL -1) blocked the effects on PP, RVR, %TNa +, and %TK +, but was not able to reverse the effects in UF and GFR promoted by venom alone. However, the highest concentration of D. marsupialis serum (30 μg mL -1) reversed all the renal effects induced by the venom. In conclusion, B. erythromelas venom altered all the renal functional parameters evaluated and the anti-bothropic factor from D. marsupialis was able to inhibit the effects induced by the venom in isolated kidney. © 2005 Elsevier Ltd. All rights reserved.

Rat Atrial Responses To Bothrops Jararacussu (jararacuçu) Snake Venom.

Envenoming by the pitviper Bothrops jararacussu produces cardiovascular alterations, including coagulopathy, systemic hemorrhage, hypotension, circulatory shock and renal failure. In this work, we examined the activity of this venom in rat isolated right atria. Incubation with venom (0.025, 0.05, 0.1 and 0.2mg/ml) caused concentration-dependent muscle contracture that was not reversed by washing. Muscle damage was seen histologically and confirmed by quantification of creatine kinase-MB (CK-MB) release. Heating and preincubation of venom with p-bromophenacyl bromide (a phospholipase A2 inhibitor) abolished the venom-induced contracture and muscle damage. In contrast, indomethacin, a non-selective inhibitor of cyclooxygenase, and verapamil, a voltage-gated Ca(2+) channel blocker, did not affect the responses to venom. Preincubation of venom with Bothrops or Bothrops/Crotalus antivenom or the addition of antivenom soon after venom attenuated the venom-induced changes in atrial function and tissue damage. These results indicate that B. jararacussu venom adversely affected rat atrial contractile activity and muscle organization through the action of venom PLA2; these venom-induced alterations were attenuated by antivenom.

Therapeutic effects and possible mechanisms of a snake venom preparation in the fibrotic rat liver

The effects of a Chinese snake venom preparation from Agkistrodon halys pallas, used for treatment of hepatic fibrosis/cirrhosis in China, was investigated in an {in vivo} rat model and using in situ hepatic perfusion. Four groups were used in the experiments: (i) healthy, (ii) healthy/venom-treated, (iii) carbon tetrachloride (CCl4)-treated, and (iv) CCl4/venom-treated. Treatment effects were assessed by determining hepatic histopathology, biochemistry and fibrosis index parameters, bile production, biliary taurocholate recovery, hepatic mRNA expression of four bile salt transporters (Ntcp, Bsep, Oatp-1, and Oatp-3), comparison of hepatic microcirculation, fibrinolytic activity, and antithrombotic effects. Liver histopathology, biochemistry, and fibrosis index showed a dramatic improvement in venom-treated animals. There were significant differences in bile production between healthy/venom-treated and all other experimental groups and between CCl4/venom-treated and CCl4-treated animals, but no significant differences were found between CCl4/venom-treated and healthy animals. Biliary taurocholate recovery was significantly increased in healthy/venom-treated and CCl4/venom-treated animals. The expression of mRNA levels of the four bile salt transporters showed an increase after venom treatment. The hepatic microcirculation studies showed normalized sinusoidal beds in CCl4/venom-treated animals compared to healthy animals, whereas CCl4-treated animals showed abnormal profiles to the healthy and the CCl4/AHPV-treated animals. The fibrinogen and plasma thromboxane B-2 levels of healthy rats decreased with increasing dose after venom treatment. It was concluded that snake venom treatment may be therapeutic in treatment of hepatic fibrosis/cirrhosis by possibly a combination of increased bile flow and improved hepatic microcirculation, changes in bile salt transporter expression, and fibrinolytic and antithrombotic effects of the snake venom preparation.

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom

Background: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. Objective: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. Methods: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. Results: Platelet counts were markedly diminished in rats administered with either ARPI (+/- 88%) or busulfan (+/- 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. Conclusions: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

Ultrastructure of Motor Nerve Terminals in the Anterior Third of Wistar Rat Tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi`s apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech. 72:464-470, 2009. (C) 2009 Wiley-Liss. Inc.

Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NF kappa B and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NF kappa B and AP-1. Activation of NF kappa B and AP-1 depended on phospholipase C and protein kinase A. Activation of NF kappa B also depended on protein kinase C. Isoprenaline activated both NF kappa B and AP-1, and phenylephrine activated NF kappa B and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.

The Snake Venom Peptide Bj-PRO-7a Is a M1 Muscarinic Acetylcholine Receptor Agonist

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (rat M1 muscarinic subtype. The activation curve established by microfluorimetry in CHO-M1 cells using increasing concentrations of Bj-PRO-7a reached the maximum response in the presence of 3 mu M Bj-PRO-7a (EC(50) = 0.25 +/- 0.07 mu M). The variation observed by calcium imaging in these cells ranged from 52 to 1218 nM (EC(50) = 0.31 +/- 0.12 mu M). [Ca(2+)](i) responses in CHO-M1 cells were largely inhibited by pirenzepine, a specific M1 antagonist. Neural-differentiated P19 cells expressing endogenous M1 receptors were also responsive to Bj-PRO-7a application, whereas no such response was observed in undifferentiated P19 cells not expressing muscarinic receptors. As further support for its specific action on M1 receptors, the peptide did not activate M3 subtypes in transfected CHO cells. Our findings provide a novel M1 muscarinic receptor agonist that could be used for basic research and even for pharmacological applications. (C) 2010 International Society for Advancement of Cytometry

Effects of morin on snake venom phospholipase A(2) (PLA(2))

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA(2) isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to. study the effect of flavonoids on PLA(2). We observed that a treatment of PLA(2) with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA(2) possess a second pharmacological site which does not affect or depend on the enzymatic activity. (c) 2005 Elsevier Ltd. All rights reserved.

Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activity

(1) Venom pools from Bothrops neuwiedi (Bn) and from two subspecies, namely Bothrops neuwiedi pauloensis (Bnp) and Bothrops neuwiedi urutu (Bnu), collected in the States of São Paulo (SP) and Minas Gerais (MG), Brazil, were electrophoretically examined. Basic toxins with different isoelectric points were identified in the venom collected in São Paulo (BnSP). These toxins were absent in the corresponding pools from Minas Gerais (BnMG, BnpMG and BnuMG). (2) BnSP, but not BnMG, BnpMG or BnuMG, showed two myotoxins (pI congruent to 8.6 and 8.8, respectively) which were isolated by ion-exchange chromatography on CM-Sepharose. (3) From BnMG, three myotoxic isoforms (pI congruent to 8.2 and M-r = 13600) were isolated by chromatography on CM-Sepharose followed by reversed-phase high-performance liquid chromatography. (4) the chemical and biological characterization of these toxins showed a high similarity with the Lys-49 myotoxins from other bothropic venoms. (5) Doses up to 5 LD50 (i.p.) of p-bromophenacyl bromide alkylated BnSP-7 caused a total loss of lethality in 18-22-g mice, thus indicating that the LD50 was increased by greater than 5-fold. At this dose myotoxicity was also not detectable, but the edematogenic activity on the rat paw apparently did not change. (C) 1998 Elsevier B.V. All rights reserved.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Use of fibrin glue derived from snake venom in testicular biopsy of rams

Sequelae due to testicular biopsy such as hemorrhage, adhesion and fibrosis may be limiting factors to the use of this surgical procedure. Fibrin glue (FG) derived from snake venom was used to minimize these sequelae, as well as to evaluate its healing property in tunica vaginalis and scrotal skin of rams. Applicability of fibrin glue derived from snake venom was tested in different tissues of other animals such as in sciatic nerve and colon of rats and skin of rabbits. In the present study, 30 healthy adult rams were used. They were divided into 3 groups of 10 animals each as follows: G1: fibrin glue group (application of fibrin glue on puncture sites and skin incisions after bilateral testicular biopsy with a Tru-Cut needle); G2: swab/nylon group (hemostasis by compression with a swab on puncture sites and skin suturing with nylon after biopsy) and G3: control group (the animals were not subjected either to biopsy or to surgery). On the 20th day after biopsy, the presence of adhesion strands between the sites of skin incision and testicle was evaluated by palpation Adhesion strands were found in three testicles (15%) in G1 and in two testicles (10%) in G2. One hundred days after biopsy, orchiectomy was carried out and the material collected was assessed for subcutaneous (SC) and/or tunica vaginalis adhesions. G3 did not present any abnormality. Groups G1 and G2 presented four testicles each (20%) with adhesion between the tunics at biopsy site. On the other hand, subcutaneous adhesions were found once (5%) in G1 and three times (15%) in G2. Fibrin glue showed to be of easy application, required short postoperative monitoring, presented fast and good-quality healing property and tended to reduce formation of subcutaneous adhesion.