Ability of suramin to antagonize the cardiotoxic and some enzymatic activities of Bothrops jararacussu venom.

We have investigated the cardiotoxic effect of Bothrops jararacussu crude venom and the ability of suramin to antagonize this effect in the heart of rats, as well as the proteolytic and phospholipase A(2) (PLA(2)) venom activities. Continuous perfusion in an isolated heart of a rat on a Langendorff preparation with a Ringer's solution with B. jararacussu crude venom (2.5-10.0 microg/mL) induces stoppage and a decrease in the cardiac tension, which were time- and concentration dependent. The analysis of the heart perfusate solution showed an increase in the rate of creatine kinase induced by the venom. Pre-incubation with suramin (1.0-30.0 microM) protected against the venom cardiotoxic effect in a concentration-dependent way, reaching up to 90% with 30.0 microM, and prevent the heart stoppage and decrease the tension. These protective effects were increased by the association with polyvalent antibothropic antivenom, suggesting a synergic effect. The PLA(2) and proteolytic activities of B. jararacussu crude venom were also inhibited in a concentration-dependent way by suramin, showing that this polyanion antivenom activity has therapeutic potential to be used as an antivenom.

Increase of the cytotoxic effect of Bothrops jararacussu venom on mouse extensor digitorum longus and soleus by potassium channel blockers and by Na(+)/K(+)-ATPase inhibition.

We investigated the myotoxicity of Bothrops jararacussu crude venom and other cytolytic agents on mouse isolated extensor digitorum longus (EDL) and soleus (SOL) muscles, which present distinct properties: EDL is a fast-twitch, white muscle with predominantly glycolytic fibers, while SOL is slow-twitch, red muscle with predominantly oxidative fibers. Muscles were exposed to B. jararacussu crude venom (25 microg/ml) and other crotaline venoms (Agkistrodon contortrix laticinctus; Crotalus viridis viridis; Crotalus durissus terrificus) at the same concentration. Basal creatine kinase (CK) release to bathing solution was 0.43+/-0.06 for EDL and 0.29+/-0.06 for SOL (U g(-)(1) h(-)(1), n=36 for each muscle). Sixty minutes after exposure to B. jararacussu venom, EDL presented higher increase in the rate of CK release than SOL, respectively, 13.2+/-1.5 and 2.9+/-0.7 U g(-)(1)h(-)(1), n=10-12. Muscle denervation, despite decreasing CK content, did not affect sensitivities to B. jararacussu venom. Ouabain and potassium channel blockers (TEA; clotrimazole; glibenclamide) increased the rate of CK release by B. jararacussu in EDL and SOL muscles, decreasing and almost abolishing the different sensitivity. When we exposed EDL or SOL muscles to Naja naja, Apis mellifera venoms (25 microg/ml), or Triton X-100 (0.01%), they showed similar rate of CK release. Our present data suggest that a mechanism involving intracellular calcium regulation or potassium channels may participate in the different sensitivity of EDL and SOL to B. jararacussu venom.

Biochemical and structural investigations of Bothropstoxin-II, a myotoxic Asp49 phospholipase A2 from Bothrops jararacussu venom.

Bothropstoxin-II a calcium-dependent enzyme from Bothrops jararacussu venom causes tissue damage and several haemostatic disorders including platelet aggregation. In order to elucidate the structural determinants of its multiple pharmacological activities, we have studied the effects of suramin on Bothropstoxin-II and present details concerning the mode of binding.

Interfacial surface charge and free accessibility to the PLA2-active site-like region are essential requirements for the activity of Lys49 PLA2 homologues.

Lys49 phospholipase A2 homologues are highly myotoxic and cause extensive tissue damage but do not display hydrolytic activity towards natural phospholipids. The binding of heparin, heparin derivatives and polyanionic compounds such as suramin result in partial inhibition (up to 60%) of the myotoxic effects due to a change in the overall charge of the interfacial surface. In vivo experiments demonstrate that polyethylene glycol inhibits more than 90% of the myotoxic effects without exhibiting secondary toxic effects. The crystal structure of bothropstoxin-I complexed with polyethylene glycol reveals that this inhibition is due to steric hindrance of the access to the PLA2-active site-like region. These two inhibitory pathways indicate the roles of the overall surface charge and free accessibility to the PLA2-active site-like region in the functioning of Lys49 phospholipases A2 homologues. Molecular dynamics simulations, small angle X-ray scattering and structural analysis indicate that the oligomeric states both in solution and in the crystalline states of Lys49 phospholipases A2 are principally mediated by hydrophobic contacts formed between the interfacial surfaces. These results provide the framework for the potential application of both clinically approved drugs for the treatment of Viperidae snakebites.

Evaluation of three Brazilian antivenom ability to antagonize myonecrosis and hemorrhage induced by Bothrops snake venoms in a mouse model.

Despite preventing death after snakebites, there is little evidence that polyvalent antivenoms (PAVs) protect against myotoxicity and local damages. We evaluated antibothropic Brazilian PAVs from three manufacturers against the myotoxicity and hemorrhagic activity of Bothrops jararacussu and B. jararaca venoms, respectively, by using two protocols: preincubation of PAVs with venom, and i.v. pretreatment with PAVs, prior to the venom inoculation. In this investigation, we used doses of PAVs ranging from 0.4 to 4.0mL/mg of venom equivalent up to 10 times the amount recommended by the producers for the clinical practice in Brazil. In our preincubation protocol in vivo, PAVs antagonized myotoxicity of B. jararacussu venom by 40-95%, while our pretreatment protocol antagonized myotoxic activity by 0-60%. Preincubation of antivenoms with B. jararaca venom antagonized its hemorrhagic activity by 70-95%, while pretreatment antagonized hemorrhagic activity by 10-50%. Although all PAVs demonstrated partial antagonism against both venoms, the magnitude of these effects varied greatly among the manufactures. The results suggest that the current clinical doses of these PAVs may have negligible antimyotoxic effect.

Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin.

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity.

Protective Response Mechanisms to Heat Stress in Interaction with High [CO2] Conditions in Coffea spp.

Modeling studies have predicted that coffee crop will be endangered by future global warming, but recent reports highlighted that high [CO2] can mitigate heat impacts on coffee. This work aimed at identifying heat protective mechanisms promoted by CO2 in Coffea arabica (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown at 25/20°C (day/night), under 380 or 700 µL CO2 L(-1), and then gradually submitted to 31/25, 37/30, and 42/34°C. Relevant heat tolerance up to 37/30°C for both [CO2] and all coffee genotypes was observed, likely supported by the maintenance or increase of the pools of several protective molecules (neoxanthin, lutein, carotenes, α-tocopherol, HSP70, raffinose), activities of antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and the upregulated expression of some genes (ELIP, Chaperonin 20). However, at 42/34°C a tolerance threshold was reached, mostly in the 380-plants and Icatu. Adjustments in raffinose, lutein, ß-carotene, α-tocopherol and HSP70 pools, and the upregulated expression of genes related to protective (ELIPS, HSP70, Chape 20, and 60) and antioxidant (CAT, CuSOD2, APX Cyt, APX Chl) proteins were largely driven by temperature. However, enhanced [CO2] maintained higher activities of GR (Icatu) and CAT (Icatu and IPR108), kept (or even increased) the Cu,Zn-SOD, APX, and CAT activities, and promoted a greater upregulation of those enzyme genes, as well as those related to HSP70, ELIPs, Chaperonins in CL153, and Icatu. These changes likely favored the maintenance of reactive oxygen species (ROS) at controlled levels and contributed to mitigate of photosystem II photoinhibition at the highest temperature. Overall, our results highlighted the important role of enhanced [CO2] on the coffee crop acclimation and sustainability under predicted future global warming scenarios.

Different effects of myotoxins bothropstoxin-I and II from Bothrops snake venom on cation transport ATPases from murine fast twitch skeletal muscle.

Bothrops jararacussu venom drastically decreases sarcoplasmic Ca(2+)-ATPase (SERCA) protein expression in vivo and inhibits its activity in vitro, in contrast to a slight increase of Na(+)/K(+)-ATPase expression in murine EDL. We investigated the effect of myotoxins bothropstoxin-I and/or -II (BthTX-I, BthTX-II and BthTX-I+II) on this model. No changes were seen in SERCA1, SERCA2 and Na(+)/K(+)-ATPase α1 protein expression as well as (2+)Ca-ATPase activity, but BthTX-II (1 µg/g) reduced Na(+)/K(+)-ATPase α2 expression by 50% one day after perimuscular injection. Interestingly, BthTX-II inhibited Ca(2+)-ATPase activity (IC50 around 6 nM). Our findings suggest that only BthTX-II affects ion transport ATPases, being a potent SERCA inhibitor and a putative target for antivenom drug development.

Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake.

Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and inflammatory cells were all decreased as compared to venom injection alone. Altogether, data show that fucCS was able to inhibit myotoxicity and inflammation induced by B. jararacussu venom and its phospholipase toxins, BthTX-I and BthTX-II. Thus, fucosylated chondroitin sulfate is a new polyanion with potential to be used as an adjuvant in the treatment of snakebites in the future.

Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

BACKGROUND: The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. METHODOLOGY/PRINCIPAL FINDINGS: Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. CONCLUSIONS: This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

Counteraction of Bothrops snake venoms by Combretum leprosum root extract and arjunolic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Serotherapy against snakebite is often unavailable in some regions over Brazil, where people make use of plants from folk medicine to deal with ophidic accidents. About 10% of Combretum species have some ethnopharmacological use, including treatment of snakebites. MATERIALS AND METHODS: We evaluated the ability of the extract of Combretum leprosum and its component arjunolic acid to reduce some in vivo and in vitro effects of Bothrops jararacussu and Bothrops jararaca venoms. The protocols investigated include phospholipase, proteolytic, collagenase, hyaluronidase, procoagulant, hemorrhagic, edematogenic, myotoxic and lethal activities induced by these venoms in Swiss mice. RESULTS: Oral pre-treatment with arjunolic acid reduced the Bothrops jararacussu lethality in up to 75%, while preincubation prevented the death of all the animals. Hemoconcentration effect of Bothrops jararacussu venom was confirmed two hours after i.p. injection, while preincubation with arjunolic acid preserved the hematocrit levels. Both Combretum leprosum extract and arjunolic acid abolished the myotoxic action of Bothrops jararacussu venom. Preincubation of Bothrops jararacussu venom with the extract or arjunolic acid prevented the increase of plasma creatine kinase activity in mice. The hemorrhagic activity of Bothrops jararaca crude venom was reduced down to about 90% and completely inhibited by preincubation with 10 mg/kg or 100 mg/kg Combretum leprosum extract, respectively, while the preincubation and the pretreatment with 30 mg/kg of arjunolic acid reduced the venom hemorrhagic activity down to about 12% and 58%, respectively. The preincubation of the venom with both extract and 30 mg/kg arjunolic acid significantly reduced the bleeding amount induced by Bothrops jararacussu venom. The extract of Combretum leprosum decreased the edema formation induced by Bothrops jararacussu venom both in preincubation and pretreatment, but not in posttreatment. Similarly, arjunolic acid preincubated with the venom abolished edema formation, while pre- and posttreatment have been partially effective. Some enzymatic activities of Bothrops jararacussu and Bothrops jararaca venoms, i.e. phospholipase A2, collagenase, proteolytic and hyaluronidase activities, were to some extent inhibited by the extract and arjunolic acid in a concentration-dependent manner. CONCLUSIONS: Altogether, our results show that Combretum leprosum extract can inhibit different activities of two important Brazilian snake venoms, giving support for its popular use in folk medicine in the management of venomous snakebites.

Sustained photosynthetic performance of Coffea spp. under long-term enhanced [CO2].

Coffee is one of the world's most traded agricultural products. Modeling studies have predicted that climate change will have a strong impact on the suitability of current cultivation areas, but these studies have not anticipated possible mitigating effects of the elevated atmospheric [CO2] because no information exists for the coffee plant. Potted plants from two genotypes of Coffea arabica and one of C. canephora were grown under controlled conditions of irradiance (800 µmol m(-2) s(-1)), RH (75%) and 380 or 700 µL CO2 L(-1) for 1 year, without water, nutrient or root development restrictions. In all genotypes, the high [CO2] treatment promoted opposite trends for stomatal density and size, which decreased and increased, respectively. Regardless of the genotype or the growth [CO2], the net rate of CO2 assimilation increased (34-49%) when measured at 700 than at 380 µL CO2 L(-1). This result, together with the almost unchanged stomatal conductance, led to an instantaneous water use efficiency increase. The results also showed a reinforcement of photosynthetic (and respiratory) components, namely thylakoid electron transport and the activities of RuBisCo, ribulose 5-phosphate kinase, malate dehydrogenase and pyruvate kinase, what may have contributed to the enhancements in the maximum rates of electron transport, carboxylation and photosynthetic capacity under elevated [CO2], although these responses were genotype dependent. The photosystem II efficiency, energy driven to photochemical events, non-structural carbohydrates, photosynthetic pigment and membrane permeability did not respond to [CO2] supply. Some alterations in total fatty acid content and the unsaturation level of the chloroplast membranes were noted but, apparently, did not affect photosynthetic functioning. Despite some differences among the genotypes, no clear species-dependent responses to elevated [CO2] were observed. Overall, as no apparent sign of photosynthetic down-regulation was found, our data suggest that Coffea spp. plants may successfully cope with high [CO2] under the present experimental conditions.

Mesenchymal stem cells in a polycaprolactone conduit promote sciatic nerve regeneration and sensory neuron survival after nerve injury.

Despite the fact that the peripheral nervous system is able to regenerate after traumatic injury, the functional outcomes following damage are limited and poor. Bone marrow mesenchymal stem cells (MSCs) are multipotent cells that have been used in studies of peripheral nerve regeneration and have yielded promising results. The aim of this study was to evaluate sciatic nerve regeneration and neuronal survival in mice after nerve transection followed by MSC treatment into a polycaprolactone (PCL) nerve guide. The left sciatic nerve of C57BL/6 mice was transected and the nerve stumps were placed into a biodegradable PCL tube leaving a 3-mm gap between them; the tube was filled with MSCs obtained from GFP+ animals (MSC-treated group) or with a culture medium (Dulbecco's modified Eagle's medium group). Motor function was analyzed according to the sciatic functional index (SFI). After 6 weeks, animals were euthanized, and the regenerated sciatic nerve, the dorsal root ganglion (DRG), the spinal cord, and the gastrocnemius muscle were collected and processed for light and electron microscopy. A quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers in the group that received, within the nerve guide, stem cells. The number of neurons in the DRG was significantly higher in the MSC-treated group, while there was no difference in the number of motor neurons in the spinal cord. We also found higher values of trophic factors expression in MSC-treated groups, especially a nerve growth factor. The SFI revealed a significant improvement in the MSC-treated group. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase enzyme, suggesting an improvement of reinnervation and activity in animals that received MSCs. Immunohistochemistry documented that some GFP+ -transplanted cells assumed a Schwann-cell-like phenotype, as evidenced by their expression of the S-100 protein, a Schwann cell marker. Our findings suggest that using a PCL tube filled with MSCs is a good strategy to improve nerve regeneration after a nerve transection in mice.

Effect of heparin treatment on the expression and activity of different ion-motive P-type ATPase isoforms from mouse extensor digitorum longus muscle during degeneration and regeneration after Bothrops jararacussu venom injection.

Ca(2+) ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca(2+) homeostasis, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/K(+)-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p<0.05) while SERCA2 and Na(+)/K(+)-ATPase alpha(1) isoform expression increased at the same time (3-6- and 2-3-fold, respectively; p<0.05). Although not significant, Na(+)/K(+)-ATPase alpha(2) isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca(2+)-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca(2+)-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na(+)/K(+)-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression.

Ability of a synthetic coumestan to antagonize Bothrops snake venom activities.

We investigated a synthetic coumestan named LQB93 and similar compounds abilities to antagonize activities of Bothrops jararacussu and Bothrops jararaca crude venoms in different protocols. The antimyotoxic activity was evaluated in vitro by the rate of release of creatine kinase (CK) from isolated mouse extensor digitorum longus muscle (EDL) induced by B. jararacussu (25 g/ml). For in vivo studies, B. jararacussu venom (1.0 mg/kg) was preincubated with LQB93 (0.1-30 mg/kg), during 30 min, for later injection in mouse tight and evaluation of the antimyotoxic and anti-edematogenic effects. LQB93 antagonized in vitro, the increase of CK release from the EDL muscle (IC(50)=0.0291 M). It also showed in vivo, antimyotoxic and anti-edematogenic effects that were dose-dependent with ID50 of 0.17 mg/kg and 0.14 mg/kg, respectively. The hemorrhage induced by B. jararaca (1.0 mg/kg) venom in the mouse skin, was abolished by LQB93 (10.0 mg/kg) preincubated with venom. Like wedelolactone, LQB93 protected rat isolated heart on a Langendorff preparation, from the cardiotoxicity of B. jararacussu venom. LQB93 inhibit the effects of Bothrops venoms like wedelolactone, a natural compound isolated from the plant Eclipta prostrata.