Antivenomic Assessment of the Immunological Reactivity of EchiTAb-Plus-ICP,an Antivenom for the Treatment of Snakebite Envenoming in Sub-Saharan Africa

The immunoreactivity of EchiTAb-Plus-ICP, an antivenom developed for the treatment of snakebite envenoming in sub-Saharan Africa, to venoms of seven Echis and Bitis species, was assessed by “antivenomics.” This proteomic approach is based on the ability of an antivenom to immunodeplete homologous or heterologous venom proteins. Our results show an extensive cross-reactivity of this antivenom against all Echis and Bitis venoms studied, as revealed by the complete immunodepletion of the majority of venom components, including metalloproteinases, serine proteinases, C-type lectin-like proteins, some phospholipases A₂ and L-amino acid oxidase. However, some phospholipases A₂, disintegrins and proteinase inhibitors were immunodepleted to only a partial extent. These results support the hypothesis that immunizing horses with a mixture of the venoms of Echis ocellatus, Bitis arietans, and Naja nigricollis generates antibodies capable of recognizing the majority of components of medically-relevant homologous and heterologous viperid venoms of the genera Bitis and Echis from sub-Saharan Africa.     

Proteomic characterization of venom of the medically important Southeast Asian Naja sumatrana (Equatorial spitting cobra)

The proteome of Naja sumatrana (Equatorial spitting cobra) venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the venom while the chromatographic approach identified 37 venom proteins. The results indicated that, like other Asiatic cobra venoms, N. sumatrana contains large number of three finger toxins and phospholipases A₂, which together constitute 92.1% by weight of venom protein. However, only eight of the toxins can be considered as major venom toxins. These include two phospholipases A₂, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6–27.2% venom proteins and together account for 89.8% (by weight) of total venom protein. Other venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, cobra venom factors, venom nerve growth factor and cobra serum albumin. The proteome of N. sumatrana venom is similar to proteome of other Asiatic cobra venoms but differs from that of African spitting cobra venom. Our results confirm that the main toxic action of N. sumatrana venom is neurotoxic but the large amount of cardiotoxins and phospholipases A₂ are likely to contribute significantly to the overall pathophysiological action of the venom. The differences in toxin distribution between N. sumatrana venom and African spitting cobra venoms suggest possible differences in the pathophysiological actions of N. sumatrana venom and the African spitting cobra venoms, and explain why antivenom raised against Asiatic cobra venom is not effective against African spitting cobra venoms.     

Present tests for detection of snake venom: clinical applications

Immunologic tests for detection of snake venom and venom antibodies have important clinical applications. Enzyme-linked immunoassay (ELISA) and radioimmunoassay (RIA) provide adequate specificity and sensitivity. The former is much more widely used because it is inexpensive, relatively easy to perform, and uses stable reagents. Some ELISA systems will detect 0.5 ng of venom; however, a sensitivity of 10 to 100 ng is more usual. Minimum running time is 30 to 45 minutes; with longer times, greater sensitivity can be attained. Wound aspirate, serum, and urine are the most suitable materials for venom detection. ELISA has been used for clinical diagnosis of snakebite, to monitor antivenom dose, to study clinical syndromes associated with envenomation, to detect venom in forensic cases, and to evaluate first aid techniques. The indirect ELISA usually is used for detecting and titrating venom antibody. This is potentially useful in epidemiological studies of snakebite incidence, in evaluating potency and paraspecific activity of antivenoms, and in studying response to venom immunogens. Current ELISA systems involving snake venoms have low specificity, and most cannot reliably differentiate venoms of related snakes. Venom antibody detection assays are less satisfactory than those for venom; nonspecific reactions and cross-reactivity are unacceptably high. Methods for improvement of snake venom immunodiagnosis are discussed.     

Studies on effects of Andrographis paniculata (Burm.f.) and Andrographis lineata nees (Family: Acanthaceae) extracts against two mosquitoes Culex quinquefasciatus (Say.) and Aedes aegypti (Linn.)

ObjectiveTo investigate the studies on effects of Andrographis paniculata (A. paniculata) (Burm.f.) and Andrographis lineata (A. lineata) nees (Family: Acanthaceae) extracts against two mosquitoes Culex quinquefasciatus (Cx. quinquefasciatus) (Say.) and Aedes aegypti (Ae. aegypti) (Linn.).MethodsThe aqueous and petroleum ether extracts of two plant species, A. paniculata and A. lineate were examined against the larvae of A. aegypti (L.) and Cx. quinquefasciatus with gradually increasing concentration ie. from 50 to 200 ppm of solvent extracts and to test their activity in combination with each other.ResultsIn a 24 h bioassay experiment with plant extracts, highest mortalities were recorded at 200 ppm of concentrations for leaves of A. lineta and A. paniculata individually. For combination effect, only 150 ppm of the mixture of solvent extracts of petroleum ether: aqueous (1:1) extracts showed 100% mortality after 24 h of exposure.ConclusionsThe results show that, insecticides of plant combination is ecofriend and has better larvicidal activity compared to individual extracts.     

Cross neutralization of common Southeast Asian viperid venoms by a Thai polyvalent snake antivenom (Hemato Polyvalent Snake Antivenom)

Snake envenomation is a serious public health threat in many rural areas of Asia and Africa. Antivenom has hitherto been the definite treatment for snake envenomation. Owing to a lack of local production of specific antivenom, most countries in these regions fully depend on foreign supplies of antivenoms. Often, the effectiveness of the imported antivenoms against local medically important species has not been validated. This study aimed to assess cross-neutralizing capacity of a recently developed polyvalent antivenom, Hemato Polyvalent Snake Antivenom (HPAV), against venoms of a common viper and some pit vipers from Southeast Asia. Neutralisation assays showed that HPAV was able to effectively neutralize lethality of the common Southeast Asian viperid venoms examined (Calloselasma, Crytelytrops, Popeia, and Daboia sp.) except for Tropidolaemus wagleri venom. HPAV also effectively neutralized the procoagulant and hemorrhagic activities of all the venoms examined, corroboratively supporting the capability of HPAV in neutralizing viperid venoms which are principally hematoxic. The study also indicated that HPAV fully prevented the occurrence of hematuria and proteinuria in mice envenomed with Thai Daboia siamensis venom but was only partially effective against venoms of Myanmar D. siamensis. Thus, HPAV appears to be useful against its homologous venoms and venoms from Southeast Asian viperids including several medically important pit vipers belonging to the Trimeresurus complex. Nevertheless, the effectiveness of HPAV as a paraspecific antivenom for treatment of viperid envenomation in Southeast Asian region requires further assessment from future clinical trials     

Mosquito adulticidal and repellent activities of botanical extracts against malarial vector, Anopheles stephensi Liston (Diptera: Culicidae)

ObjectiveTo determine the adulticidal and repellent activities of different solvent leaf extracts of Eclipta alba (E. alba) and Andrographis paniculata (A. paniculata) against malarial vector, Anopheles stephensi (An. stephensi).MethodsAdulticidal efficacy of the crude leaf extracts of E. alba and A. paniculata with five different solvents like benzene, hexane, ethyl acetate, methanol and chloroform was tested against the five to six day old adult female mosquitoes of An. stephensi. The adult mortality was observed after 24 h under the laboratory conditions. The repellent efficacy was determined against An. stephensi mosquito species at three concentrations viz., 1.0, 2.5 and 5.0 mg/cm² under laboratory conditions.ResultsAmong the tested solvents the maximum efficacy was observed in the methanol extract. The LC₅₀ and LC₉₀ values of E. alba and A. paniculata against adults of An. stephensi were 150.36, 130.19 ppm and 285.22, 244.16 ppm, respectively. No mortality was observed in controls. The chi-square values were significant at P<0.05 level. Methanol extract of E. alba and A. paniculata was produce maximum repellency against An. stephensi.ConclusionsFrom the results it can be concluded the crude extract of E. alba and A. paniculata was an excellent potential for controlling An. stephensi mosquitoes.     

Protective effects of melatonin against oxidative damage induced by Egyptian cobra (Naja haje) crude venom in rats

Naja haje envenomation is one of the leading causes of death due to snakebite. Antiserum therapy sometimes fails to provide enough protection against venom toxicity. In this study, we investigated the protective effects of melatonin against N. haje venom in rats. The animals were injected with venom (0.25 mg/kg) and/or melatonin (10 mg/kg) and compared with vehicle-treated rats. There was oxidative/nitrosative damage and apoptosis in the liver, heart, and kidneys of venom-injected rats. Melatonin counteracted the increased lipoperoxidation and nitric oxide, prevented decreased glutathione peroxidase and reductase activity, reduced the glutathione disulfide/glutathione (GSSG/GSH) ratio, and maintained the GSH pool. Furthermore, melatonin administration was associated with a reduction of apoptosis, which was increased in venom-injected rats. Overall, these results suggest that melatonin mitigates oxidative/nitrosative stress in venom-induced cardio-hepato-renal injury in rats. Our results suggest that melatonin treatment may ameliorate some of the effects of N. haje envenomation.     

Evaluation of Andrographis paniculata Burm.f. (Family:Acanthaceae) extracts against Culex quinquefasciatus (Say.) and Aedes aegypti (Linn.) (Diptera:Culicidae)

ObjectiveTo investigate the larvicidal and ovicidal efficacy of different extracts of Andrographis paniculata (A. paniculata) against Culex quinquefasciatus (Cx. quinquefasciatus) Say and Aedes aegypti (Ae. aegypti) L. (Diptera: Culicidae).MethodsLarvicidal efficacy of the crude leaf extracts of A. paniculata with five different solvents like benzene, hexane, ethyl acetate, methanol and chloroform was tested against the early third instar larvae of Cx. quinquefasciatus and Ae. aegypti. The ovicidal activity was determined against two mosquito species to various concentrations ranging from 50-300 ppm under the laboratory conditions.ResultsThe benzene, hexane, ethyl acetate, methanol and chloroform leaf extract of A. paniculata was found to be more effective against Cx. quinquefasciatus than Ae. aegypti. The LC₅₀ values were 112.19, 137.48, 118.67, 102.05, 91.20 ppm and 119.58, 146.34, 124.24, 110.12, 99.54 ppm respectively. Among five tested solvent, methanol and ethyl acetate crude extract was found to be most effective for ovicidal activity against two mosquito species. The extract of methanol and ethyl acetate exerted 100% mortality at 200 ppm against Cx. quinquefasciatus and at 250 ppm against Ae. aegypti.ConclusionsFrom the results it can be concluded the crude extract of A. paniculata was a potential for controlling Cx. quinquefasciatus and Ae. aegypti mosquitoes.     

Regulation of Acetylcholine Receptors in Relation to Acetylcholinesterase in Neuroblastoma Cells

Purified alpha-toxin from Naja nigricollis snake venom labeled by [(3)H]acetylation binds specifically to the acetylcholine receptors of mouse neuroblastoma cells. Toxin binding was inhibited by inhibitors for nicotinic and muscarinic acetylcholine receptors. Clones of neuroblastoma cells were selected for low acetylcholinesterase (EC 3.1.1.7) activity with antibodies against this enzyme. Selection for an 80-fold decrease in acetylcholinesterase activity was not associated with any decrease in the number of acetylcholine receptors (3.4 x 10(7) per cell). Removal or inactivation of 80% of the acetylcholine receptors by proteolytic enzymes or by compounds that block sulfhydryl groups did not change the activity of acetylcholinesterase on the cell surface. In addition to these results on the separation between acetylcholine receptors and acetylcholinesterase, a common regulation was found in that both the number of acetylcholine receptors and the activity of acetylcholinesterase were increased 5- to 10-fold when the cells stopped to multiply or were induced to differentiate by dibutyryl-cyclic AMP. It is suggested that there are different genes for the acetylcholine receptor and acetylcholinesterase, and that both are regulated during growth and differentiation by a common regulatory gene.     

蛇毒多肽X在巨噬细胞泡沫化过程中的Ca2+拮抗作用

为研究由中华眼镜蛇毒分离纯化而得的蛇毒多肽X在巨噬细胞泡沫化过程中的Ca2+拮抗作用。将C57BL/6J小鼠腹膜巨噬细胞置于10mg/L氧化型低密度脂蛋白中培养,制备泡沫样细胞;应用Ca2+荧光指示剂技术,检测蛇毒多肽X对泡沫样细胞Ca2+水平的缓慢与即刻影响。结果发现,在10mg/L蛇毒多肽X中孵育的泡沫样细胞Ca2+水平为3.61×10-7mol/L,为对照细胞的40.2%;而蛇毒多肽X对细胞Ca2+水平的即刻影响不明显。结果提示,在巨噬细胞泡沫化过程中,蛇毒多肽X可拮抗细胞Ca2+水平的增高,从而阻止泡沫细胞的形成。     

Oxidative stress‐induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin

Oxidative stress‐induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole‐blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin‐induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia‐inducible factor‐3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia‐inducible factor‐3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.     

Influence of arbuscular mycorrhizal fungi on the nematicidal properties of leaf extracts of Thymus vulgaris L.

The effect of arbuscolar mycorrhizal fungi (AMF) on the nematicidal activity of Thymus vulgaris against the rootknot nematodes Meloidogyne incognita and M. javanica was investigated in two in vitro experiments. In the first experiment egg masses of M. incognita and M. javanica were immersed for 3 weeks in aqueous leaf extracts of thyme plants non-inoculated or previously inoculated with Glomus mosseae or mixed AMF strains (Sclerocystis sinuosa, Glomus claroideum-1, G. claroideum-2 and G. claroideum-3). Thereafter the hatching test continued in distilled water for five more weeks. In the second experiment egg masses of both Meloidogyne species were exposed to the different thyme extracts for 4, 8 and 16 hours and then incubated in distilled water for 8 weeks. Distilled water and 5 mg/ml aqueous solution of fenamiphos nematicide were used as controls. Numbers of second stage juveniles emerging weekly were expressed as cumulative percentages of the total egg content of the egg masses. In the first experiment juvenile emergence from eggs of both Meloidogyne species immersed in thyme extracts for three weeks was completely suppressed since the first week. Hatching of eggs of M. incognita in all the extracts was significantly lower than that in water control, although emergence in the extract from uninoculated thyme plants was significantly higher than the others and no statistical different from that of aqueous fenamiphos solution. Emergence of M. javanica juveniles was significantly lower after immersion in all the extracts than in distilled water control and aqueous fenamiphos solution. In the second experiment a 4-hour exposure to the extract from thyme inoculated with G. mosseae and mixed AMF population significantly reduced the final hatch of M. incognita in comparison to distilled water. A 16-hour exposure to the extract from mixed AMF inoculated plants resulted in a significantly lower egg hatch than the shorter exposure times, whereas no statistical difference was found between 4 and 8 hour exposure to both extracts. Emergence of M. javanica juveniles was statistically lower than in water only after 16 hours exposure to the extracts from mixed AMF strains inoculated plants, but no difference was found among the different exposure times. Growth of T. vulgaris was significantly increased only by the infections of mixed AMF strains.     

Absence of anti fungicidal activity of few Indian medicinal plants methanolic extracts

ObjectiveThe present study was aimed at evaluating the In vitro antimicrobial activity of few Indian medicinal plants H. tiliaceus L., N.arbortristis Linn, S. rhombifolia Linn., T. procumbens Linn methanolic extracts against A. alternata, A. flavus, A. niger, M. phaseolina, R. solani using agar well diffusion technique.MethodsSoxhlet extraction method was used to get the extracts of methanol. The length of zone of inhibition was measured in millimeters from the edge of the well to the edge of the inhibition zone.ResultsIt showed zero results with H. tiliaceous, N. arbortristis with all three 100, 300, 500 mg/ml DMSO concentrations.ConclusionsVarious medical plants have been used for years in daily life to treat disease all over the world throughout the history of mankind. Plant Species H. tiliaceous and N. arbortristis may not be useful in controlling diseases against A. alternata, A. flavus, A. niger, M. phaseolina, R. solani. This study give an idea to avoid work of activity of these medicinal plants extracts on mentioned fungal for screening.     

Surveillance of multidrug resistance of two Gram-positive pathogenic bacteria in a teaching hospital and in vitro efficacy of 30 ethnomedicinal plants used by an aborigine of India

ObjectiveTo record hospital- and community-acquired accounts of multidrug resistance (MDR) of two Gram-positive pathogens, Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis), by surveillance, and to evaluate antibacterial potencies of 30 plants with information on ethnomedicinal uses for infectious ailments by the aborigine Kandha tribe of Kalahandi district, Odisha (India), against both pathogens.MethodsOver a period of 6 months bacteria/ strains of S. aureus and E. faecalis were isolated from clinical samples in a teaching hospital and their antibiograms were ascertained using 17 antibiotics of 9 different groups. S. aureus strains were further tested for confirmation if they were methicillin and vancomycin resistant, similarly, E. faecalis strains for vancomycin resistance. Concentrated aqueous and ethanolic extracts of leaves/barks of 30 plants were used for monitoring their antimicrobial potencies, by the agar-well diffusion method, along with qualitative phytochemical analyses.ResultsFrom the surveillance, both pathogens were found MDR and it was evident that the distribution of MDR strains was more in hospital-acquired than community-acquired samples. Both aqueous and ethanolic extracts of plants, Diospyrous melanoxylon, Woodfordia fruticosa (W. fruticosa), Oroxylum indicum (O. indicum), Dalbergia paniculata and Lantana camara had the most significant in vitro controlling capacity against MDR strains of both bacteria. Further, extracts of Holarrhena antidysenterica, Aspidopterys tomentosa and Argyreia speciosa had moderate antibacterial activities. Ethanolic extracts of L. camara, O. indicum and W. fruticosa contained all the phytochemicals, alkaloids, glycosides, terpenoids, reducing sugars, saponins, tannins, flavonoids and steroids, which could be attributed to the recorded significant antibacterial activity.ConclusionS. aureus strains have been found as the most widely prevailing pathogens in nosocomial settings, than in community. Plants, L. camara. W. fruticosa, O. indicum and P. santalinus, particularly could be useful for a use as complementary/ supplementary/alternative therapeutic agents against Gram-positive pathogens.     

Hepatoprotective activity of Terminalia paniculata against paracetamol induced hepatocellular damage in Wistar albino rats

ObjectiveTo evaluate the hepatoprotective activity of Terminalia paniculata against paracetamol induced hepatic damage in rats.MethodsThe plant material was shade dried, powdered and extracted with ethanol. Liv 52 and silymarin were used as standard drugs and 2% gum acacia as a control (vehicle). Alteration in the levels of biochemical markers of hepatic damage like AST, ALT, ALP and lipid peroxides were tested, and phytochemical tests were also performed.ResultsParacetamol (2 g/kg) increased the serum levels of alanine aminotransfer (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and the lipid peroxides. Treatment of Liv 52, silymarin and ethanolic extract of Terminalia paniculata (200 mg/kg) altered levels of biochemical marker and showed significant hepatoprotective activity. Ethanolic extract revealed the presence of phenolic compound and flavanoids. Our findings suggested that ethanolic bark extract of Terminalia paniculata possessed hepatoprotective activity in a dose dependent manner.ConclusionsTerminalia paniculata possesses hepatoprotective activity. It could be an effective and promising preventive agent against PCT induced hepatotoxicity.     

A survey on Hibiscus rosa—sinensis,Alcea rosea L. and Malva neglecta Wallr as antibacterial agents

ObjectiveTo guide for selection of plants with antibacterial activity for further phytochemical works on the isolation and identification of the active compounds.MethodsEthanolic extracts of 3 species from Malvaceae family were evaluated by agar disc diffusion method for antibacterial activity against some gram-positive and gram-negative bacteria (Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Bacillus cereus, Bacillus anthracis, Escherichia coli Streptococcus pyogenes). The extracts were obtained from aerial parts of Hibiscus rosa (H. rosa)-sinensis (leaf and flower), Alcea rosea (A. rosea) L. (leaf and flower) and Malva neglecta (M. neglecta) Wallr (flower).ResultsThese extracts had inhibitory effects at different concentrations (0.05, 0.10, 0.20 and 0.40 g/mL) against above mentioned bacteria. Escherichia coli was the most resistant strain. The highest inhibitory zone was showed by ethanolic extract of M. neglecta against Staphylococcus epidermidis (22 mm) and followed by ethanolic extract from flower of H. rosa against Staphylococcus epidermidis and Staphylococcus aureus (20 mm). The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values against Staphylococcus epidermidis were equal (MIC=MBC=5 mg/mL for M. neglecta extract and for H. rosa extract MIC=MBC=20 mg/mL).ConclusionsThese findings suggest that these native plants have good antibacterial properties that can be used for infection control and treatment and could also be as new source for antibiotics discovery and infection treatment.     

A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

Innovation and coevolution play major roles in the evolution of biological diversity. Evolutionary innovations (novelties) enable organisms to adopt new lifestyles and exploit new niches and can trigger major radiations while coevolutionary interactions among organisms (e.g., predator–prey, plant–herbivore, and host–parasite) are thought to spur evolutionary innovation and drive diversification (1, 2). Thus, the genetic origins of evolutionary novelties and the mechanistic bases of coevolutionary interactions are of central interest in evolutionary biology.Animal venoms in general and snake venoms in particular offer rich models for exploring the molecular bases of novelty and coevolution. Most snake venoms contain an arsenal of toxin proteins that facilitate the immobilization, killing, and initial digestion of prey (3). Resistance to specific venoms has evolved in both snake prey as well as animals that prey on snakes, and this resistance appears to be adaptations to predation pressures (reviewed in (4, 5, 6). In many instances, resistance is conveyed by specific serum proteins that inhibit the biological activity of major toxins such as snake venom metalloproteinases (SVMPs) (5, 7). For example, the Mexican ground squirrel (8), California ground squirrel (9, 10), and New Mexican rock squirrel (11) have evolved resistance to particular rattlesnake venoms via SVMP inhibitors (12).The ability of prey to evolve venom resistance can trigger a coevolutionary arms race in which snake predators in turn evolve toxins or venom compositions that circumvent resistance, as has been shown, for example, in the case of California ground squirrels and the Northern Pacific rattlesnake (C. oreganus) (11, 13). Indeed, it is widely thought that the diversity of snake venom composition reflects adaptation to the diversity and susceptibility of prey in their diets (14, 15, 16, 17).However, the potential for venom–prey coevolution raises yet another facet of possible coevolutionary pressure on venomous snakes that has not yet received much attention. Many venomous snakes are themselves resistant to their own venom (18, 19, 20, 21), reviewed in refs. 5 and 22, which has been proposed to protect the snakes from exposure to venom toxins during feeding and digestion (23). Autoresistance in many viperids has been shown to involve serum proteins that inhibit venom hemorrhagic and protease activities (24, 25, 26). The specific proteins responsible for venom resistance have been identified in a few crotalids such as Protobothrops flavoviridis (27, 28), Gloydius blomhoffi (29), and Bothrops jararaca (30) that each express a Fetuin-related cystatin-type metalloprotease inhibitor (named “serum factor” by Omari-Satoh 1972 (27)) that inhibits autologous SVMP activities.The process of venom–prey coevolution raises the question of how snake autoinhibitors coevolve to maintain inhibition of evolving venoms? One possibility is that the diversification of venom proteins is accompanied by the expansion and diversification of autoinhibitors. Alternatively, autoinhibitors may maintain their inhibitory activity despite the diversification of toxin sequences and structures.To explore this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum-borne SVMP inhibitors in the Western Diamondback rattlesnake Crotalus atrox. We selected this species because we have previously shown that it possesses by far the largest known battery of SVMP genes (30 loci) of any crotalid (31); other rattlesnake species studied possess 5 to 15 SVMP genes (31–33). We anticipated that the greater expansion and diversification of SVMPs in this lineage may have in turn influenced the evolution of the number and/or activity of potential SVMP inhibitors. We found, however, that the number of Fetuin A-related metalloproteinase inhibitor family members was only slightly greater in Crotalus than in other crotalid genera, and no greater in C. atrox than in other Crotalus species with smaller SVMP gene batteries. Rather, we discovered that the activities of two family members have shifted dramatically in the evolution of the C. atrox lineage. One family member FETUA-3 is the major, broad-spectrum inhibitor of venom SVMP activities in C. atrox, but its orthologs are not major inhibitors in other crotalid genera. Conversely, a second family member FETUA-2 exhibits limited activity against C. atrox SVMPs, but its orthologs are the major serum SVMP inhibitors in Asian and South American crotalid genera. We conclude that there has been an evolutionary shift in the major serum SVMP inhibitor as the SVMP family expanded and diversified in the Crotalus lineage. The broad activity of this inhibitor may be promising for the potential treatment of crotalid envenomation.     

In vitro larvicidal potential against Anopheles stephensi and antioxidative enzyme activities of Ginkgo biloba, Stevia rebaudiana and Parthenium hysterophorous

ObjectiveTo investigate in vitro larvicidal and antioxidant enzymes potential of the medicinal plants Ginkgo biloba (G. biloba), Stevia rebaudiana (S. rebaudiana) and Parthenium hysterophorous (P. hysterophorous) against Anopheles stephensi (An. stephensi) 4th instars larvae.MethodsFor evaluation of larvicidal potential, the ethanolic, methanolic and dichloromethane leaves extracts of three different plants were used in dose-dependent experiments in two media, while the antioxidant enzymes activities were investigated using four different methods viz., superoxide dismutase, peroxidase, ascorbate and catalase.ResultsAn. stephensi has developed resistance to various synthetic insecticides, making its control increasingly difficult. The comparative performance of ethanolic extracts (65%-90%) was found better than the methanolic extract (70%-87%) and dichloromethane extract (60%-70%). Among the three plants extracts tested in two media, S. rebaudiana exhibited higher larvicidal activity with LC₅₀ (24 h) in methanolic extract than P. hysterophorous and G. biloba. G. biloba and P. hysterophorous exhibited the strongest antioxidative enzymes activity and S. rebaudiana were less active and no significant difference was observed.ConclusionsThese three plants exhibit larvicidal potential and can be further used for vector control alternative to synthetic insecticide due to eco-friendly and diseases control, furthermore these plant species have potent antioxidative enzyme activities, therefore, making them strong natural candidate particularly for diseases which are caused due to free radicals.     

Direct nephrotoxicity of Russell's viper venom demonstrated in the isolated perfused rat kidney

Envenoming by Russell's Viper (Vipera russelli) is an important cause of acute renal failure. The mechanism of renal damage is unresolved. It is difficult to obtain evidence of a direct nephrotoxic action because of the coincidental disturbance to the systemic circulation. We studied the action of Russell's Viper venom on the function of the isolated perfused rat kidney. Direct nephrotoxic action was indicated by a dose dependent decrease in inulin clearance and an increase in fractional excretion of sodium seen at venom concentrations down to 50 ng/ml, a concentration likely to be achieved in the human circulation after envenoming. The isolated perfused kidney was also used to assess the efficiency of antivenom and for a comparison with snake venoms from the Thai cobra (Naja kauothia) and the Nigerian Saw-Scaled Viper (Echis ocellatus).