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1.
J. venom. anim. toxins incl. trop. dis ; 27: e20200066, 2021. graf
Article in English | LILACS, VETINDEX | ID: biblio-1154773

ABSTRACT

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)


Subject(s)
Animals , Snake Venoms , Antivenins , Bothrops , Serine Proteases , Peptides
2.
J. venom. anim. toxins incl. trop. dis ; 27: e20200047, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1287090

ABSTRACT

The venom of the krait (Bungarus sindanus), an Elapidae snake, is highly toxic to humans and contains a great amount of acetylcholinesterase (AChE). The enzyme AChE provokes the hydrolysis of substrate acetylcholine (ACh) in the nervous system and terminates nerve impulse. Different inhibitors inactivate AChE and lead to ACh accumulation and disrupted neurotransmission. Methods: The present study was designed to evaluate the effect of palladium(II) complex as antivenom against krait venom AChE using kinetics methods. Results: Statistical analysis showed that krait venom AChE inhibition decreases with the increase of Pd(II) complex (0.025-0.05 µM) and exerted 61% inhibition against the AChE at a fixed concentration (0.5 mM) of ACh. Kinetic analysis using the Lineweaver Burk plot showed that Pd(II) caused a competitive inhibition. The compound Pd(II) complex binds at the active site of the enzyme. It was observed that K m (Michaelis-Menten constant of AChE-ACh into AChE and product) increased from 0.108 to 0.310 mM (45.74 to 318.35%) and V max remained constant with an increase of Pd(II) complex concentrations. In AChE K Iapp was found to increase from 0.0912 to 0.025 µM (29.82-72.58%) and did not affect the V maxapp with an increase of ACh from (0.05-1 mM). K i (inhibitory constant) was estimated to be 0.029µM for snake venom; while the K m was estimated to be 0.4 mM. The calculated IC50 for Pd(II) complex was found to be 0.043 µM at constant ACh concentration (0.5 mM). Conclusions: The results show that the Pd(II) complex can be deliberated as an inhibitor of AChE.(AU)


Subject(s)
Animals , Bungarus , Elapid Venoms/toxicity , Synthetic Biology , Palladium , Acetylcholinesterase
3.
J. venom. anim. toxins incl. trop. dis ; 27: e20200180, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1287094

ABSTRACT

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)


Subject(s)
Animals , Oxidoreductases , Bothrops/physiology , Angiogenesis Inhibitors , Crotalid Venoms , Metalloproteases
4.
J. venom. anim. toxins incl. trop. dis ; 27: e20200196, 2021. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1346436

ABSTRACT

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)


Subject(s)
Animals , Oxidoreductases , Peptides , Viper Venoms , Proteome , Neurotoxins
5.
Tropical Biomedicine ; : 111-118, 2021.
Article in English | WPRIM | ID: wpr-904638

ABSTRACT

@#In 2000, an equine Yamakagashi (Rhabdophis tigrinus) antivenom (Lot 0001) was testmanufactured as an unapproved drug for treatment of Yamakagashi bites. It was stocked on the premise of super-legal use from the viewpoint of emergency health crisis management. The antivenom showed a strong neutralizing ability against the hemorrhagic and coagulation activity of the Yamakagashi venom in its potency test. One vial of the antivenom can effectively neutralize at least about 4 mg of Yamakagashi venom. Its efficacy has also been confirmed in patients with severe cases of R. tigrinus bite that has been used in emergency. In 2020, this antivenom (Lot 0001) has reached 20 years after its production. To evaluate the integrity and potency of the antivenom, quality control, safety and potency tests had been conducted almost every year since 2012. Physical and chemical tests (property test, moisture content test, insoluble foreign matter test, osmotic pressure ratio test, pH test, protein content test, endotoxin test, sterility test) of the antivenom, showed no significant changes throughout the years, when compared to the results immediately after its production in 2000. All the parameters measured were also within the standard values. In animal safety tests (test for absence of toxicity and pyrogen), there was no change in the test results during the storage period and no abnormalities were observed. The potency test (anti-coagulant activity) after 20 years of the product, showed the same potency as those recorded immediately after production. Therefore, in all of the stability monitoring tests conducted so far, the product did not show any significant change compared to the results immediately after production. This confirms the stability of the product during the stockpiling period to the present, that is, 20 years after production.

6.
Braz. arch. biol. technol ; 64: e21210109, 2021. tab, graf
Article in English | LILACS-Express | LILACS | ID: biblio-1355807

ABSTRACT

Abstract The genus Eucalyptus present high content of essential oil (EO). This study evaluated the pharmacological properties of Eucalyptus grandis (EG) and Eucalyptus microcorys (EM) EOs. The major component in both EOs was 1,8-Cineole. Both essential oils prevented thrombus dissolution and reduced clotting, hemolysis, and genotoxicity induced by snake venoms. 50% (EM) and 73% (EG) were the greatest inhibitions obtained in the thrombolytic assay - thrombolysis induced by B. moojeni venom. Increases in clotting time were also observed, with values considered significant between 10-27 seconds. Lysis values 50% lower than the negative control were observed in both EOs. The EOs also protected fibrinogenolysis induced by snake venom. EM EO was more effective in reducing venom-induced DNA fragmentation in the comet assay, with arbitrary unit values 66.15% lower than the positive control. These oils present wide application potential considering the pharmacological properties observed in this study.

7.
Article | IMSEAR | ID: sea-204674

ABSTRACT

Background: Every year about 50,000, people die of snake bites in India. Anti-snake venom and mechanical ventilation is mainstay of treatment in cases with severe neurotoxic envenomation. ASV is costly and scarce resource. There is lack of universal consensus towards the optimal dose of ASV in management protocol for children with severe neurotoxic snake envenomation. Objective was to compare the difference in outcome between two fixed doses of ASV, 10vials versus 20 vials, in children with severe neurotoxic snake envenomationMethods: This comparative observational study was carried out for a period of 3 years in Department of Pediatrics of SVS Medical College, Mahabubnagar, Telangana, India. Children with history of snake bite and clinical evidence of neuroparalysis were included. In addition to the mechanical ventilation and other supportive measures, every alternate patient was administered with 10vials (low dose) and 20 vials (high dose) of ASV over 1 hour. Outcome was compared between the two groups.Results: Of the 62 patients, 32 were in each group. The median time to extubation was 41 hours and 39.5 hours and mean duration of the hospital stay was 4.6 days and 4.5 days among the low dose and high dose groups, respectively. There were three deaths, one from low dose group and two from high dose group.Conclusions: There was no significant difference in outcome between the 10 vials vs 20 vials of ASV in addition to mechanical ventilation in treatment of children with severe neurotoxic snake envenomation. So, 10 vials of ASV can be utilized to reduce the cost of treatment.

8.
Article | IMSEAR | ID: sea-204633

ABSTRACT

Background: Pediatric snake bite cases are frequently encountered at health care centers of Himachal Pradesh. It is a medical emergency which require early hospitalisation and immediate medical intervention. This study determines the epidemiological and envenomation details along with clinical profile and management of pediatric snake bite patients in Himachal Pradesh, India.Methods: This prospective clinico-epidemiologic study included 30 paediatric snake bite cases reported at Department of Paediatrics, IGMC, Shimla. Demography, envenomation details, first aid and treatment, antivenom administration and outcome were recorded for all patients.Results: Of the total 30 cases, 56.6% were males and 43.3% were females. All patients were resident of rural areas and most of them belonged to middle socioeconomic status. Most (56.7%) snake bites occurred between 6pm to 12am and peak time was during rainy season. More cases of hemotoxic envenomation were observed as compared to neuroparalytic envenomation. Most common hematologic abnormalities were hematuria (4.8%) and epistaxis (4.8%). Respiratory paralysis, ptosis and opthalmoplegia were the most common presentation in patients with neuroparalytic envenomation. Clinical profile of patients showed thrombocytopenia (26.7%), prolonged PT/INR (76.6%) and 36.7% had prolonged activated partial thromboplastin time. All symptomatic patients were given ASV as primary treatment out of which 79.2% patients required more than 10 vials of ASV. Most common complication observed in neuroparalytic envenomation was respiratory failure (33.3%) and in hemotoxic envenomation was coagulation failure (66.7%). Only 3.3% case fatality was observed in this study.Conclusions: Snake bite in children is a medical emergency in hilly state of Himachal Pradesh where people still follow traditional first aid methods and treatment protocol. A widespread awareness programme is needed to propagate the newly advised first aid methods to prevent mortality by early hospitalization and administration of ASV.

9.
Article | IMSEAR | ID: sea-204586

ABSTRACT

Background: Snake bite is a neglected and underestimated public health problem in tropical and subtropical region. Snake envenomation is a well-known cause of morbidity and mortality in India. In 2009, WHO declared snake bite a neglected disease. Ophitoxaemia is an exotic term characterising the clinical spectrum of snake bite. Objective of this study was to know the outcome in paediatric snake bite cases in a tertiary care PICU (Paediatric Intensive Care Unit) with special reference to envenomation time and also to evaluate and analyse few atypical presentations of snake envenomation by proper history taking, physical examination and help start early intervention to prevent morbidity and mortality.Methods: A prospective observational study was done in Paediatric Intensive care unit of the hospital from June 2017 to June 2019.Results: Around 56.8% of 109 cases were non-poisonous snake-bites. Out of 47 cases admitted to PICU, 68.08% developed cellulitis at the site of bite with Staphylococcus aureus being the commonest organism isolated (56.25%). Anaerobes (bacteroides and clostridium) were also isolated in few cases. Edema at site of bite (hematotoxic) and ptosis(neurotoxic) were most common initial presentation. 36.17% of patients received ASV (Anti- Snake Venom) and first aid within 6 hours of snake bite. The morbidity and mortality were significantly less (p<0.05) as compared to those who didn't receive ASV. 12.76% of cases with normal CRT (Clot Retraction Time) presented with features of coagulopathy. DIC (58.33% of hematotoxic bites) and respiratory paralysis 68 (75% of neurotoxic bites) were the commonest complications. Renal replacement therapy was required in 6.38%, transfusion in 10.63% cases and case-fatality-rate was 12.7%. There were a few atypical presentations of snake bite mimicking Gullain-Barre syndrome, acute onset encephalitis with absent brainstem and pupillary reflexes, intracerebral hemorrhage and cortical blindness.Conclusions: Most snake bites are non-poisonous. Early first aid and ASV administration has better outcome. Fibrinogen levels are more reliable than CRT to diagnose coagulopathy. Acute presentations i.e, altered sensorium, paralysis, blindness and stroke like features should always be evaluated for snake envenomation in suspected cases to prevent morbidity and mortality.

10.
Article | IMSEAR | ID: sea-214866

ABSTRACT

According to the World Health Organization, snakebites cause more death and disability and are more notorious than some tropical diseases. Snakebite is a leading medical emergency in Asia/Pacific. It is one of the major causes of mortality in India. The objective of this study was to determine the clinico-epidemiological profile of a neuroparalytic snake bite at the Department of Medicine, Krishna Institute of Medical Sciences, Karad, one of the tertiary care centers of Western Maharashtra, in India.METHODSA descriptive study of 80 patients was carried out at the Department of Medicine, KIMS, Karad, a tertiary care center of Western Maharashtra, India. Present study showed outcome, delay in arrival, and epidemiology of patients with low dose of ASV in neuroparalytic snakebites and ventilator support. Descriptive statistics were shown by using MS Excel and SPSS Version 25.RESULTSOut of 80 cases 56.2% were males and 43.8% were females with a mean age of 28.16 years. 63.7% of the snakebite victims were bitten outside and 85% of the snakebites occurred on the lower extremity. On an average, 15.24 vials of ASV were administered. 80% patient were in Intensive Care and they were on ventilator support and 91% patients recovered from these snake bites. 51.25% of the patients reached the hospital within the interval of 3-7 hours.CONCLUSIONSStudy signifies the importance of snakebite threat to the community. Delay in reaching a hospital in time where snakebite patients can be treated, was the most important cause of death. Public health programs should be strengthened. Administration of Low dose ASV and ventilator support can provide sufficient cure if patients reach on time. Lack of awareness, delay in reaching the hospital, and treatment by non-medical persons are important factors that should to be addressed.

11.
Article | IMSEAR | ID: sea-214794

ABSTRACT

Snake bite is an important life-threatening emergency but often neglected public health problem in India. India being a subtropical country, has been a land of poisonous snakes, especially in Southern India poisonous snakes like Russell’s Viper, Cobra, Krait and Saw Scaled Viper are commonly encountered. In India around 50,000 people are dying every year from snake bites. In spite of these, the true scale of mortality and morbidity from snakebite remains uncertain. We wanted to study the pattern of envenomation and dry bite among the snake bite cases admitted in Government Vellore Medical College Hospital for the period Jan 2018- Jan 2019, describe the clinicoepidemiological profile and outcome of snake bite cases. We also wanted to evaluate the predictive factors of mortality among snakebite cases admitted in a tertiary care centre in Vellore, South India.METHODSA retrospective descriptive analysis of data extracted from medical records of 522 snakebite cases admitted in the Medical wards and IMCU at Government Vellore Medical College and Hospital, Vellore, Tamil Nadu, during the period extending from January 2018 to January 2019 was done, to describe Clinico –epidemiological profile and management of snake bite cases. We described age/gender distribution, seasonal trends of snake bite cases. We computed proportion of envenomation, dry bite and case fatality rate. Among envenomation we analysed the trends in antisnake venom (ASV) usage and its clinical outcome. Statistical analysis was done using Epi Info – Version 7.2 software.RESULTSOf the total 522 snake bite cases admitted in the hospital during the period from Jan 2018 to Jan. 2019, it was found that the median age of the snake bite victim was 40 years (range 13-76). Snakebite cases were higher among male (56%), agriculture worker (47%), rural population (92%) and during monsoon July-October (65%). Sixty percent of the bites occurred in ankle and foot. Species of snake was identified in 23 (4%) cases. The proportion of dry bite and envenomation were 46% and 54% respectively. Of the envenomation cases 20% were haemotoxic, 24% were neurotoxic and 10% were local envenomation. The median duration of hospital stay among envenomation cases was 4 (range 1-20 ) days and interval between bite and ASV administration was 4 (range 1-48) hours. Sixty cases (13%) were allergic to ASV. Case fatality was 2.5 %( 13/522).CONCLUSIONSEffective management of snakebites would include early admission to a hospital, appropriate ASV administration and early identification of the warning signs. Delay in hospitalization is associated with poor prognosis and increased mortality rate due to consumptive coagulopathy, renal failure and respiratory failure.

12.
Article | IMSEAR | ID: sea-214679

ABSTRACT

Bites by snakes represent an important health problem in the tropical world including India. The true incidence of snakebites is difficult to assess and often is under reported. Though the exact number of snake bites is unknown, an estimated 5.4 million people are bitten each year with up to 2.7 million envenoming. Around 81,000 to 1,38,000 people die each year because of snake bites, and around three times as many amputations and other permanent disabilities are caused by snakebites annually. We wanted to determine frequency of various modes of presentation, complications and management of snakebite.METHODSAll patients presenting to emergency department of Basaveshwara Teaching and General Hospital with history of snakebite and features of envenomation were included in the study. All these patients were given Anti-Snake Venom. Symptomatic treatment of complications was done. Semi-structured questionnaire was filled during hospital stay to analyse various characteristics of snakebite and people’s attitude towards this problem.RESULTS75 cases of snakebite admitted to Basaveshwara Teaching & General Hospital, Gulbarga between January 2018 to November 2019 were studied. Males [50 (62.5%)] were affected more than females [25 (31.5%)] in the ratio of 2:1. Most common age group is 20-40 years [45 (60%)]. Snakebite is a common health hazard in rural areas [55 (73.34%)]. Agricultural labourers (68.6%) are the major sufferers with majority of bites occurring outdoor 60 (80%). 71 (94.66%) bites occurred over limbs, out of which 38 (50.66%) were in lower limbs. Most bites occur between 12 Noon to 12 Midnight 60 patients (81.2%). Out of 75 patients, 60 (80%) were poisonous snakebites, 20 (20%) were non-poisonous snakebites. Among 60 (80%) poisonous bites, 32 (53.34%) had neurotoxic manifestations and 28 (46.66%) had hemotoxic manifestations.CONCLUSIONSSnakebite is common in rural setup, in agricultural labourers. Most common victims are males in the age group between 20-40 years. Poisonous bites were more common than non-poisonous snake bites in this study. In neurotoxic envenomation ptosis was the commonest and earliest symptom while in hemotoxic envenomation, most common symptoms were bleeding from bite site and hematuria. The commonest complications were acute renal failure and respiratory paralysis. Shock, infection and gangrene were also noted in some patients. Maximum mortality was observed in patients who were admitted after 24 hours. Early hospitalization and timely ASV administration were the corner stones in the treatment of snakebite.

13.
J. venom. anim. toxins incl. trop. dis ; 26: e20200076, 2020. graf
Article in English | LILACS, VETINDEX | ID: biblio-1143214

ABSTRACT

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)


Subject(s)
Phospholipids , Snake Venoms , Crotoxin , Phospholipases A2 , Anticoagulants , Biological Products , Lipopolysaccharides
14.
J. venom. anim. toxins incl. trop. dis ; 26: e20200016, 2020. graf
Article in English | LILACS, VETINDEX | ID: biblio-1135158

ABSTRACT

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)


Subject(s)
Animals , Crotalus , Elapid Venoms , Phospholipases A2 , Geographic Locations
15.
J. venom. anim. toxins incl. trop. dis ; 26: e20190073, 2020. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1135150

ABSTRACT

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)


Subject(s)
Animals , Peptides , Bothrops , Crotalid Venoms/isolation & purification , Lectins, C-Type/isolation & purification , Neuroblastoma , Neutrophils , In Vitro Techniques
16.
J. venom. anim. toxins incl. trop. dis ; 26: e20200018, 2020. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1135146

ABSTRACT

Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. Methods: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. Results: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. Conclusion: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.(AU)


Subject(s)
Animals , Snake Bites , Snake Venoms , Mass Spectrometry , Bothrops , L-Amino Acid Oxidase , Phospholipases A2 , Biological Products
17.
J. venom. anim. toxins incl. trop. dis ; 26: e20200007, 2020. graf
Article in English | LILACS, VETINDEX | ID: biblio-1135143

ABSTRACT

Pathogenesis of Bothrops envenomations is complex and despite numerous studies on the effects of this snake venom on various biological systems, relatively little is known about such effects on the male reproductive system. In the present study, the toxicological outcomes of the low molecular weight fraction (LMWF) of B. jararaca snake venom - containing a range of bioactive peptides - were investigated on the dynamics and structure of the seminiferous epithelium and 15P-1 Sertoli cells viability. Methods: LMWF (5 µg/dose per testis) venom was administered in male Swiss mice by intratesticular (i.t.) injection. Seven days after this procedure, the testes were collected for morphological and morphometric evaluation, distribution of claudin-1 in the seminiferous epithelium by immunohistochemical analyses of testes, and the nitric oxide (NO) levels were evaluated in the total extract of the testis protein. In addition, the toxicological effects of LMWF and crude venom (CV) were analyzed on the 15P-1 Sertoli cell culture. Results: LMWF induced changes in the structure and function of the seminiferous epithelium without altering claudin-1 distribution. LMWF effects were characterized especially by lost cells in the adluminal compartment of epithelium (spermatocytes in pachytene, preleptotene spermatocytes, zygotene spermatocytes, and round spermatid) and different stages of the seminiferous epithelium cycle. LMWF also increased the NO levels in the total extract of the testis protein and was not cytotoxic in concentrations and time tested in the present study. However, CV showed cytotoxicity at 10 μg/mL from 6 to 48 h of treatment. Conclusions: The major finding of the present study was that the LMWF inhibited spermatozoa production; principally in the spermiogenesis stage without altering claudin-1 distribution in the basal compartment. Moreover, NO increased by LMWF induce open of complexes junctions and release the germ cells of the adluminal compartment to the seminiferous tubule.(AU)


Subject(s)
Animals , Male , Peptides , Seminiferous Epithelium , Snake Venoms , Spermatogenesis , Bothrops , Biological Products
18.
J. venom. anim. toxins incl. trop. dis ; 26: e20190103, 2020. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1101265

ABSTRACT

The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. Conclusion: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.(AU)


Subject(s)
Animals , Oxidoreductases , Snake Bites , Snake Venoms , Bites and Stings , Antivenins , Bothrops , Proteome
19.
J. venom. anim. toxins incl. trop. dis ; 26: e20190053, 2020. graf, mapas, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1091020

ABSTRACT

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)


Subject(s)
Animals , Antivenins , Crotalus , Crotalid Venoms/analysis , Animal Distribution
20.
Article in Chinese | WPRIM | ID: wpr-855876

ABSTRACT

AIM: To investigate the effect of endoplasmic reticulum stress pathway on the apoptosis of tongue squamous cancer Tca8113 cells induced by antitumor component-I from Agkistrodon acutus venom (AAVC-I). METHODS: The in vitro experiments were performed on subculture tongue squamous cancer Tca8113 cells in their growth period. A normal control group, a DL-dithiothreitol (DTT) positive control group and different AAVC-I concentrations were set according to the experiment objective. MTT assay was used to detect the proliferation inhibition of Tca8113 cells after been treated with different concentrations of DTT and AAVC-I for 24 h. The results were used to choose appropriate concentrations of DTT and AAVC-I in DTT positive control group and AAVC-I treated group, respectively. HE staining and Annexin V-FITC/PI double fluorescence staining were used to monitor the apoptosis of Tca8113 cells. Western blot was used to identify the expression levels of apoptosis-related proteins including endoplasmic reticulum stress glucose-regulatory protein 78 (GRP78), enhance-binding protein-homologousprotein (CHOP), cysteine-containing aspartate specific protease-12 (Caspase-12), cysteine-containing aspartate specific protease-9 (Caspase-9) and cysteine-containing aspartate specific protease-3 (Caspase-3).RESULTS:The proliferation inhibition of Tca8113 cells increased with an increased concentration of AAVC-I concentration (P<0.05), causing cell shrinkage, increased cell gaps, cytonuclear condensation, cell fragmentation, the appearance of apoptotic bodies, and increased rate of apoptosis (P<0.05). In addition, the expression level of GRP78 protein, CHOP protein, proteins of Caspase-12, Caspase-9 and Caspase-3 were increased (P<0.05).CONCLUSION: Endoplasmic reticulum stress CHOP/Caspase-12 pathway plays an important role in AAVC-I induced Tca8113 cells apoptosis.

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