Purification and partial characterization of a coagulant serine protease from the venom of the Iranian snake Agkistrodon halys

Agkistrodon halys is one of several dangerous snake species in Iran. Among the most important signs and symptoms in patients envenomated by this snake is disseminated intravascular coagulation. A thrombin-like enzyme, called AH143, was isolated from Agkistrodon halys venom by gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a DEAE-Sepharose and high performance liquid chromatography (HPLC) on a C18 column. In the final stage of purification, 0.82 mg of purified enzyme was obtained from 182.5 mg of venom. The purified enzyme showed a single protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, and its molecular mass was found to be about 30 kDa. AH143 revealed clotting activity in human plasma, which was not inhibited by EDTA or heparin. This enzyme still demonstrated coagulation activity when exposed to variations in temperature and pH ranging, respectively, from 30 to 40°C and from 7.0 to 8.0. It also displayed proteolytic activities on synthetic substrate. The purified enzyme did not show any effect on casein. We concluded that the venom of the Iranian snake Agkistrodon halys contains about 0.45% single procoagulant protein which appears to be a thrombin-like enzyme

An in vitro Comparative study upon the Hemolytic, Thrombogenic, Coagulation parameters and Stability properties of the Hemiscorpius lepturus Venom

Hemiscorpius lepturus belonging to Hemiscorpiidae family is the most venomous of all types of scorpion existing in south west of Iran causing hemoglobinuria and dermal lesions by envenomation. We compare the hemolytic pattern upon time in different domestic animals upon time according to their different sphingomyelin contents. In addition other in vitro hematologic parameters, platelet lysis, coagulation changes and finally preservative factors (temperature, pH, protases) are discussed. The hemolytic activity was inhibited significantly by heating at 100 °C for 60 minutes (26%) and reached 38% via incubation with papain (10U/ml) while retained over a pH range of 4-11. Horses and sheep have the lower (61%) and upper (100%) rate of hemolysis. Calcium and magnesium ions could increase rate of hemolysis and EDTA solution had significantly decresing effect. The venom significantly changed in vitro coagulation factors (PT and APTT) from base line levels and had no effect on platelet lysis. It seems that our venom belongs to metalloproteinases due to potentiation effects of bivalent cations (calcium and magnesium) and ghost cell formation in our study indicatiing hemoglobin efflux

Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (

Hemiscorpius lepturus envenomation: Manifestations and management with specific antivenom

Scorpionism is a known significant problem of medical and social importance in many tropical and subtropical regions including the Middle East. In Iran, highest prevalence of scorpion sting about 60% of all the stings has been reported from Khuzestan province. Among the 21,000 cases of reported scorpion stung patients, 12% were caused by H. lepturus, but contributed to 95% of all moralities in scorpion stung patients. The sting of H. lepturus does not produce an immediate pain as does the sting of other scorpions, rather cause delayed swelling that may diffuse and is often accompanied by late necrosis at the sting site suggestive of less significant role of the nervous system stimulation. Since the venom from H. lepturus is cytotoxic in nature and the renal response and blood toxicity are normally simultaneously manifested, it is suggested that the toxin binds to kidney tissue and potentially induce acute renal failure in stung patients. Pharmacokinetic analysis revealed that Intramuscular) i.m) injection of antivenoms is ineffective in neutralizing the action of venoms. Although some reports mention the slow distribution rate of H. lepturus venom following sting, but since the cytotoxic effect of venom from this scorpion is irreversible by antivenom once it occurs, it is recommended to use antivenom through intravascular (i.v) route. Antivenoms of F(ab)2 fraction are the best choice of treatment for their fast extravasation, their ample distribution into the extracellular space, and their prolonged retention time

Biomacromolecular Journal Acute Effects of the Iranian Snake (Naja Naja Oxiana) Venom on Heart

ABSTRACT The myocardial effect of snake venoms is considered as one of the most common pathogenesis in many cases of snake envenomation. This study was undertaken to investigate the effects of the Iranian cobra (Naja naja oxiana) venom on cardiac function in experimental animals. The blood samples from all the rabbits were collected before venom injection, and then 140 µg kg -1 venom of snake (Naja naja oxiana) was injected intramuscularly to the rabbits. Following venom injection the blood was collected again at 1, 3 and 24 h. Electrocardiogram (ECG) was recorded during the experiment. The levels of serum enzymes lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and creatine phosphokinase (CPK) were determined. Statistical analyses were carried out by SPSS version 21 software. CPK enzyme showed a significant increase at 1 and 3 h after venom injection. The level of CK-MB also rose significantly after 1 h following venom injection. However, even at 24 h the level of CPK was not changed significantly, and the rise in CK-MB at 3 and 24 h following venom injection was not significant statistically. Although there was a rise in LDH level following venom injection but it was not significant. The ECG also confirmed changes in heart rhythmic and showed bradycardia and T tall. Based on the results obtained in the present study, it seems that the Naja naja oxiana venom has acute effect on cardiac system during the first few hours of snake bite

Final 3.indd

Cell culture The HUVEC cell line was cultured in DMEM:F12 supplemented with 7% heat-inactivated FBS along with 100 IU/mL penicillin G and 100 g/mL streptomycin. The culture was incubated at 37°C with humidi ed air that contained 5% CO 2 . Abstract Background: Agiogenesis is the development of new blood vessels from pre-existing vasculatures. Although essential in the physiological process, it becomes pathological in various diseases including cancer. Preventing the formation of new blood vessels causes reductions in tumor size and metastasis. This study has been undertaken to elucidate the anti-angiogenesis effects of ICD-85 (derived peptides from venom). Methods: We evaluated the ICD-85 anti-angiogenesis activity by the in vivo CAM assay and in vitro tube formation assay of human umbilical vein endothelial cells (HUVECs). The anti-proliferative activity of ICD-85 was also determined through MTT assay on HUVECs. Results: Results of this study revealed the anti-proliferative activity of ICD-85 on the HUVEC cell line with an IC50 of 12 g/mL. The in vivo CAM assay also clearly showed the prevention of new vascular formation when the chick embryos were exposed to 0.15 g/disc of ICD-85. In vitro tube formation assay of HUVECs also showed the complete prevention of capillary tube formation on 18 g/mL. Conclusion: Based on the results obtained in this study, ICD-85 has anti-angiogenesis activity as shown by the prevention of capillary tube formation and the CAM assay

Two-step purification and partial characterization of an extra cellular α-amylase from Bacillus licheniformis

The aim of this study was production and partial purification of α-amylase enzyme by Bacillus licheniformis. B. Licheniformis was allowed to grow in broth culture for purpose of inducing α-amylase enzyme. Optimal conditions for amylase production by B. Licheniformis are incubation period of 120 h, temperature of 37 °C and pH 7.0. The α-amylase enzyme was purified by ion exchange chromatography on DEAE-sepharose CL-6B and sephadex G-100 gel filtration with a 19.1-fold increase in specific activity as compared to the concentrated supernatant and with a specific activity of 926.47 U/mg. The α-amylase had the highest activity at pH 7.0 and 65 °C. According to the data on native polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was 72 kDa

Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment) and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims

Cytotoxic effect of ICD-85 (venom-derived peptides) on MDA-MB-231 cell line

Since 1987, when chemopreventive testing programs began, more than 1,000 agents and agent combinations have been selected and evaluated in preclinical studies of chemopreventive activity against various types of cancers. In the present study we aimed to provide quantitative and qualitative characterization of biological and pharmacological activities of ICD-85 on MDA-MB-231 cell line (a highly invasive breast cancer cell line) in order to gain a better understanding of the cytotoxic and apoptotic effects of this compound. For this study, the MDA-MB-231 cell line was used and the effect of ICD-85 was assayed by measuring the activity of the cytosolic enzyme lactate dehydrogenase (LDH), released into the culture medium after membrane damage. Morphological alterations of cells were investigated in the control group and cells incubated with ICD-85 as cytotoxic agent. Results showed, in the test group, that cells incubated with 16 µg/mL of ICD-85 had decreased cytoplasmic branching. Some cells were had ruptured and lost the continuity of their surrounding membranes while some had shrunk. Cells incubated with higher doses (above16 µg/mL) showed similar changes towards cellular normality with more severity. Results obtained from the ICD-85 stability test reveal that the effect of ICD-85 on MDA-MB-231 cell line in culture medium is stable throughout the incubation time period (24 hours). It appears that ICD-85 at higher concentrations acts at the membrane level, which allows the passage of ions down the concentration gradient, resulting in osmotic changes in organelles followed by several unidentified mechanisms leading to cell death. At lower concentrations, it appears that ICD-85 can prevent cell growth by another mechanism, which may be one of the causes for apoptosis in the cell line