A new species of saw‐scaled viper of the Echis coloratus complex (Ophidia: Viperidae) from Oman,Eastern Arabia

A study of 353 museum specimens of the Echis coloratus complex from its entire range of distribution revealed an undescribed species in the United Arab Emirates and northern Oman. The results of UPGMA clustering and principal coordinate analysis of 138 male and 142 female specimens yielded for both sexes two major clusters, one with specimens from the UAE and northern Oman and one from southern and western Arabia, the Levant and Egypt. The new species has a longer tail with higher subcaudal counts; the lower prenasal scale is often missing and the upper prenasal is frequently fused with the nasal; the subnasal is often missing or fused with the nasal. The gular scales between the chin‐shield and the preventrals are round or only slightly elongate, not elongate as in Echis coloratus, and their number is higher. Other differences in characters of the gular area indicate a different scale structure of the ventral surface of the head. The new species is allopatric or parapatric with E. coloratus, but sympatric with Echis carinatus sochureki.     

Separation and purification of Echis coloratus venom and some biological and biochemical effects of the proteins

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1-F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37 degrees C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters.     

Molecular Cloning of Echis ocellatus Disintegrins Reveals Non-Venom-Secreted Proteins and a Pathway for the Evolution of Ocellatusin

We report the cloning and sequence analysis of Echis ocellatus cDNAs coding for dimeric disintegrin subunits and for the short disintegrin ocellatusin. All the dimeric disintegrin subunit messengers belong to the short-coding class, indicating that short messengers may be more widely distributed than previously thought. Mass spectrometric analysis of the HPLC-separated venom proteins was performed to characterize the dimeric disintegrins expressed in the venom proteome. In addition to previously reported EO4 and EO5 heterodimers, a novel dimeric disintegrin containing RGD- and KGD-bearing subunits was identified. However, a WGD-containing polypeptide encoded by clone Eo1-1 was not detected in the venom, suggesting the occurrence of larger genomic than proteomic diversity, which could represent part of a non-venom-secreted reservoir of disintegrin that may eventually acquire physiological relevance for the snake upon changes of ecological niches and prey habits. On the other hand, the realization of the existence of two distinct messengers coding for the short disintegrin ocellatusin reveals key events of the evolutionary emergence of the short disintegrin ocellatusin from a short-coding dimeric disintegrin precursor by two nucleotide mutations. [Reviewing Editor: Dr. Bryan Grieg Fry]     

Possible recovery from an acute envenomation in male rats with LD50 of Echis coloratus crude venom: I-A seven days hematological follow-up study

The effect of an acute LD₅₀ dose of Echis coloratus crude venom in male albino rats was tested on blood parameters: white blood cells (WBCs), red blood cells (RBCs), platelets count, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), also serum glucose, total protein, triglycerides with alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) enzyme activities. The effect of the LD₅₀ dose was monitored over a period of seven days, with time intervals of 1, 3, 6, 12, 24, 72 h. All of the tested parameters show fluctuations with time and with tendency to regain normal control level after 12 h. At 12–24 h it seems to be crucial for the process of physiological recovery, in spite of the irreversible damage and tissue distraction. The process of physiological adaptation and recovery from the lethal destructive venom effect seems to stabilize after one week, leaving the animal alive with several biochemical altered metabolisms and disturbed physiological profile.     

Microangioarchitecture of the islets of Langerhans in the snakes,Naja naja,Vipera russelli,andEchis carinatus

Summary Due to a decided lack in the literature of reports on the microangioarchitecture of the pancreas of snakes, an analysis was performed in three different species of two different ophidian families with the use of cast preparations and complementary scanning electron microscopy. The vascular architecture reflects the lobular substructure of the pancreas; the organ is supplied by branches of the superior mesentric artery. Coiled lobular arteries and arterioles continue into a meshwork of capillaries. Dilated capillaries of the endocrine portion of the pancreas are an integral component of this fine capillary network. A few very small capillaries establish a connection between the endocrine and the exocrine pancreas. The other capillaries drain into venules, which ultimately join their respective veins. No interspecific differences were noted in the vascularization of the pancreas of the three ophidian species examined. The present results suggest the existence of arterio-venous anastomoses and speak against the presence of a portal system, but establish a feed-forward capillary connection from the endocrine to the exocrine component of the ophidian pancreas.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Time-course of lipid peroxidation in different organs of mice treated with Echis pyramidum snake venom

This study examined the effect of Echis pyramidum (EP) venom on time-course of lipid peroxidation in different vital organs of mice. Adult male Swiss albino mice were injected with EP venom (2 mg/kg, i.p.); control mice received vehicle alone (normal saline). Mice were killed at 1, 3, 6, 12, and 24 h post-envenomation. The liver, lung, kidney, heart, and brain (cerebrum and cerebellum) were collected for the estimation of malondialdehyde (MDA), an index of lipid peroxidation. The results of this study showed that a single injection of EP venom caused a significant lipid peroxidation in all the organs studied. The onset of lipid peroxidation was as early as 1 h and persisted for several hours, suggesting an important role of oxidative stress in the cytotoxicity of EP venom.     

The effect of Echis carinatus crude venom and purified protein fractions on carbohydrate metabolism in rats

Echis carinatus crude venom was fractionated into 11 protein fractions by preparative native polyacrylamide gel electrophoresis (PAGE). All fractions except fractions 5 and 10 appeared as a single band on analytical native PAGE. Purified venom fractions 1, 4, 8, 10 and 11 appeared as single bands on SDS-PAGE whereas fractions 2, 3 and 7 contained two bands and fraction 6 contained three bands. Fractions 1 and 3 exhibited basic pI (7.3 and 7.6) respectively, while fractions 2, 4, 6, 8, 10 and 11 showed an acidic pI. Amino acid analysis also showed that crude venom is rich in acidic amino acids. A significant hyperglycaemia was produced by i.p. injection of E. carinatus crude venom, after 15 min of envenomation which persisted even after 24 h. Along with hyperglycaemia there was a significant decrease of liver glycogen at 15 min and 1, 12 and 24 h. A significant decrease of plasma [pyr + lac] levels was found from 15 min to 24 h. The liver [pyr + lac] levels increased significantly after 24 h. Skeletal muscle [pyr + lac] level was significantly decreased after 24 h of envenomation. Fractions 2 and 6 produced the highest increase in plasma glucose after 12 h and fraction 7 after 24 h. The plasma insulin level was significantly decreased by these three fractions (2, 6 and 7). So it can be hypothesized that the hyperglycaemia may result from a direct effect of a venom component on plasma insulin. Fractions 7, 8 and 11 caused the highest decrease in plasma [pyr + lac] while fractions 1, 2, 3, 4 and 8 produced the most significant decrease in liver [pyr + lac]. The most significant increase in lactate dehydrogenase level was also produced by fractions 1, 2, 3, 4 and 8.     

Histological,molecular and biochemical detection of renal injury after Echis pyramidum snake envenomation in rats

Nephrotoxicity is a common sign of snake envenomation. The present work aimed to clarify the effect of intraperitoneal injection of 1/8 LD₅₀ and 1/4 LD₅₀ doses of Echis pyramidum snake venom on the renal tissue of rats after 2, 4 and 6 h from envenomation. Histopathological examination showed intense dose and time dependent abnormalities, including swelling glomerulus and tubular necrosis and damage as well as signs of intertubular medullary hemorrhage at early stages of envenomation. However, at late stages of envenomation by any of the doses under investigation, no intact renal corpuscles were recorded and complete lysis in renal corpuscles with ruptured Bowman’s capsules was observed. Immunohistochemistry by immunohistochemical staining was used to test the protein expression of Bax in renal tissue of rats. The result showed that the expression of Bax in renal tissue sections of envenomated rats was increased according to dose and time-dependant manner. The isolation of DNA from the renal cells of envenomed rats pointed out to the occurrence of DNA fragmentation, which is another indicator for renal tissue injury especially after 6 h of 1/4 LD₅₀ of E. pyramidum envenomation. Oxidative stress biomarkers malondialdehyde and nitrite/nitrate levels, antioxidant parameters; glutathione, total antioxidant capacity and catalase were assayed in renal tissue homogenates. The venom induced significant increase in the levels of malondialdehyde and nitrite/nitrate while the levels of glutathione, total antioxidant capacity and catalase were significantly decreased, especially after 6 h of envenomation. The results revealed that the E. pyramidum induced dose and time-dependant significant disturbances in the physiological parameters in the kidney. We conclude that the use of the immunohistochemical techniques, the detection of DNA integrity and oxidative stress marker estimations are more specific tools that can clarify cellular injury and could point out to the defense activity of the renal tissue at envenomation.