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Chlorotoxin (Cltx), a cell-penetrating peptide from the venom of the scorpion Leiurus quinquestriatus has selective activity on a range of neuroectodermal tumours and related cell lines. Although the precise mechanism of Cltx action is not clear, Cltx has been shown to be a useful tumour imaging agent and radiolabelled Cltx has undergone clinical trials for targeted glioma radiotherapy. The native peptide (and synthetic material, TM601) is amidated at its C-terminus (Cltx-CONH2). A recombinant form produced in E. coli is not processed and has a free C-terminal carboxyl group (Cltx-COOH). The amidated form appears to have no effects on proliferation and cytotoxicity, whereas studies on the free carboxyl form are contradictory. Cltx-CONH2 and Cltx-COOH have been used interchangeably over many years and this has resulted in confusion in the literature; the significance of any difference is extremely important one with respect to the use of the toxin in a clinical diagnostic or therapeutic setting. This study was carried out to investigate, in parallel, the effects of Cltx-CONH2 and Cltx-COOH on cell proliferation and cytotoxicity on a variety of representative tumour cell lines (U87MG, MCF-7, PC3 and A549) using cell Titer Glo and cell Tox Green assays, respectively. Neither Cltx-CONH2 nor Cltx-COOH affected cell proliferation or cytotoxicity, indicating that terminal arginine amidation does not play an important role in cytotoxic or cytostatic properties of Cltx.

Neurotoxic and cytotoxic effects of venoms from Scorpio maurus palmatus taken from different populations were assessed for geographic based variability in toxicity, and to evaluate their insecticidal potency. Scorpions were collected from four regions. Three locations were mutually isolated pockets in the arid area of Southern Sinai. The fourth sample was collected from a population inhabiting the semi-arid environment of Western Mediterranean Coastal Desert. The neurotoxic (paralytic) effect of the venom from each population was assayed by its ability to induce permanent disability in adult cockroaches within 3h. Venom was applied using microinjection techniques through an intersegmental membrane. Probit analysis was used to calculate the Paralytic Effective Dose (PED(50), ng/100mg). Levels of glutathione, lipid peroxidation, protein carbonyl content and nitric oxide, as well as the activities of superoxide dismutase, catalase and cholinesterase, were measured to assess the cytotoxicity of the venom. The results show that the injected venom from each population induced obvious spasticity, followed by flaccid paralysis. All the tested biochemical parameters, except glutathione content, revealed significant differences in toxicity in venom taken from the different scorpion populations. We conclude that (i) the venom of this scorpion has significant neurotoxic and cytotoxic effects on insect cells, (ii) its efficacy, as assessed by the PED(50) unit, exhibited variation across its geographic range, and (iii) components in the venom may have the potential for being developed into effective and environmentally friendly bioinsecticides.

The Buthid scorpion Leiurus quinquestriatus is divided into two sub-species whose region of contact occurs at the Suez Isthmus. L.q. quinquestriatus, found in the North Africa is distinguished from L.q. hebraeus by minor differences in morphology. Despite the very high similarity in body plan, out of the 24 toxins isolated from this species none is common to both sub-species, suggesting a degree of molecular divergence not apparent at the morphological level. We show, by comparison with two sets of reference proteins from humans and Old-World monkeys, that divergence of toxins with common structures and functions in these two sub-species has occurred under conditions of strong negative selection. Potential selection pressures responsible for toxin divergence are discussed, including the role of conflicting interests during courtship.

Leiurus quinquestriatus is a single species genus scorpion of Saharan origin, which has penetrated the Mediterranean region. The Sinai Isthmus is believed to be the region in which the subspecies L...

Calotropis procera (Ait.) R.Br. (Asclepiadaceae) is a shrub or small tree that grows wild in Egypt. Calotropis acts as a purgative, anthelmintic, anticoagulant, palliative (in problems with respiration, blood pressure), antipyretic, and analgesic, and induces neuromuscular blocking activity. Little research has been done to study the electrophysiological effects of this plant's extracts on cardiac, smooth, and skeletal muscle activities.The present study was conducted to determine the phytochemical composition and the effect of the total alcohol extract of the shoot of the plant, which contains almost all of C. procera's cardiac glycosides, flavonoids, and saponins. Also, this study attempted to throw more light on the electrophysiological effects of the plant extracts on cardiac, smooth, and skeletal muscle activities and to clarify the mechanism(s) of their observed action(s).The aerial parts of the plant were air dried and their ethanol extracts partitioned with successive solvents. Cardiac, smooth, and skeletal muscles were used in this study to investigate the physiological and pharmacological effects of the plant extracts from different solvents. The data were analyzed by paired t-test.The phytochemical investigation of Calotropis procera revealed the presence of cardenolides, flavonoids, and saponins. The effects of ethanol, n-butanol, and ethyl acetate (EtOAc) extracts were each evaluated on isolated toad heart and their mechanisms of action determined. Perfusion with 2 μg/mL ethanol, 0.2 μg/mL butanol, and 0.2 μg/mL EtOAc extracts caused a significant decrease in heart rate (bradycardia), significant increase in the force of ventricular contraction, and increase in T-wave amplitude. In addition, the effects of different extracts of the studied plant on smooth muscle and skeletal muscle were investigated in this study. The different extracts and latex of C. procera induced a negative chronotropic effect and decreased the heart rate (HR) of isolated toad heart. The different extracts increased the power of contraction of the duodenum (trace a). Pretreatment with atropine sulfate as a muscarinic receptor blocker abolished the stimulatory effect of the different plant extracts and latex of C. procera (trace b).The present data suggest that ethanol, butanol, and EtOAc extracts of Calotropis procera have negative chronotropism and positive inotropism. Verapamil could abolish the inotropic effect of ethanol as well as that of butanol and EtOAc extracts. Meanwhile, atropine did not abolish the observed negative chronotropic effect. The ethanol extract increased the power of contraction of rabbit duodenum, but atropine abolished this effect. It also decreased the skeletal muscle contraction; this effect could be through blocking of the nicotinic receptors. Butanol and EtOAc extract data for smooth and skeletal muscles are very close to those for the corresponding ethanol extract of the studied plant. The present data for C. procera indicate its direct action on the myocardium, its increase of smooth muscle motility, and its relaxation of skeletal muscle contraction. The chemical constituents could directly affect the cell membrane probably through receptors coupling to G proteins. They regulate the ion channel physiology as in the myocardium.The present data on the extracts of C. procera indicate a direct action on the myocardium, stimulatory effect on smooth muscle motility, and relaxant action on skeletal muscle contraction. Chemical constituents could directly affect the cell membrane probably through receptors coupling to G proteins. They regulate the ion channel physiology as in the myocardium.

The present study was conducted to explore the following hypotheses: (i) do scorpions (Scorpio maurus palmatus) from different biotopes exhibit intraspecific diversity in their venom? (ii) if so, is this variation associated with ecological or genetic factors, geographical distance, and/or multiple interrelated parameters? To address these questions, scorpions were collected from four geographically isolated localities in Egypt. Three of these locations are from mutually isolated pockets in the arid biotope of Southern Sinai (Wadi Sahab, El-Agramia and Rahaba plains). The fourth population was sampled from the semiarid biotope of Western Mediterranean Costal Desert (WMCD). Using reducing gel electrophoresis (SDS-PAGE), we have shown biotope-specific variation in the expression of peptides from scorpions collected from these distinct areas. WMCD sourced venom samples contain higher molecular weight protein components (219, 200, 170, 139, 116 kDa) than Southern Sinai scorpion venom samples. The Southern Sinai venom is characterized by the presence of 11 protein bands (93-0.58 kDa) that are not mirrored in the individual venom samples of WMCD. Bands of 33 and 3.4 kDa were characteristics of all individual venom samples of the scorpion populations. Even within Southern Sinai area, Sahab venom contains five fractions that are not detected in both El-Agramia and Rahaba venom samples. Moreover, male and female venom analysis revealed some sex-related proteomic similarities and differences between scorpion populations. Female venom appears to be more complicated than the male venom. Female venom samples showed bands of 219, 200, 77.5, 55.5, 45, 39, 37, 24 and 16 kDa which were absent in the male venom. The random amplified polymorphic DNA (RAPD) technique was used to estimate the genetic distance between the four scorpion populations. The RAPD data confirmed the genetic diversity at molecular level among the sampled populations. More than 77 RAPD bands (ranging in size from 125 to 15,000 bp) were defined from the four scorpion populations. Of the 77 bands, 57 (76.2%) were polymorphic and 20 were monomorphic among the populations. The similarity coefficient data of venom and DNA were used to construct separate dendrograms, which grouped together the Southern Sinai populations and these were some distance away from the WMCD population. Taken together, we suspect that a combination of local environmental conditions, geographical separation and genetic separation may play a major role in the intraspecific variation of venom of S. m. palmatus.

We investigated the in vitro process of cell death caused by Egyptian cobra venom on primary human embryonic kidney (293T) and mouse myoblast (C2C12) cell lines. The aim of these studies was to provide further information about triggering cell death, and suggest methods for eliminating unwanted cells, such as tumour cells. Both cell lines were treated with 10, 20, and 50 m g/ml of Egyptian cobra (Naja haje) venom in serum free media (SFM) and incubated for 8 hours. Total activities of the lactate dehydrogenase (LDH) and creatine kinase (CK) released in the culture during venom incubation were used as an indicator of the venom in vitro cytotoxicity. Cell injury was morphologically recognized and apoptosis determined by a Fluorescing Apoptosis Detection System and confirmed by staining nuclear DNA with DAPI. Our data clearly demonstrated marked cytotoxic effects and acute cell injury for both cell lines. Release of LDH and CK into the culture media induced by the venom correlates well with the morphological changes and extent of cell death. Mostly, these consequences were time and dose-dependent in both cell lines. The results obtained from this study indicated that cobra venom cause cell death by two different mechanisms: necrosis and induction of apoptosis. The apoptotic mechanism, accompanied by cell necrosis, mediated cell destruction of both tested cell lines; however, necrosis was predominant in the C2C12 cell line while apoptosis, in 293T cells. This unusual form of cell death induced by cobra venom may represent a combination of apoptosis and necrosis within the same cell. This is a first-hand investigation showing the apoptotic effects of N. haje venom at the cellular level. However, the contribution of the apoptotic pathway may be dependent on concentration and/or time of exposure to snake venom.

Scorpion venom toxicity is of major concern due to its influence on human activities and public health. The cytotoxicity and apoptosis induced by scorpion L. quinquestriatus venom on two established eukaryotic cell lines (293T and C2C12) were analyzed. Both cultured cell lines were incubated with varying doses (10, 20, and 50 µg/ml) of scorpion venom in serum free medium (SFM) for 0.5, 1, 2, 4, and 8 hours at 37°C. The percentage of total lactate dehydrogenase (LDH) released in the culture during venom incubation was used as an index of cell damage. Control culture was treated with an equal amount of SFM. Cell injury was recognized morphologically and apoptosis was researched by a Fluorescing Apoptosis Detection System using the principle of TUNEL (TdT-mediated dUTP Nick-End Labelling) assay and confirmed by another assay concerning nuclear DNA staining with DAPI stain. Cytotoxicity was remarkable and cell survival highly reduced at the highest tested concentration (50 µg/ml). These effects were rapid and observed within 30 minutes. The apparent initial damage to the nucleus and lysis of the plasmalemma and/or organelle membranes, which was evident by a significant increase in cytosolic LDH release, suggested that this toxin acts at the membrane level. The morphological changes that occurred in apoptotic cells include condensation and compartmentalization of nuclear and cytoplasmic materials into structurally preserved membrane-bound fragments or blebs. The cytotoxic effects are dose and time dependent and cell death by apoptosis was more characteristic of 293T cells than C2C12 cells. The apoptotic effects were more prominent and clear in the early stages of toxicity, while other forms of cell damage such as swelling, rupture, and/or necrosis occurred at later stages.

1 The effect of Egyptian cobra (Naja haje) venom on the normal electrical activity of the cardiac muscles (ECG) and arterial blood pressure of envenomated rats were investigated in this study. 2 Rats were divided into three groups. The first group was injected im with saline and considered as control group. Rats of the second and third groups were injected IM with 0.02 μg and 0.04 μg cobra venom/ gim b.wt, respectively. 3 Mean blood pressure (MBP), heart rate (HR) and four different ECG parameters (PR and QT intervals, R and T wave amplitudes) were measured over 1 h following envenomation. 4 The low dose (0.02 μg/g) of N. haje venom caused hypotension accompanied by an increase in the HR, whereas hypertension and bradycardia developed after injection of the high dose (0.04 μg/g) of venom. 5 There was a decrease in the P-R interval after administration of the low dose and prolongation of it after the high dose. The Q-T interval and R-wave amplitude were significantly increased after injection of both doses. T-wave amplitude was significantly elevated only after injection of the high dose. 6 The present results indicate that the Egyptian cobra (N. haje) venom significantly alters the arterial blood pressure and ECG parameters of envenomated rats. This suggests that impairment of the electrical activity of cardiac muscle may be one of the reasons why victims of cobra bite die.

The effects of the scorpion Leiurus quinquestriatus (H.&E.) venom on electrocardiogram (ECG) parameters such as P-R and Q-T intervals and R and T wave amplitudes were investigated in anesthetized rats. Venom was administered intramuscularly (i.m.) at three doses (100, 200 and 400 micrograms/kg). ECG limb lead II was recorded for 4-h sessions. Because autonomic nervous system tone plays an important role in influencing ECG findings, another study was completed with concomitant pharmacologic autonomic nervous system blockade. Propranolol or atropine was injected 20 min before venom administration in two groups of rats. The results indicated that the venom has drastic effects on the electrical activity of the heart. The Q-T interval developed a dose-response relationship after venom administration. Propranolol abolished the toxic effects of the venom on P-R and Q-T intervals as well as on R wave amplitude, while atropine had no effect on the ECG changes produced by the venom.

Egyptian scorpion venom was collected by electrical stimulation of the telson. Rats were injected with the lyophilized venom in 3 different doses (100, 200 and 400 μg ml ). Blood samples were drawn by heart puncture before and 4 h after venom administration. Serum was separated and collected for determination of glucose, blood urea nitrogen (BUN), creatinine, uric acid (UA), total proteins, cholesterol, sodium, potassium, calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase (AST, GOT), alanine aminotransferase (ALT, GPT), lactate dehydrogenase and creatine phosphokinase (CPK). Serum glucose, creatinine, GOT, GPT and LDH were increased significantly in all treatments. At the same time serum BUN and CPK were elevated significantly with a dose-response relationship. On the other hand, serum total proteins, uric acid, cholesterol, calcium and potassium were significantly decreased 4 h after administration of the 3 doses. These changes in clinical chemistry parameters are most probably related to the toxic effect of the venom on the target organs.

The effects of scorpion Leiurus quinquestriatus (H&E) venom collected from the South Sinai region, Egypt, on heart rate (HR) and mean arterial pressure (MAP) were studied after intramuscular administration of 3 different doses (100, 200 and 400 micrograms/kg) to anesthetized rats. The effects of adrenergic and cholinergic blocking agents on the venom-induced HR and MAP changes were also evaluated. In two groups of rats, propranolol or atropine were given before the venom administration. In the third group the venom was given before the injection of propranolol and atropine in combination. HR was measured by using a cardiotachometer coupler connected to an ECG coupler. MAP was calculated from the recorded arterial blood pressure (ABP) after catheterization of the left common carotid artery. Venom doses of 100 and 200 micrograms/kg produced tachycardia with a dose-response relationship, whereas 400 micrograms/kg evoked sinus tachycardia followed by bradycardia then tachycardia. MAP was elevated after the administration of each dose and reached its maximum value after 60 min with a dose-response relationship. Sinus, atrial and ventricular arrhythmias were observed from the recorded ECG during the time studied. This study revealed that the venom has pressor and depressor effects which are mediated through the autonomic nervous system. Propranolol reduced the stimulatory effects of the highest dose of the venom while atropine was effective in eliminating the depressor effect of the venom on HR. The arrhythmias induced by the venom were blocked by the injection of the two blockers and are assumed to be due to the release of catecholamines and acetylcholine.