A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies.

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K+-channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named alpha-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

The regulation of the ratio of the cardiopulmonary blood volume to stroke volume in sheep.

Volume expansion with dextran was used in unanaesthetised sheep to investigate the mechanisms which are responsible for keeping the ratio of the cardiopulmonary blood volume to stroke volume (CPFI) constant under physiological conditions. For animals with heart rates lower than 80, active pulmonary vasoconstriction was mainly responsible for the regulation obtained with volume expansion, while the Starling mechanism was responsible for keeping the ratio constant for animals with heart rates above 79. The involvement of a regulatory mechanism was shown by the absence of large changes in the CPFI after inducing mechanical stress on the cardiopulmonary system as a result of experimental hypertension. Results obtained with receptor blockade showed that active pulmonary vasoconstriction may be the result of a reflex mechanism with parasympathetic efferents. It is suggested that the receptors for this reflex are situated in the pulmonary arteries and are activated by the pressure changes as a result of heart contractions.

Ratio of cardiopulmonary blood volume to stroke volume as an index of cardiac function in animals in man.

A scintillation probe was used to record a radiocardiogram as 99mTc flowed through the heart with the first pass after an intravenous injection. The ratio of cardiopulmonary blood volume to stroke volume, which could be determined indirectly from the simultaneous recording of a radiocardiogram and an electrocardiogram, was found to be a very sensitive and reliable index to describe the efficiency of the pump function of the heart with the different models of heart disease induced in experimental animals. It was found that left ventricular, right ventricular and biventricular failures were reflected by a large increase and pulmonary emboli by a decrease in the value of this index, which was named the cardiopulmonary flow index (CPFI). The technique has also been tested on a number of control and heart patients. The general principles found for animals could also be applied to man. As this is a noninvasive bedside technique it may have an important implication for the diagnosis of heart disease.

Purification and partial characterization of a 'short' insectotoxin-like peptide from the venom of the scorpion Parabuthus schlechteri.

A disulfide-rich, low-molecular-mass toxin-like peptide has been isolated from Parabuthus schlechteri venom using gel filtration, ion exchange, and reversed phase chromatography. Partial characterization of this peptide reveals a relationship with four-disulfide bridge proteins belonging to the family of 'short' insectotoxins (44% residue identity). In recognition hereof, the peptide was named PBITx1 (sITx10). Our work also reports on the deduced sequences of two other 'short' insectotoxins from Buthus eupeus, I3 and I4, and it provides a consensus sequence and nomenclature for all known 'short' insectotoxins. Finally, sequence similarities with K+ channel blockers (charybdotoxin, kappa-conotoxin), and a Cl- channel blocker (chlorotoxin) are highlighted.

Neurospecificity of phyto-bufadienolides is not related to differences in Na+/K+ pump inhibition.

The aim of the present study was to investigate the effects of neuro- (cumulative) and cardiotoxic (non-cumulative) bufadienolides originating from plants (phyto-bufadienolides) on the Na+/K+ pump current (Ip) in cardiac (rat and guinea pig) and dorsal root ganglion cells (guinea pig), and on Ca2+ currents in cardiomyocytes (guinea pig). All bufadienolides tested (non-cumulative drugs: thesiuside, tyledoside C; lanceotoxin B and tyledoside F for the neurotoxic group) were potent blockers of Ip at concentrations in the micro- and submicromolar range. K0.5 values for Ip inhibition in dorsal root ganglion neurones were slightly lower compared to cardiomyocytes, but the order of potency was similar in both cell types. Both classes of bufadienolides were equipotent in suppressing Ip, generated by high- and low-affinity pump isoforms. Phenomena related to pump inhibition, as hypercontracture and increase in T-type Ca2+ current in cardiomyocytes, were influenced to the same extent. Therefore, from these results, neurospecificity of some bufadienolides could not be explained by differences in Na+/K+ pump affinity.

Lafora-body disease with optic atrophy, macular degeneration and cardiac failure.

A 17-year-old boy was admitted to hospital in acute cardiac failure and psychosis. The clinical course, EEG records and tissue diagnosis, including biopsies of brain, skin, skeletal muscle, peripheral nerve and liver were compatible with Lafora-body disease (LBD). Unusual features were those of optic atrophy and macular degeneration, signs generally regarded as negative criteria for the diagnosis of this disease. We also present the findings on endomyocardial biopsy which was performed because cardiac failure as an early symptom of LBD has not been previously described. The patient died in status epilepticus a few months after discharge from hospital.

Comparison and characterization of the venoms of three Parabuthus scorpion species occurring in southern Africa.

Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion species occurring in southern Africa which can cause severe envenoming among people. In contrast to many other genera, no data is available on the venom composition of scorpions belonging to the genus Parabuthus. Here we have investigated the components which may contribute to the venomous potential. The constancy of venom composition within each of the three species and between the three species was investigated by means of gel filtration chromatography. The venoms of the three species each were characterized by a constant and typical elution pattern, resulting in a 'gel filtration fingerprint' which allows distinction between each species. It appears that certain components in the venoms are common to either all three species, or to two of the three species. This points to a clear interspecies relationship within the genus. We also describe the isolation and characterization of some of the polypeptide toxins present in the venoms of P. villosus, P. transvaalicus and P. granulatus by means of reversed phase chromatography and screening of the toxic components on voltage-activated potassium and sodium channels. Our results confirm that toxins which inhibit potassium channels and alter sodium channel gating are present in the venoms studied.

The biological activity of selected cyclic dipeptides.

Cyclic dipeptides are widely used as models for larger peptides because of their simplicity and limited conformational freedom. Some cyclic dipeptides have been shown to be antiviral, antibiotic and anti-tumour. The aim of this study was to determine the biological activity of four cyclic dipeptides synthesized in this laboratory: cyclo(L-phenylalanyl-L-prolyl), cyclo(L-tyrosyl-L-prolyl), cyclo(L-tryptophanyl-L-prolyl) and cyclo(L-tryptophanyl-L-tryptophanyl). The enhancement or inhibition of calcium channels in ventricular myocytes from rats and delayed-rectifier potassium channels in ventricular myocytes from guinea-pigs were determined by use of the whole-cell patch-clamp technique. The induction of differentiation in HT-29 cells was assessed by assaying for an increase in the expression of alkaline phosphatase. Antibiotic properties against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilus and Streptococcus sp. were determined by use of the Kirby-Bauer disc-diffusion assay. Results from these assays indicate that the cyclic dipeptides have biological activity in both prokaryotes and eukaryotes. Three of the dipeptides block cation channels in ventricular myocytes and all increase the expression of alkaline phosphatase. All the dipeptides have concentration-dependent antibacterial properties. These results suggest that with increased solubility the cyclic dipeptides might have potential as muscle relaxants, anti-tumour compounds and antibiotics.

A study on the function of some subcellular systems of the sheep myocardium during gousiekte. I. The energy production system.

In order to determine the status of the energy production system of the heart during cardiac failure of sheep with gousiekte, observations were made of the heart tissue levels of adenosine triphosphate (ATP), creatine phosphate (CrP), inorganic phosphate, reduced nicotine adenine dinucleotide (NADH) and lactate. Some measurements on oxidative phosphorylation were also made. A significant decrease in ATP and CrP levels coincided with a simultaneous rise in the ATP:CrP ratio and lactate levels in gousiekte hearts. No significant deviations in inorganic phosphate and NADH levels could be demonstrated. These abnormalities were accompanied by a decreased uptake of oxygen by isolated mitochondria of gousiekte hearts. There was a marked increase in the anaerobic state of the hearts of dying gousiekte sheep, while the values of NADH and the ATP:CrP ratio at a presymptomatic stage indicated a possible early derangement in the energy metabolism of sheep fed the toxic material. No hypertrophy could be detected for the failing ventricles of gousiekte sheep after being corrected for a significant amount of oedema found in the heart tissue of these animals. It was concluded that the depressed ATP and CrP levels in the heart tissue of gousiekte sheep during cardiac failure could at least in part, be attributed to a depressed aerobic energy production. It is not possible, however, to state whether this is a primary or a secondary response due to intoxication and also whether it could be seen as a cause or effect of cardiac failure.

A study on the function of some subcellular systems of the sheep myocardium during gousiekte. II. The contractile protein system.

Two groups of Merino sheep were intoxicated separately and at different times with "gousiektebossie" (Pachystigma pygmaeum) until definite symptoms of heart failure were auscultated. Cardiectomy was carried out and some ventricular muscle from 1 group was stored in 50% glycerol at -20 degrees C for about 4 months. Natural actomyosin (n-actomyosin) was subsequently extracted and tested for magnesium, calcium and adenosine triphosphate (ATP)-dependent adenosine triphosphatase (ATP-ase) activity as well as for superprecipitation characteristics. Muscle strips were taken from the other group and stored for 2 weeks in 50% glycerol at -20 degrees C, whereafter it was analysed for an isometric tension-calcium response. The data showed no difference between gousiekte and control sheep in the sensitivity of the contractile system to the activating effect of calcium ions with respect to isometric tension development. A significant reduction of the magnesium dependent ATP-ase was found for gousiekte n-actomyosin in either the presence or absence of calcium ions. A depressed sensitivity for this enzyme to increasing concentrations of ATP in comparison to controls was also found ([ATP] less than 1 mM, [MgCl2] = 1 mM). No significant difference could be detected in the sensitivity of the n-actomyosin:ATP-ase system to magnesium. n-Actomyosin:ATP-ase of gousiekte hearts revealed a depressed sensitivity to calcium ions. Gousiekte n-actomyosin also showed a significant depression in the rate of superprecipitation with a concomitant increase in the duration of the clearing phase. We conclude from these observations that a definite biochemical lesion is induced in the contractile proteins of heart muscle obtained from sheep intoxicated with "gousiektebossie" at the stage of cardiac failure. This condition is characterized by abnormal superprecipitation characteristics and a depressed n-actomyosin:ATP-ase activity, showing a reduced sensitivity to the activating effect of calcium ions.

The toxic effects on cardiac myocytes of tyledoside F, a cumulative neurotoxic cardiac glycoside isolated from Tylecodon grandiflorus.

To investigate the cardiac cellular effects of tyledoside F, a cumulative neurotoxic bufadienolide and ouabain, a non-cumulative cardenolide, the whole-cell clamp method was used to measure the Na-K pump current after the Na-K pump had been activated by high intracellular Na2+. The toxic effects of tyledoside F and ouabain on cardiac myocytes were also investigated by observing the effect of the Ca2+ overload on the variability of myocytes during a period of 75 min. From the results it is clear that there are similarities in the direct effects of tyledoside F and ouabain on the Na-K pump. It was found that ouabain inhibited the Na-K pump current more than that of tyledoside F. With regard to Ca2+ overload, there are differences in their mode of production of Ca2+ overload because cinnarizine protects the myocytes against ouabain-induced Ca2+ overload but not against tyledoside-induced Ca2+ overload. This study shows that with the whole-cell clamp technique tyledoside F inhibited the Na-K pump in a manner similar to inhibition of the pump by ouabain. Viability studies with myocytes indicated that tyledoside F also has other effects which are different from these of ouabain.

A comparison of haemodynamic and vasoconstrictory responses in sheep with a toxic fraction from Pachystigma pygmaeum and with the plant material.

ST-segment changes in the ECG, which are an indication of acute myocardial ischaemia, are obtained when a small quantity of an extract from dried Pachystigma pygmaeum is injected intravenously in sheep. When the fraction was injected subcutaneously, animals reached a crisis after about 5 h, with low values of stroke volumes and high values for pulmonary arterial pressures, pulmonary vascular resistances and heart rates. The haemodynamic changes are an indication of the development of pump failure of the heart. In sheep, injected subcutaneously with the toxic fraction, as well as for sheep dosed with plant material through rumen fistula, increased serum levels for thromboxane and increased or decreased levels for prostacycline were observed. The experimental results are interpreted as being an indication that these prostaglandines may be involved in the development of gousiekte by impeding cardiopulmonary function as a result of coronary and pulmonary vasoconstriction. The sudden death observed in some gousiekte sheep may be due to myocardial ischaemia and associated arrhythmias.

In vivo effects of a novel calcium antagonist (R56865) against induced epoxyscillirosidin and tulp poisoning in sheep.

Two anaesthetized sheep were intoxicated with epoxyscillirosidin, the main cardio-active bufadienolide, extracted from Homeria pallida (Natal yellow tulp). The epoxyscillirosidin was injected intravenously as a bolus of 50 micrograms/kg, followed 30 min later by a continuous infusion in a normal saline drip (0.9% NaCl) at 25 micrograms/kg/h. In addition, another two conscious sheep were poisoned by intraruminal dosing of 1,25 g/kg of dried H. pallida plant material. Electrocardiograms, heart and respiratory rates and venous-acid-base levels were recorded prior to and at approximately 30-60 min intervals during the course of the experiment. Additional recordings were made when animals showed signs of intoxication. R56865 (Janssen Pharmaceutica, Pty Ltd), a novel Ca++ antagonist, was administered at the first distinct signs of cardiac disturbances in the sheep given epoxyscillirosidin and after development of tachycardia and dyspnoea in those that received plant material. Activated charcoal was drenched at 3 g/kg to both sheep that received H. pallida about 1 h after the initial administration of R56865. All H. pallida sheep and one of the epoxyscillirosidin sheep survived. The signs of intoxication with H. pallida, namely groaning and tachypnoea, abated within minutes of treatment with R56865, but returned c. 30 min later in both animals. The treatment apparently had little effect on heart rate and EKG changes. One of the epoxyscillirosidin sheep was treated while exhibiting paroxysmal ventricular tachycardia. Although a transient improvement in conduction disturbance was recorded, the animal died soon afterwards. The results of this study indicate that the in vivo response of R56865 against induced bufadienolide cardiac disturbance in sheep is not as evident as that observed with R56865 against similar cardiac disturbance in vitro. The potential use of R56865 together with activated charcoal is discussed.

Effects of derivatives of NGP 1-01, a putative calcium channel antagonist, on electrically stimulated guinea-pig papillary muscle.

In earlier work we have reported calcium antagonistic properties for the polycyclic compound NGP 1-01. We now have derivatized NGP 1-01 by side-chain substitution to obtain ten novel aromatic and aliphatic imino-keto and amino-ether compounds. Electrophysiological tests were conducted on these compounds using isolated guinea-pig papillary muscle preparations to record calcium-mediated (slow) action potentials (APs). The lipophilicities of the compounds, expressed as chromatographically determined RM values, were measured and the molecular surface areas calculated. Several derivatives showed increased activity compared with NGP 1-01. All compounds with aromatic side-chains (benzyl, phenethyl, phenylpropyl) were active (concentrations required for complete suppression of the AP varied between 1 x 10(-5) M and 5 x 10(-5) M) and compounds with shorter (methyl, butyl) aliphatic side-chains were inactive whilst activity increased dramatically in those compounds with octyl side-chains. Lipophilicity and calculated molecular volumes correlated linearly and bulkier, more lipophilic molecules had increasing activities in the electrophysiological assay. We therefore conclude that bulky substituents on the nitrogen atom increase calcium antagonistic activity in this series of compounds.

Enantiomeric resolution of the calcium channel antagonist 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]unde cane (NGP 1-01).

We report the enantiomeric resolution and results of studies designed to probe possible enantiospecific calcium channel activity of 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undec ane (NGP 1-01), a known polycyclic benzylamino calcium channel antagonist. R-(-)-acetylmandeloylchloride was used to generate the two diastereomers which were separated by conventional column-chromatography. Hydrolysis of the separated amide diastereomers was achieved in 10% oxalic acid/dichloromethane with silica gel as catalyst and yielded the resolved enantiomers of NGP 1-01. Enantiomeric purity was assessed by in situ derivatization of the individual enantiomers with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (TAGIT) and HPLC analysis. A standard C18 column was used and the enantiomeric purity was found to be 98.5% and 96.63% for the (+)- and (-)-enantiomer, respectively. The enantiomers exhibited similar activity profiles, for calcium channel antagonism and also did not differ from the racemic mixture, suggesting that NGP 1-01 has very weak or no stereo-selectivity in the calcium channel assay used.

The ratio of cardiopulmonary blood volume to stroke volume as an index of cardiac function in horses.

A method was developed for determining the ratio of cardiopulmonary blood volume to stroke volume, in horses. The radioisotope 99 Tc (technetium 99m pertechnetate) was injected into the jugular vein as a bolus, which was then detected in the right and left ventricles consecutively by a scanning device consisting of a Na I crystal, a collimator, an amplifier and a discriminator. The radiocardiogram (RCG) and the ECG were recorded simultaneously by a two-channel writing device. The ratio of cardiopulmonary blood volume to stroke volume (cardiopulmonary flow index = CPFI) was then determined from the RCG and ECG tracings. Five categories of horses were examined, viz. Thoroughbreds in training, showjumpers in training, horses not in training, horses with cardiovascular disease and horses with chronic lung disease. The mean CPFI of the above categories were respectively 7.0 +/- 0.39, 7.3 +/- 0.45, 6.7 +/- 0.61, 9.8 +/- 1.30 and 6.2 +/- 0.47. The mean CPFI of the subjects with heart disease was significantly greater than the mean values of the other four categories (P less than 0.001). It was concluded that the CPFI was a reproducible physiological parameter in horses and that the value was significantly increased in our series of subjects with heart disease.

Development and optimization of a purification strategy for the venom of the scorpion Parabuthus transvaalicus.

In search for the toxic components constituting the venom of the southern African scorpion Parabuthus transvaalicus we compared and optimized several chromatographic separation protocols resulting in an efficient venom purification strategy. We found that the sequential combination of gelfiltration (Superdex) together with reversed phase chromatography (C2/C18)-but not cation exchange (Mono S)-stood surety for the best purification.