Carlos Sevcik

Relationship between plasmatic levels of various cytokines, tumour necrosis factor, enzymes, glucose and venom concentration following Tityus scorpion sting.

A sandwich enzyme-linked immunosorbent assay was developed for measuring Tityus venom levels in plasma. The method proved capable of distinguishing patients with only local symptoms from controls, and was used to quantify venom in 205 accidental human envenomations. Our results show that the severity of envenoming is related to the patient plasma venom concentration. This depends on time elapsed between the sting and when the plasma was drawn. We observed that 46 and 49% of patients with moderate to severe symptoms (MS, n=41) showed hyperamylasemia and hyperglycemia, respectively. In addition, 39% of cases with MS symptoms had partial thromboplastin time values prolonged or shorted and 6.5% of patients with local symptoms (LS, n=164) had only prolonged prothrombin time values. Interleukin 6 (IL6) increased significantly in patients with MS symptoms. IL6 values increased with hyperamylasemia, envenoming severity and time hyperamylasemia.

Molecular cloning and nucleotide sequence analysis of genes from a cDNA library of the scorpion Tityus discrepans.

Tityus discrepans is a Venezuelan scorpion known to cause severe human envenomations. It contains toxins that impair proper ion channels function, affect coagulation pathways and interfere with the immunological system, leading to a widespread inflammatory syndrome. This communication reports the results of genes cloned from a cDNA expression library of venomous glands from T. discrepans. A full-length cDNA phagemid library was prepared from which 127 genes were cloned and grouped in 22 clusters showing more than one EST (expressed sequence tag) (74%), and 29 singlets (26%). The identified putative proteins were assorted into two groups. One conformed by precursors similar to gene products implicated in common cellular processes, accounting for 13.4% of transcripts and other comprising putative toxins, representing 50% of total ESTs. A total of 14 sequences are thought to be peptides that recognize or affect Na(+)-channel function and 6 peptides that affect K(+)-channels. Among these two classes of venom components are several for which the peptides were previously isolated and characterized. However, based on sequence similarities, three distinct classes of peptides were also identified and are reported: a bradykinin-potentiating peptide, a defensin-like peptide and an acidic peptide of unknown function. The N-terminal amino acid sequence of several peptides is reported here for the first time. A phylogenetic tree analysis is also reported, as well as three three-dimensional models of representative toxins.

Tityus discrepans venom produces a respiratory distress syndrome in rabbits through an indirect mechanism

It is well known that scorpion venom induces lung lesions and respiratory distress which are usually classified as pulmonary oedema (PO). Tityus discrepans is a scorpion that lives in the north-central area of Venezuela, is the most common source of human envenomation here and produces PO. We studied the action of the venom of Tityus discrepans on whole rabbits and on their isolated lungs perfused with Krebs saline with 1 g/l of bovine serum albumin (Krebs-BSA saline). Two milligram of venom were diluted in 250 ml of solution (approximately the rabbits total blood volume) and used to perfuse isolated lungs. Lung oedema occurred in rabbits which received 1 mg/kg of scorpion venom i.p., heparin prevented the production of this lung oedema. T. discrepans venom produced PO, in rabbits pretreated with 15 mg/kg of ajoene. Yet, Tityus venom had no effects on isolated lungs perfused with citrated or heparinized blood, and in lungs perfused with Krebs-BSA with normal Ca2+. These result show that Tityus venom does not act directly on lungs. Otherwise, we have observed that abundant microthrombi occurred in all rabbit lungs exposed to venom in vivo, suggesting that these clotting alterations are fundamental to produce PO. The presence of intravascular microthrombi is not characteristic of the usual PO hinting that scorpion venom induced pulmonary alterations are a different clinical entity. We thus propose that the use of the term pulmonary oedema in scorpionism should abandoned in favor of scorpion venom respiratory distress syndrome.

Antibacterial activity of six novel peptides from Tityus discrepans scorpion venom. A fluorescent probe study of microbial membrane Na+ permeability changes.

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1-6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2-6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of gram positive and gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 microM depending on the bacteria and peptide tested. Effects on membrane Na(+) permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 microM CoroNa Red. CoroNa Red fluorescence leakage from bacteria was observed after exposure to 0.3 microM of any bactridine tested, indicating that they modified Na(+) membrane permeability. This effect was blocked by 10 microM amiloride and by 25 microM mibefradil drugs that affect Na(+) and Ca(2+) channels respectively. We found no evidence of changes of K(+) or Ca(2+) concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 microM) and PBFI (20 microM).

Subtype specificity interaction of bactridines with mammalian, insect and bacterial sodium channels under voltage clamp conditions.

The present work demonstrates that bactridines (Bacts) possess different selectivities for neuronal and muscular voltage‐dependent sodium (NaV) channels, with subtle differences on channel isoforms. Bacts 2, 3, 4, 5 and 6 (100 nm) reduced the peak current of several skeletal and neuronal channel isoforms selectively. Bacts 2 and 3 were more potent on NaV1.4, Bacts 4 and 6 on NaV1.3 and Bact 5 on NaV1.7. Bactridines (except Bacts 1 and 5) caused a hyperpolarizing shift in the V1/2 of activation and inactivation of NaV1.3, NaV1.4 and NaV1.6. Voltage shifts of Boltzmann curves fitted to activation and inactivation occurred with a decrease in κ. Since the slope is proportional to κ = RT/zF, changes in κ probably express changes in z, the valence, in a voltage‐dependent manner. Changes in z may express toxin‐induced changes in the channel ionic environment, perhaps due to surface charges of the molecules. Bact 2 induced a NaV1.2 voltage shift of the activation curves but no shift of the mutant NaV1.2 IFM/QQQ; peak INa was reduced in both channel forms, suggesting that channel blockage resulted from toxin binding to a site partially distinct from the α subunit binding site 4. Bactridines emerge as potential research tools to understand sodium channel isoform structure–function relationships and also as pharmacologically interesting peptides.

The presynaptic effect of fractions isolated from the sponge Tedania ignis

Two fractions from the sponge Tedania ignis which have presynaptic effects were isolated. In neuromuscular junctions of Rana pipiens, the crude fraction decreases the amplitude of the evoked endplate potential and increases the frequency of miniature endplate potentials (MEPP), without effects on their amplitude or shape. Elution with 1 M acetic acid through Sephadex G15 produces three peaks, only one of which contains the biological activity. The compounds in this peak possess a molecular weight close to 900. Elution with 1M acetic acid through BioGel P2 produces 9 peaks, only 2 of which are biologically active. One of these fractions (f alpha) increases the frequency of MEPP and another fraction (f beta) inhibits the evoked release of neurotransmitter. None of the fractions change the amplitude or shape of MEPP, nor do they modify the resting membrane or action potentials in frog muscle. The action of f alpha occurs in low (15 microM) Ca2+, while the effect of f beta is antagonized by raising the extracellular Ca2+ concentration above 1.8 MM. Fraction beta antagonizes the increase of acetylcholine release produced by the venom of the black widow spider Latrodectus mactans mactans. This antagonistic action of f beta is reversible and the effect of the spider venom reappears if the sponge toxin is washed out with normal Ringers solution.

LD50 determination: Objections to the method of Beccari as modified by Molinengo

The efficiency of the method of Beccari as modified by Molinengo is compared with the sequential technique of Dixon and Mood. It is concluded that the sequential technique is less constrained, gives estimates that are less disperse and is simpler to analyze statistically than the regression of Beccari-Molinengo. Both techniques are comparable in the number of animals required, as well as in the time invested in each assay.

Identification of Enterobacter bacteria as saxitoxin producers in cattle's rumen and surface water from Venezuelan Savannahs

We have previously shown that a paralytic toxin able to block sodium channels in nerve is associated with a cattle disease known as bovine paraplegic syndrome (BPS) [Toxicon. 31 (1993) 1581]. We have now identified this as saxitoxin (STX) using HPLC by either the methods of [Toxicon. 31 (1993) 1581], or [Toxicon. 25 (1987) 1105]. In recent experiments we were able to collect and cultivate facultative anaerobic bacteria growing on rumen, grass and ponds of corrals with high incidence of BPS; the cultured bacteria produce compounds indistinguishable from STX under both HPLC procedures described above. Two species of the Enterobacter genus (E. asburiae and E. cloacae) and a strain of Klebsiella pneumoniae, were identified using standard biochemical criteria as well as gas chromatography of bacterial lipids. All these bacteria produced STX in aerobic cultures.

Blockage of resting potassium conductance in frog muscle fibers by a toxin isolated from the sponge Haliclona viridis

A fraction able to irreversibly depolarize sartorius muscle fibers was isolated from the marine sponge. The resting potential is decreased from -84 (-85, -83) mV (median and its 95% confidence interval) to -40 (-46, -30) mV. The fibers depolarized by the sponge toxin are restored to -54 (-57, -49) mV when external sodium is replaced by Tris or to -52 (-57, -47) mV when calcium is removed from the saline solution in the presence of 1 mM EDTA and 1 microM tetrodotoxin. Tetrodotoxin alone (1 micron) has no effect on the depolarization [-43 (-50, -37) mV] produced by the sponge and 5 mM manganese only repolarizes the fibers to -48 (-55, -40) mV. The depolarization is potentiated [-28 (-33, -23) mV] when chloride is replaced by glutamate in the external solution. The access resistance of the muscle fibers is not significantly changed from its control value of 2.74 (2.28, 3.30) M omega when toxin is added. By contrast 20 mM K+ superfused to the fibers changes membrane potential to -44 (-46, -42) mV and decreases access resistance to 1.99 (1.38, 2.87) M omega. The toxin is devoid of any effect on the endplate, since the depolarization of the postsynaptic membrane is identical to the extrajunctional area, and miniature endplate potentials of normal shape and high frequency are easy to record from toxin treated fibers. The action potential is not modified by the toxin. The toxin is a small polar compound insoluble in acetone and is likely to act on a receptor located on the outer phase of the membrane. The biological activity appears as a peak on elution with 1 M acetic acid on Bio Gel P2.

Fibrin(ogen)olytic enzymes in scorpion (Tityus discrepans) venom

Several fibrin(ogen)olytic enzymes from Tityus discrepans (Buthidae, Buthoidea) venom (TdV) were partially purified on a Sephadex G-50 column, by affinity and molecular exclusion high-performance chromatography. Fractions SB1-I and SB1-II had fibrinolytic, fibrinogenolytic (Aα-chains degradation) and tissue plasminogen activator (t-PA)-like activities. SB1-III was only fibrinogenolytic (fast degradation of Aα-chains and slower degradation of fibrinogen Bβ-chains). These results showed the presence of α-fibrinogenases in TdV. The fibrino(geno)lytic activity in these fractions was abolished by metalloprotease inhibitors (MPI). Fractions SB3-I and SB3-II contain fibrinogenolytic (Aα-chains degradation) and fibronectinolytic activities. Also fraction SB3-I had a t-PA-like activity. Activities in SB3-I and SB3-II were abolished by serine protease inhibitors (SPI). None of the fractions degraded fibrinogen γ-chains. Fibrinogen degradation by active fractions is associated with an anticoagulant effect supported by a reduced coagulant activity. The overall outcome suggests that metalloproteases and serine proteases in TdV are responsible for fibrin(ogen)olytic activity because MPI and SPI inhibited these activities.