Biochemical characterization and insecticidal activity of isolated peptides from the venom of the scorpion Centruroides tecomanus.

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 µg/100 mg of cricket weight, but low amounts of peptides (0.8 µg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant ß-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.

The normal electrocardiogram of four species of conscious raptors.

The aim of this study was to describe normal ECG patterns and values in four species of conscious raptors (Eurasian kestrel, Griffon vulture, Little owl, and Eurasian Eagle owl). Electrocardiograms were carried out in 75 conscious birds belonging to four species of raptors. Lead II waveforms were analysed to determine amplitudes and durations of waves and intervals. Morphologic patterns of P-QRS-T deflections were analysed in the six limb leads. Rhythm, heart rate, mean electrical axis, presence of Ta wave, ST slurring, and P-on-T phenomenon were also studied. The influence of species, body weight and heart rate in electrocardiographic variables were statistically analysed (P < 0.05). Sinus rhythm was present in all tracings, showing sinus arrhythmia in four cases. Ta wave was present in six tracings and P-on-T phenomenon in four. ST segment could be identified in all tracings, being mainly high above baseline. Significant differences between species were found for all the electrocardiographic parameters. The heart rate and body weight were also found to be a significant influence in most parameters. This study provides electrocardiographic data for four species of raptors that can be used to establish comparisons for clinical purposes.

Interaction of ivermectin with gamma-aminobutyric acid receptors in Trichinella spiralis muscle larvae.

The value of the gamma-aminobutyric acid (GABA) receptor of nematodes as a target for ivermectin's mode of action remains unclear. Using binding assays, we examined extracts from Trichinella spiralis muscle larvae for the presence of [3H]-ivermectin and [3H]-GABA binding sites. Tissue preparations displayed affinity binding sites for [3H]-ivermectin with a dissociation constant (Kd) of 83 nM and a receptor density (Bmax) of 145 fmol/mg protein. We also identified a specific [3H]-GABA binding activity with a Kd of 1.2 microM and a Bmax of 4.78 pmol/mg protein. In competition studies, ivermectin was found to be a competitive inhibitor of specific [3H]-GABA binding activity with an inhibition constant (K(i)) of 3.39 nM, suggesting that GABA receptors could be implicated in the mechanism of action of ivermectin in nematodes.

Characterization of levamisole binding sites in Trichinella spiralis.

Characterization of the levamisole receptor was performed with total extracts of Trichinella spiralis muscle larvae using binding assays with tritiated levamisole ([3H]LEV, 291 GBq/mmol). We detected a specific [3H]LEV binding activity with a dissociation constant (Kd) of 4.76 microM and a receptor density (Bmax) of 2.14 pmol/mg of protein. In inhibition studies, only dimethylphenylpiperazinium iodide (DMPP) and hexamethonium were found to be competitive inhibitors of the [3H]LEV binding with an inhibition constant (Ki) of 31.04 and 4.43 microM, respectively, whereas d-tubocurarine and alpha-bungarotoxine had no effect on [3H]LEV binding activity, and procaine and atropine potentiated the [3H]LEV-receptor binding. All these data support the idea that levamisole acts as a cholinergic agonist in T. spiralis.

Levamisole binding sites in Haemonchus contortus.

Larval and adult extracts from isolates of Haemonchus contortus were assayed for specific [3H]levamisole binding activity. All of the tissue preparations displayed [3H]levamisole binding sites. The sensitive isolate SE and resistant isolate RJ showed no differences in larval and adult binding data. Larval SE extracts had higher receptor density (Bmax = 648 fmol mg-1) and dissociation constant (Kd = 1.28 microM) for [3H]levamisole than larval extracts of the American isolate RUSA (Bmax = 87 fmol mg-1 and Kd = 0.15 microM). Extracts of adult SE and RUSA isolates contain as much as 327 fmol mg-1 of protein and 205 fmol mg-1 of protein, respectively, and similar dissociation constants (Kd = 0.77 microM and Kd = 0.81 microM, respectively). There was a good correlation between specific binding activity of larval and adult extracts in both SE and RUSA isolates. The nicotinic cholinergic antagonist alpha-bungarotoxin had no effects in either isolate on [3H]levamisole binding activity. The results confirm that levamisole acts at a cholinergic receptor in H. contortus, and suggest that target site modification could be involved in the development of levamisole resistance.