Comparative Study of the Effect of Snake Venoms on the Growth of Ciliates Tetrahymena pyriformis: Identification of Venoms with High Antiprotozoal Activity.

To search for compounds with antiprotozoal activity, effects of snake venoms on the ciliates Tetrahymena pyriformis was studied. T. pyriformis from subkingdom of Protozoa, including the protozoal pathogens, was used as a model organism to select the venoms that are the most active against parasitic protozoans. Various concentrations of venoms were added to the cells, and the cells that survived after 24 h were counted. Among the six snake species from the Viperidae family, the venom of the viper Vipera berus, which completely killed the cells at 49 µg/mL, was the most active. Among four species from the Elapidae family, the previously studied cobra venoms containing cytotoxins with strong antiprotozoal activity as well as the venom of krait Bungarus multicinctus (10 µg/mL) were the most active. The venoms of the pit vipers and Nikolsky's viper did not show any activity at 12.5 mg/mL. Thus, the venoms of V. berus and B. multicinctus are promising for the isolation of new antiprotozoal compounds.

New Plant Species Showing Antiprotozoian Activity.

The effects of extracts of ten plant species from Russia and five species from Vietnam on the growth and survival of ciliates Tetrahymena pyriformis were studied. T. pyriformis belongs to the subkingdom Protozoa, which also includes pathogens of protozoan infections. Extraction of dried plants was carried out with acidic and alkaline aqueous solutions, as well as with an aqueous ethanol. Various amounts of extracts were added to the ciliate cells, and the number of cells survived after incubation for 1 and 24 h was recorded. We found that our samples of several plants, including wormwood, harmala, and licorice, similarly to those studied earlier, exhibit antiprotozoal activity, which may indicate that the secondary metabolites are the same in plants from different regions. Using the ciliate T. pyriformis as a model organism, the presence of antiprotozoal activity in extracts of lilac, chondrilla, cinquefoil, hop, and elm was shown for the first time.

Dimeric Disintegrins from the Steppe Viper V. ursinii Venom.

Four dimeric disintegrins were isolated from the venom of the steppe viper V. ursinii using liquid chromatography. Disintegrins prevented adhesion of MCF7 cells to fibronectin, which indicates their interaction with integrin receptors of the αVß1 type. According to mass spectrometry data, the molar masses of disintegrins are about 14 kDa. The method of peptide mapping established the structure of a new heterodimeric disintegrin weighing 13 995.5 Da and shows that it belongs to the class of RGD/KGD-containing disintegrins.

Encapsulation of Neurotoxins, Blockers of Nicotinic Acetylcholine Receptors, in Nanomaterials Based on Sulfated Polysaccharides.

Three-finger snake neurotoxins are selective antagonists of some nicotinic acetylcholine receptor subtypes and are widely used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective blocker of muscle-type nicotinic acetylcholine receptor. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.

New paradoxical three-finger toxin from the cobra Naja kaouthia venom: Isolation and characterization.

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of "orphan" toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.

Arginine derivatives of dicarboxylic acids from the parotid gland secretions of common toad Bufo bufo-New agonists of ionotropic γ-aminobutyric acid receptors.

Compounds activating γ-aminobutyric acid type A receptor were isolated from the toad Bufo bufo venom as a result of chromatographic separation. Analysis of the structure of these compounds by mass spectrometry and nuclear magnetic resonance showed that they are arginine derivatives of dicarboxylic acids and represent suberylarginine, pimeloylarginine, and adipoylarginine.

N-methyl serotonin analogues from the Bufo bufo toad venom interact efficiently with the α7 nicotinic acetylcholine receptors.

Two low-molecular-weight compounds were isolated from the parotid gland secret of the toad Bufo bufo, which by absorption spectra and HPLC-MS/MS chromatography data correspond to di- and trimethyl derivatives of serotonin (5-hydorxytryptamine): bufotenine (confirmed by counter synthesis) and bufotenidine (5-HTQ). In experiments on competitive radioligand binding, these compounds showed a higher affinity and selectivity for neuronal α7 nicotinic acetylcholine receptors compared with the muscular cholinergic receptors. The most efficient compound in terms of binding value was bufotenine, the efficiency of 5-HTQ was an order of magnitude lower, and the minimal activity was exhibited by serotonin.

Nonconventional three-finger toxin BMLCL from krait Bungarus multicinctus venom with high affinity interacts with nicotinic acetylcholine receptors.

Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

[Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

[New weak toxins from the cobra venom].

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by muscle nicotinic cholinoreceptors and alpha7 nicotinic cholinoreceptors.

First tryptophan-containing weak neurotoxin from cobra venom.

With the purpose of studying structure-function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated alpha-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.

Muscarinic toxin-like proteins from cobra venom.

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.

[MALDI-mass spectrometry for identification of new proteins in snake venoms]. / MALDI-mass-spektrometriia dlia identifikatsii novykh belkov v iade zmei.

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7-25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.