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Previously, from the plant Thymus armeniacus a new lignan sevanol was isolated, it's structure was elucidated and was shown that it effectively inhibits the acid-sensing channel ASIC3 and also exhibits a pronounced analgesic and anti-inflammatory effect. In this work biological activity of the sevanol analog obtained by chemical synthesis from simple precursors, the stereoisomer of sevanol and a precursor molecule represents a half of sevanol was measured in electrophysiological experiments on human ASIC3 channels expressed in Xenopus laevis oocytes. Measured inhibitory activity of a synthetic analogue coincided with the activity ofthe natural molecule. Stereoisomer showed inhibitory activity drop by about a third part, and the precursor molecule showed much less significant activity. In result the significance of functional groups and a spatial configuration of sevanol in order to biological activity was shown that is important to take into account for the optimal synthesis design as well as for new drugs development on its base.

Scorpion venom is a complex multicomponent mixture of biologically active substances, some of which possess very interesting properties and are used in quite unexpected fields. The family of chlorotoxin (CTX)-like peptides serves a good example. These toxins exhibit insecticidal activity, however, their molecular mechanism of action on insect organism remains elusive. Nevertheless, CTX-like peptides attracted considerable research effort due to their ability to specifically interact with cells of brain tumors, i.e. gliomas. In the future these compounds may considerably aid anticancer therapy. This review summarizes the results obtained during the past 40 years of CTX-like peptides investigation. Both biological function aspects and the applied field related to gliomas are considered.

Noninstrumental methods of qualitative rapid test for detection of staphylococcal enterotoxin A (SEA) in milk foods sample and brothof using immunochromatography (IC) and dot-assay has been developed. Monoclonal antibodies to SEA with colloidal gold forimmunochromatography; monoclonal antibodies to SEA with colloidal gold or biotinylated monoclonal antibodies and streptavidin-peroxidase conjugate for dot-assay were used to visualize the results. The detection limits, ng/mL: 10 (IC), 20 (dot-assay with antibody-colloidal gold), 10 (dot-assay with STR-HRP), 4 (ELISA). Time of assay, min: 25 (IC), 60 (dot-assay with antibody-colloidal gold), 70 (dot-assay with STR-HRPO, 150 (ELISA).

Polypeptide toxin pi-AnmTX Hcr 1b-1 with a molecular weight 4537 Da was isolated from the whole body extract of sea anemone by a multistage liquid chromatography. The BLAST search algorithm revealed homology of the novel toxin amino acid sequence to the group of the known sea anemone toxins including BDS and APETx with similarity less then 50%. The toxin pi-AnmTX Hcr 1b-1 inhibited the amplitude of the fast component of integral ASIC3 current in electrophysiological studies on receptors expressed in Xenopus laevis oocytes. The calculated IC50 value was 5.5 +/- 1.0 microM. Among the known polypeptide toxins interacted with ASICs channels, the micro-AnmTX Hcr 1b-1 toxin is the least potent inhibitor that in our opinion correlates with a small amount of charged amino acid residues in its structure.

Anthrax is the widespread acute infection disease, affecting animals and humans, refers to the bioterrorist threat agents of category A, because of the high resistance of Bacillus anthracis spores to adverse environmental factors and the ease of receiving them. We obtain a representative panel of 20 monoclonal antibodies against the key component of pathogenic exotoxins, anthrax protective antigen. Quantitative sandwich-ELISA for protective antigen with antibody obtained was developed. Six pairs of monoclonal antibodies showed the detection limit up to 1 ng/ml concentration of the protective antigen in blood serum.

One-step rapid immunochromatographic method for detection of diphtheria toxin in different water samples (phosphate buffer, milk, human nasopharyngeal swab) with the conjugate of monoclonal antibodies labeled with colloidal gold was developed. The limit of visible detection of the diphtheria toxin is 10 ng/ml and 15 min time analysis. The use of silver sensitivity enhancement and scanning equipment decreased the detection limit to 1.25 ng/ml.

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.

A novel antifungal peptide, LAMP-Ia, was isolated from sand-elymus (Leymus arenarius) seeds. Expression of a synthetic gene encoding this peptide in Escherichia coli cells was obtained. The target peptide was expressed as a fusion with thioredoxin. Identity of the recombinant peptide to native LAMP-Ia was confirmed by chromatography, mass spectrometry, and amino acid sequencing. LAMP-Ia displayed a high inhibitory activity in respect of a number of phytopathogenic fungi in in vitro assays, which opens up possibilities for the gene encoding it to be used for genetic transformation of plants and for engineering pathogen-resistant crops.

An electron microscopy study of large-sized DNA microparticles produced in PCR with different gene-specific primers and plasmid DNAs is described. DNA microspheres of two distinct types were revealed in the all studied samples, namely smooth moderately electron-dense microspheres, and highly electron-dense particles with large thorns and offshoots. Singular microspheres have the average diameter of 1 mum, and their aggregates were up to 3 mum in dimensions. In addition, rare so-called three-dimensional net-like structures with various size (up to several micrometers) were observed. They consisted of different amounts of DNA nanoparticles, having the special compact topology. In some studied samples the discs (nanodiscs) of several dozens nm in thickness and up to 3 mum in diameter were revealed. It was shown that the quantity of net-like structures and nanodiscs sharply increases in asymmetric PCR. We also observed DNA nanowires of different length and thickness, nanodots, nanoparticles in the form of shits of paper as well as electron-dense spherical nanoparticles of big size. Aqueous suspensions of DNA microparticles were heated at 94 degrees C for 5 min and analyzed by electron microscopy. It was shown that microspheres in heated suspensions underwent partial melting; they lost a part of DNA, therefore details of their structure (ultrastructure) can be recognized. At the some time numerous tangles of nanowires appeared. Molecular mechanisms of the DNA micro- and nanoparticles formation are discussed.

It has been previously found that in a PCR with yeast genomic DNA as a template, microparticles of condensed DNA are formed in the presence of KlenTaq polymerase. In the present work, the study of these microparticles was continued using electron microscopy. It was shown that along with standard electron-dense microspheres, microspheres of a low electron density with a few thorns or without any thorns are formed. Various types of nanoparticles were detected in the samples: nanowires, dot-like electron-absorbing particles (nanodots), and compact nanoparticles (nanoscales) of different shape and size. It was found that increasing the number of PCR cycles above the optimum leads to an abrupt rise in the amount of nanoparticles in the PCR mixture. Suspensions of microparticles after quick (5 min) heating at 94 degrees C were examined. The partial melting of the microspheres in the heated samples was established: they lost part of the DNA and decreased in size; simultaneously, abundant clusters of nanowires appeared. The effect of nuclease S1 on the DNA of microspheres was studied. The molecular mechanisms of the formation of micro- and nanoparticles are discussed.

Spider venoms are peculiar combinatory libraries of polypeptide molecules that specifically affect various cell targets. However, the question has remained up to now regarding whether the observed diversity of the polypeptides results from the synthesis of the complete library of these molecules by each individual spider or is due to the peculiarity of each zooid producing a limited set of components. We studied the composition of the mixed venom taken from several dozens of zooids of the Central Asian species of the Agelena orientalis and compared it with the venoms of 20 individual spiders of this species. The venoms were qualitatively and quantitatively analyzed by HPLC, mass spectrometry, and amino acid sequencing. It was shown that the individual venoms contain a lesser number of polypeptide components in comparison with the mixed venom and, in addition, differ from each other by the component composition. The set of components produced by single zooids is relatively narrow, and on the whole is a set identical to that of the mixed venom. The polypeptides with a high content in the venom were structurally characterized and compared with the amino acid sequences deduced from the cDNA library of this species.

Two new polypeptide components which exhibited an analgesic effect in experiments on mice were isolated from the Heteractis crispa sea tropical anemone by the combination of chromatographic methods. The APHC2 and APHC3 new polypeptides consisted of 56 amino acid residues and contained six cysteine residues. Their complete amino acid sequence was determined by the methods of Edman sequencing, mass spectrometry, and peptide mapping. An analysis of the primary structure of the new peptides allowed for their attribution to a large group of trypsin inhibitors of the Kunitz type. An interesting biological function of the new polypeptides was their analgesic effect on mammals, which is possibly realized via the modulation of the activity of the TRPV1 receptor and was not associated with the residual inhibiting activity towards trypsin and chymotrypsin. The analgesic activity of the APHC3 polypeptide was measured on the hot plate model of acute pain and was significantly higher than that, of APHC2. Methods of preparation of the recombinant analogues were created for both polypeptides.

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

A novel lipid transfer protein called Ec-LTP was isolated from resting caryopsis of weed barnyard grass Echinochloa crusgalli (L.) Beauv.; its molecular weight, amino acid content and N-terminal amino acid sequence were determined. Ec-LTP was a 9150 Da protein, containing eight cysteine residues, which formed four disulfide bonds. The isolated protein could significantly inhibit the development of pathogenic fungi Phytophthora infestans and Helminthosporium sativum, causing the late blight of potato and tomato and the root rot of herbs, respectively.

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.

A novel lipid-transporting protein (Ns-LTP1) has been isolated from seeds of the garden fennel flower Nigella sativa. The molecular mass, N-terminal amino acid sequence, and amino acid composition of the protein have been determined. Ns-LTP1 has a molecular mass of 9602 Da and contains eight cysteine residues which form four disulfide bridges. The protein is capable of suppressing the development of some phytopathogenic fungi and oomycetes.

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3-4 microm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg2+ cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.

Analogues of latarcins Ltc1 and Ltc3b, antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi capable of formation of amphiphilic structures in membranes without involvement of disulfide bonds, were synthesized. The amino acid sequences of the analogues correspond to immature forms of these peptides, each of them containing an additional C-terminal amino acid residue. It is concluded from the study of the biological activity of the synthesized peptides that the posttranslational C-terminal amidation of Ltc3b is a functionally important modification that ensures a high activity of the mature peptide. The lipid composition was shown to affect the interaction of synthesized peptides with artificial membranes. The analogue of Ltc3b manifested the highest activity on cholesterol-containing membranes. The mechanism of action of the studied antimicrobial peptides on membranes is discussed.

The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably permeate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.

The venom of South American ant Paraponera clavata and its low-molecular-mass fraction were shown to possess insectotoxic and pore-forming activities. A number of glycophospholipid components were isolated from this ant venom by means of gel filtration and reversed-phase chromatography. Some of the compounds cause conductivity fluctuations in lipid bilayer membranes within the ranges 3-25 pS and 200-400 pS at concentrations of 10(-6) to 10(-7) M. N-Acetylglucosamine, a fatty acid, and phosphoric acid residues were found in their structures. A full structure, 3-myristoyl-2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate, was elucidated for one of the compounds by the use of 1H, 13C, and 31P NMR spectroscopy and mass spectrometry.

An internal DNA fragment (approximately 2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5'- and 3'-ends (5'- and 3'-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (approximately 6000 nt) differ in the structures of both the sites encoding signal peptides and 5'-terminal untranslated regions. They encode large proteins (approximately 1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane regions followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severly truncated at their C-termini (approximately 900 aa). They are composed of only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3'-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.

A simple and rapid procedure for the preparation of yeast and fungal DNA samples useful in PCR amplification was developed. The DNA was purified from proteins, lipids, polysaccharides, and other impurities by their high-temperature extraction (in a boiling water bath) with buffer solutions containing chaotropic salts. Under these conditions, yeast and fungal cell envelopes remain unbroken and retain the original DNA and RNA that could be used for direct PCR amplification. We called the proposed PCR technique as the PCR using DNA-containing cell envelopes.

The overlapping fragments of the chromosomal DNA from black widow spider Latrodectus mactans carrying genes for high-molecular-mass protein neurotoxins, alpha- and delta-latroinsectotoxins (alpha-LIT and delta-LIT) and alpha-latrotoxin (alpha-LTX), were PCR-amplified and cloned. Restriction analysis of the PCR products showed that the distribution and sizes of the restriction fragments coincided with those deduced from the earlier sequencing of cDNAs of the corresponding genes. It thus followed that the alpha-LIT and delta-LIT genes are intronless. Along with our data on the structure of the alpha-latrocrustotoxin (alpha-LCT), this implies that the lack of introns is a common feature of the black widow spider genes encoding high molecular mass neurotoxins.

alpha-Latrotoxin (alpha-LTX) binding sites to functionally active monoclonal antibodies (MA) A4 and A24 were localized using three approaches: hydrolysis of the toxin followed by the N-terminal sequencing of immunoreactive peptides; the study of antibody interaction with several recombinant alpha-LTX fragments; and Western immunoblotting of synthetic overlapping peptides (6-8 aa) whose structures correspond to that of the immunoreactive alpha-LTX fragment. It was shown that the MA A4 epitope is located within the F234-M294 protein fragment and that of MA A24 interacts with the fragment 347FDKDIT352.

Desmosome-like contacts (DLC) in afferent chemical synapses of the Mauthner cells (MC) were investigated after application of low and high molecular mass peptide fractions 6 and 9, correspondingly, from the Central Asiatic black scorpion Orthochirus scrobiculosus. Besides, the DLC were examined in condition of a training induced morpho-functional stability of the MC (adaptation) mediated by transformation of actin monomers into polymers. In addition, the structure of DLC was studied after cytochalasin application which disrupts F-actin. Fraction 6 was shown to increase the length of DLC and osmiophily of fibrous material. Similar changes in DLC were caused by adaptation. Fraction 9 decreased the osmiophily of the fibrous material, made DLC asymmetric, but did not influence their length. Similar changes in DLC were seen also after cytochalasin D application. Taking into account our previous data on the role of F-actin in the MC functioning, which were obtained following specific pharmacological treatments, the similarity of ultrastructural changes in DLC after both adaptation and fraction 6 application, on the one hand, and after both cytochalasin D and fraction 9 application, on the other one, enabled us to suggest that these fractions may contain peptides able to exert influence of the actin cytoskeleton.

The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).

To study the structural variability of the Os3 neurotoxin from the venom of scorpion Orthochirus scrobiculosus inhabiting Uzbekistan and Turkmenia, a cloning method for the family of cDNAs that encode the highly homologous Os3-like polypeptides was developed. The Turkmenian scorpion venom was shown to contain a family of closely related Os3-like polypeptides. Mass-spectrometry indicated that crude venoms from scorpions in-habiting these two regions of Central Asia differ in the amino acid composition of their toxic components depending on the habitat.

A fragment of the structural gene of alpha-latrocrustotoxin, a new representative of latrotoxins from black widow spider venom, was cloned. The fragment (1191 bp) was obtained by means of PCR based on the data obtained by sequencing tryptic peptides of the toxin. The fragment codes for a 397-aa sequence. The encoded polypeptide is the C-terminal fragment of the toxin central domain that presumably contains a site responsible for the toxin species specificity. The structural similarity of this fragment to the corresponding fragments of other latrotoxins was studied.

The effect of long-term natural stimulation resulting in prolonged fatigue and structural disorders as well as the black scorpion poison influence on these processes were studied in gold fish Mauthner neurons (MN). The poison previously applied to MN significantly protects their function and structure from the stimulatory effect and subsequently allows the neurons to recover more fast. Appearance of numerous desmosome-like (actin-containing) contacts and proliferation of subsurface cisterns, the accumulators of calcium ions was noted immediately after the poison action and stimulation and one day later in afferent synapse ultrastructure. The poison interaction with neuronal actin, mediated by alteration of calcium-accumulating systems is probably one of the mechanisms of its protective effect.

Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.

Toxic components of the Latrodectus pallidus spider venom were isolated and characterized. The venom was shown to contain a toxin specific for mammals and at least one insectospecific toxin. Partial amino acid sequences of both toxins were determined, and their high structural homology with previously studied alpha-latrotoxin and alpha-latroinsectotoxin from L. mactans tredecimguttatus was found.

Hybridoma lines producing anti-idiotypic monoclonal antibodies (AImAbs) were prepared by fusing splenocytes of mice immunized with alpha-latrotoxin (LT) and P3-X63Ag8.653 myeloma line cells. AImAbs (1) bind to the rat brain synaptosomes, (2) do not affect the LT binding to the high-affinity receptor, and (3) do not react with LT in solution. The effect of AImAbs on the LT-induced 45Ca2+ influx into rat brain synaptosomes was studied. Some antibodies (6.6D11 and 11.7B7) were found to strongly inhibit this process. The result obtained indicate that the presynaptic membrane contains unidentified components interacting with LT. The distortion of the interaction of LT with these unknown components affects the LT-induced calcium influx into synaptosomes.

The possibility of the existence of a new type of ionotropic glutamate receptor differing from NMDA and non-NMDA receptors was confirmed by cloning cDNA for XenNR1, a channel-forming subunit of the glutamate receptor from the Xenopus laevis brain. Cotransfection of the HEK-293 cells with the cDNAs for XenNR1 and XenU1 (a non-NMDA receptor subunit from X. laevis brain) resulted in the biosynthesis of a functionally active heterosubunit receptor composed of NMDA and non-NMDA subunits. The functional co-expression of the XenNR1 and XenU1 cDNAs is the first direct evidence for the existence of a new type of glutamate receptor.

The structural gene of delta-latroinsectotoxin was cloned and its nucleotide sequence was determined. The gene contains an open reading frame of 3642 bp. The deduced amino acid sequence is homologous to the sequences of latrotoxins studied earlier.

Protein compositions of venoms of South-American stinging ants, Ectatomma tuberculatum, Paraponera clavata (subfamily Ponerinae), and "tangarana" were analyzed. The venom of E. tuberculatum displayed the most complex protein composition (more than 15 polypeptides). The water-soluble fraction of the venoms of P. clavata and "tangarana" contained acidic proteins (pI < 3.5 to 5.2), whereas the venom of E. tuberculatum contained predominantly basic proteins (pI 8 to > 9.5). N-Terminal residues and N-terminal sequences of a number of polypeptides were determined. High-molecular-mass polypeptides of the P. clavata venom slightly stimulated the ATPase activity of mitochondrial F1-ATPase. Low-molecular-mass nonprotein components of this venom significantly inhibited the ATPase activity of submitochondrial particles and F1-ATPase from bovine heart mitochondria. The venoms of E. tuberculatum and "tangarana" produced no effect on the ATPase activity.

A toxic principle of the Ectatomma tuberculatum ant venom called ectatomin was isolated. Ectatomin is a protein with molecular weight 7928 Da. Its complete amino acid sequence and spatial structure in aqueous solution were determined by protein chemistry methods and NMR spectroscopy techniques. Ectatomin contains two highly homologous polypeptide chains linked to each other by a disulfide bond. The chains consist of 37 and 34 amino acid residues with an internal disulfide bridge in each. In aqueous solution the molecule forms a bundle of four amphipathic alpha-helices. This toxin in a concentration of 0.05-0.01 mM forms potential dependent nonselective cation channels both in cell and artificial membranes. The channel is dimeric and the mechanism of its formation can be explained in terms of the spatial structure established.

A panel of monoclonal antibodies (mAb) against alpha-latrotoxin (LT) has been produced and their main characteristics have been determined. The influence of mAb on the functional effects of LT in synaptosomes from rat brain and on the channel formation in bilayer lipid membrane has been investigated. These mAbs do not inhibit binding of LT to rat synaptosomes but modify LT-receptor interaction in terms of LT's channel-forming and secretogenic effects. Antibodies A6 and A24 block these effects, whereas A4 partially preserves the secretogenic action of LT and completely blocks its channel-forming action. Only antibodies A15 affect the LT ability to form cationic channels in BLM, inducing considerable decrease in the frequency of the channel formation. These data and their analysis allow to identify several functional (and, probably, structural) domains of LT responsible for: 1) toxin-receptor interaction; 2) channel-forming and related calcium-dependent secretogenic effects; 3) calcium-independent secretogenic effects; 4) formation of cationic channels in the artificial lipid bilayer.

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.

alpha-Latrocrustatoxin, the crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus was radioactively labelled with Bolton-Hunter reagent to the specific activity of 160 Ci/mmol with retention of the biological activity. A highly specific binding of radioactive toxin on plasmatic membranes from the crayfish Astacus astacus nerve cells with Bmax = 0.04 pmol binding toxin/mg membrane protein and Kd = 0.7 x 10(-10) M was demonstrated.

The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.

A method of the isolation of a crustacea-specific neurotoxin from the venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and hydrophobic chromatography on Phenyl-Superose column has been developed. LD50 of the toxin has been elucidated.