Effects of intranasal administration of the peptide antagonist of type I vaniloid receptor (TRPV1) in the rodent central nervous system.

Intranasal administration of the polypeptide APHC3, an antagonist of the TRPV1 receptor, had acute anxiolytic and antidepressant effects, as well as an ability to modify the microglial response to proinflammatory stress and cytokine profile of the hippocampus. However, the acute antidepressant effect of the polypeptide was not related to the attenuation of neuroiflammation and probably had a different mechanism. The use of intranasal administration of the APHC3 peptide as a therapeutic approach aimed at decreasing depression symptoms needs additional studies in order to find the mechanism of action of this polypeptide in the central nervous system (CNS).

Effect of polypeptides from sea anemone Heteractis crispa on the rodent blood pressure, heart rate, and hemostasis.

АРНС1-3 peptides, modulators of TRPV1 receptors, have been administered to SD rats to study their influence on the animal hemostatic system, heart rate, and blood pressure. None of АЗРС1-3 polypeptides have any effect on the hemostatic system. Both АРНС1 and АРНС2 polypeptides increased significantly the heart rate, but they did not affect blood pressure, which was probably caused by an ability of these polypeptides to modify animal thermoregulation.

Biological activity of a polypeptide modulator of TRPV1 receptor.

This paper presents data on the activity of a new APHC2 polypeptide modulator of TRPV1 receptors, which was isolated from the sea anemone Heteractis crispa. It has been shown that APHC2 has an analgesic activity, does not impair normal motor activity, and does not change body temperature of experimental animals, which has a great practical value for design of potent analgesics of a new generation. Further study of the characteristics of binding of the polypeptide to the TRPV1 receptor may show approaches to the development of other antagonists of this receptor that do not influence the body temperature.

Diversity of Potassium Channel Ligands: Focus on Scorpion Toxins.

Potassium (K+) channels are a widespread superfamily of integral membrane proteins that mediate selective transport of K+ ions through the cell membrane. They have been found in all living organisms from bacteria to higher multicellular animals, including humans. Not surprisingly, K+ channels bind ligands of different nature, such as metal ions, low molecular mass compounds, venom-derived peptides, and antibodies. Functionally these substances can be K+ channel pore blockers or modulators. Representatives of the first group occlude the channel pore, like a cork in a bottle, while the second group of ligands alters the operation of channels without physically blocking the ion current. A rich source of K+ channel ligands is venom of different animals: snakes, sea anemones, cone snails, bees, spiders, and scorpions. More than a half of the known K+ channel ligands of polypeptide nature are scorpion toxins (KTx), all of which are pore blockers. These compounds have become an indispensable molecular tool for the study of K+ channel structure and function. A recent special interest is the possibility of toxin application as drugs to treat diseases involving K+ channels or related to their dysfunction (channelopathies).

[The Biological Activity of the Sevanol and Its Analogues].

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.

Structure of the yellow sac spider Cheiracanthium punctorium genes provides clues to evolution of insecticidal two-domain knottin toxins.

Yellow sac spiders (Cheiracanthium punctorium, family Miturgidae) are unique in terms of venom composition, because, as we show here, two-domain toxins have replaced the usual one-domain peptides as the major constituents. We report the structure of the two-domain Che. punctorium toxins (CpTx), along with the corresponding cDNA and genomic DNA sequences. At least three groups of insecticidal CpTx were identified, each consisting of several members. Unlike many cone snail and snake toxins, accelerated evolution is not typical of cptx genes, which instead appear to be under the pressure of purifying selection. Both CpTx modules present the inhibitor cystine knot (ICK), or knottin signature; however, the sequence similarity between the domains is low. Conversely, notable similarity was found between separate domains of CpTx and one-domain toxins from spiders of the Lycosidae family. The observed chimerism is a landmark of exon shuffling events, but in contrast to many families of multidomain protein genes no introns were found in the cptx genes. Considering the possible scenarios, we suggest that an early transcription-mediated fusion event between two related one-domain toxin genes led to the emergence of a primordial cptx-like sequence. We conclude that evolution of toxin variability in spiders appears to be quite different from other venomous animals.

[Development of methods for rapid test of staphylococcal enterotoxin A in food].

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).

[Chlorotoxin and related peptides are short insect toxins from scorpion venom].

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.

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.

[Polypeptide toxin from sea anemone inhibiting proton-sensitive channel ASIC3].

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.

Effect of the format of antibodies on their specificity.

The influence of alterations in the format of antibodies on their specificity has been examined. To analyze the role of Ig constant regions in recognizing antigens, a comparison was made of the specificities of full-scale murine monoclonal antibodies and scFv single-chain miniantibodies obtained from the latter with regard to a group of closely related protein antigens - Staphylococcus enterotoxins. It was found that in the scFv format the specificity and affinity of miniantibodies diminished as compared to the full-scale ones. Specificity of antibodies may be enhanced by transforming them into full-scale antibodies. Moreover it was shown that miniantibodies within a phage particle generated from combinatorial phage libraries possess greater specificity to the antigen, however during the subsequent transformation to soluble scFv antibodies their specificity diminishes.

[Preparation and characterization of monoclonal antibodies to Bacillus anthracis protective antigen].

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.

[Monoclonal antibodies to type A, B, E and F botulinum neurotoxins].

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.

[Monoclonal antibodies labeled with colloidal gold for immunochromatographic express analysis of diphtheria toxin].

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.

Simultaneous detection of seven staphylococcal enterotoxins: development of hydrogel biochips for analytical and practical application.

A method of simultaneous analysis of staphylococcal enterotoxins using hydrogel-based microarrays (biochips) has been developed. The method allows simultaneous quantitative detection of seven enterotoxins: A, B, C1, D, E, G, and I in a single sample. The development of the method included expression and purification of recombinant toxins, production of panels of monoclonal antibodies (mAbs) against the toxins, and design and manufacturing of an experimental biochip for the screening of mAbs and selection of optimal pairs of primary and secondary antibodies for each toxin. The selected mAbs have high affinity toward their targets and no cross-reactivity with unrelated enterotoxins. Finally, a diagnostic biochip was designed for quantitative analysis of the toxins, and the analytical protocols were optimized. The sensitivity of the detection reached 0.1-0.5 ng/mL, depending on the type of enterotoxin. The evaluation of the resulting biochip using spiked food samples demonstrated that the sensitivity, specificity, and reproducibility of the proposed test system fully satisfy the requirements for traditional immunoanalytical systems. The diagnostic biochips manufactured on reflecting metal-coated surfaces shortened the time of analysis from 17 to 2 h without loss of sensitivity. The method was successfully tested on samples of food and biological media.

[Condensed DNA particles formed in PCR with plasmid DNA: electron microscopy study].

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.