Alexey V. Osipov

Magnetodielectric Substrates in Antenna Miniaturization: Potential and Limitations

We discuss patch antenna miniaturization using magnetodielectric substrates. Recent results found in the literature reveal that with passive substrates advantages over conventional dielectric substrates can only be achieved if natural magnetic inclusions are embedded into the substrate. This observation is revised and the physical background is clarified. We present a detailed discussion concerning magnetic materials available in the microwave regime and containing natural magnetic constituents. The effects of magnetic dispersion and loss are studied: constraints on the microwave permeability are used to estimate the effect of magnetic substrates on the achievable impedance bandwidth. Microwave composites filled with thin ferromagnetic films are considered as a prospective antenna substrate. We calculate the impedance bandwidth of a lambda/2-patch antenna loaded with the proposed substrate, and challenge the results against those obtained with conventional dielectric substrates. The results are verified using full-wave simulations, and it is shown that the radiation quality factor is strongly minimized with the proposed substrate even in the presence of realistic losses. Estimates for the radiation efficiency are given as a function of the magnetic loss factor

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins A PARADIGM FOR BIOLOGICAL ACTIVITY DIVERSIFICATION

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of α-cobratoxin (a long-chain α-neurotoxin) and heterodimers formed by α-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26–30 in polypeptide loop II of α-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the α-cobratoxin capacity to compete with α-bungarotoxin for binding to Torpedo and α7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that α-cobratoxin dimer not only interacts with α7 nAChR but, in contrast to α-cobratoxin monomer, also blocks α3β2 nAChR. In the latter activity it resembles κ-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric α3β2 nAChRs.

A new type of thrombin inhibitor, noncytotoxic phospholipase A2, from the Naja haje cobra venom

Thrombin is a key enzyme in the blood coagulation cascade and is also involved in carcinogenesis; therefore, its inhibitors are of fundamental and clinical importance. Snake venoms are widely used as sources of proteins that affect blood coagulation. We have isolated a new protein, called TI-Nh, from the Naja haje cobra venom. TI-Nh is a mixed-type inhibitor of thrombin (K(i) of 72.8 nM for a synthetic peptide substrate) and effectively inhibits thrombin-induced platelet aggregation with an IC(50) value of 0.2 nM. At concentrations up to approximately 50 nM, at which the thrombin-clotting time is substantially prolonged, TI-Nh exerts no detectable effects on both the intrinsic and extrinsic pathways of the coagulation cascade. It does not hydrolyze either fibrinogen or thrombin. Although TI-Nh bears structural features typical of group IB phospholipases A(2) (PLA(2)s), it possesses relatively weak enzymatic activity and is nontoxic to PC12 cells at concentrations up to 15 microM. Nevertheless, TI-Nh evokes neurite outgrowth in these cells at a concentration of approximately 1 microM, similar to cytotoxic snake PLA(2)s with strong enzymatic activity. TI-Nh is the first thrombin inhibitor found in the venom of the Elapidae snake family, and it is the first phospholipase shown to inhibit thrombin.

An unusual phospholipase A2 from puff adder Bitis arietans venom – a novel blocker of nicotinic acetylcholine receptors

The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A₂ (PLA₂s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²⁵I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC₅₀ being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC₅₀ of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA₂s from snake venoms. Indeed, the protein possessed high PLA₂ activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA₂ that contains 14 disulphide bonds and the first nAChR blocker possessing PLA₂ activity.

Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor.

α‐Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine‐binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α‐Conotoxins blocking muscle‐type or α7 nAChRs compete with α‐bungarotoxin. However, α‐conotoxin ImII, a close homolog of the α7 nAChR‐targeting α‐conotoxin ImI, blocked α7 and muscle nAChRs without displacing α‐bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α‐conotoxin ImII, its ribbon isomer (ImIIiso), ‘mutant’ ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [125I]‐α‐bungarotoxin from human α7 nAChRs in the cell line GH4C1 (IC50 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC50 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α‐bungarotoxin (Kd and IC50 2.5–8.2 μM). On Torpedo nAChR, α‐conotoxin [125I]‐ImII(W10Y) revealed specific binding (Kd 1.5–6.1 μM) and could be displaced by α‐conotoxin ImII, ImIIiso and ImII(W10Y) with IC50 2.7, 2.2 and 3.1 μM, respectively. As α‐cobratoxin and α‐conotoxin ImI displaced [125I]‐ImII(W10Y) only at higher concentrations (IC50≥ 90 μM), our results indicate that α‐conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.

cDNA cloning, structural, and functional analyses of venom phospholipases A2 and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi☆

Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom.

Dimeric α-Cobratoxin X-ray Structure LOCALIZATION OF INTERMOLECULAR DISULFIDES AND POSSIBLE MODE OF BINDING TO NICOTINIC ACETYLCHOLINE RECEPTORS

Background: α-Cobratoxin (αCT) dimer (αCT-αCT) has recently been discovered and found to bind both with α7 and α3β2 nicotinic receptors (nAChR). Results: αCT-αCT x-ray structure and intermolecular disulfides were established, intramolecular disulfides in central loops II were reduced, and interaction with distinct nAChRs was analyzed. Conclusion: Loop II disulfide is necessary for αCT-αCT binding to α7 but not α3β2 nAChR. Significance: Dimeric α-neurotoxins provide new means for distinguishing distinct nAChRs. In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys3 in one protomer and Cys20 of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys26 and Cys30 in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys26-Cys30 in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys26-Cys30 disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys26-Cys30 disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.

Cysteine-rich venom proteins from the snakes of Viperinae subfamily - molecular cloning and phylogenetic relationship.

Cysteine-rich proteins found in animal venoms (CRISP-Vs) are members of a large family of cysteine-rich secretory proteins (CRISPs). CRISP-Vs acting on different ion channels were found in venoms or mRNA (cDNA) encoding CRISP-Vs were cloned from snakes of three main families (Elapidae, Colubridae and Viperidae). About thirty snake CRISP-Vs were sequenced so far, however no complete sequence for CRISP-V from Viperinae subfamily was reported. We have cloned and sequenced for the first time cDNAs encoding CRISP-Vs from Vipera nikolskii and Vipera berus vipers (Viperinae). The deduced mature CRISP-V amino acid sequences consist of 220 amino acid residues. Phylogenetic analysis showed that viper proteins are closely related to those of Crotalinae snakes. The presence of CRISP-V in the V. berus venom was revealed using a combination of gel-filtration chromatography, electrophoresis and MALDI mass spectrometry. The finding of the putative channel blocker in viper venom may indicate its action on prey nervous system.

Nerve growth factor from cobra venom inhibits the growth of Ehrlich tumor in mice.

The effects of nerve growth factor (NGF) from cobra venom (cvNGF) on growth of Ehrlich ascites carcinoma (EAC) cells inoculated subcutaneously in mice have been studied. The carcinoma growth slows down, but does not stop, during a course of cvNGF injections and restores after the course has been discontinued. The maximal anti-tumor effect has been observed at a dose of 8 nmoles cvNGF/kg body weight. cvNGF does not impact on lifespan of mice with grafted EAC cells. K252a, a tyrosine kinase inhibitor, attenuates the anti-tumor effect of cvNGF indicating the involvement of TrkA receptors in the process. cvNGF has induced also increase in body weight of the experimental animals. In overall, cvNGF shows the anti-tumor and weight-increasing effects which are opposite to those described for mammalian NGF (mNGF). However in experiments on breast cancer cell line MCF-7 cvNGF showed the same proliferative effects as mNGF and had no cytotoxic action on tumor cells in vitro. These data suggest that cvNGF slows down EAC growth via an indirect mechanism in which TrkA receptors are involved.

Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.