Synthetic Toxin Research Articles

Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae.

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.

Natural and Selected Synthetic Toxins: Biological Implications Edited by A. T. Tu (Colorado State University) and W. Gaffield (U.S. Department of Agriculture). American Chemical Society, Washington D. C. 2000. xi + 426 pp. 15 × 22.5 cm. $140.00. ISBN 0-8412-3630-5.

ADVERTISEMENT RETURN TO ISSUEPREVBook ReviewNEXTNatural and Selected Synthetic Toxins: Biological Implications Edited by A. T. Tu (Colorado State University) and W. Gaffield (U.S. Department of Agriculture). American Chemical Society, Washington D. C. 2000. xi + 426 pp. 15 × 22.5 cm. $140.00. ISBN 0-8412-3630-5.Frank R. StermitzView Author Information Department of Chemistry Colorado State University Fort Collins, Colorado 80523Cite this: J. Nat. Prod. 2001, 64, 4, 552Publication Date (Web):March 2, 2001Publication History Published online2 March 2001Published inissue 1 April 2001https://doi.org/10.1021/np000746aCopyright © 2001 American Chemical Society and American Society of PharmacognosyRIGHTS & PERMISSIONSArticle Views98Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (13 KB) Get e-AlertsSUBJECTS:Cell signaling,Organic compounds,Toxicity,Toxicology,Toxins Get e-Alerts

Long‐term potentiation in the presence of NMDA receptor antagonist arylalkylamine spider toxins

The role of the NMDA receptor (NMDAR) in long-term potentiation (LTP) is now well established. All potent NMDAR antagonists known to date inhibit the induction of LTP at the Schaffer collateral-CA1 pyramidal cell synapse in rat hippocampus, regardless of their site and mechanism of action. Arylalkylamine toxins are noncompetitive NMDAR antagonists in the mammalian central nervous system (CNS). The synthetic toxins argiotoxin-636 (Arg-636), Joro spider toxin (JSTX-3), α-agatoxin-489 and -505 (Agel-489 and Agel-505) and philanthotoxin-433 (δ-PhTX) were found in the present study to have no effect on the induction of LTP in the Schaffer collateral-CA1 pyramidal cell pathway in rat hippocampal slices maintained in vitro. Arylalkylamine toxins represent a class of potent NMDAR antagonists that fail to affect hippocampal LTP, and thus provide novel structural leads for the development of NMDAR antagonists that do not impair cognition. Excitatory amino acid (EAA) antagonists, especially those targeting the N-methyl-D-aspartate receptor (NMDAR), have been the subject of intensive research over the past decade. It is anticipated that such agents will prove therapeutically useful in a wide variety of CNS disorders, including epilepsy, ischemic stroke, acute neural trauma, and neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. A major drawback of all such compounds examined to date, be they competitive or noncompetitive antagonists, however, is their deleterious side effects such as impairment of cognition or psychotomimetic symptoms (Willetts et al., 1990). J. Neurosci. Res. 62:177–185, 2000. © 2000 Wiley-Liss, Inc.

Chemical engineering of a three-fingered toxin with anti-α7 neuronal acetylcholine receptor activity

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ¿i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.

Long-term potentiation in the presence of NMDA receptor antagonist arylalkylamine spider toxins.

The role of the NMDA receptor (NMDAR) in long-term potentiation (LTP) is now well established. All potent NMDAR antagonists known to date inhibit the induction of LTP at the Schaffer collateral-CA1 pyramidal cell synapse in rat hippocampus, regardless of their site and mechanism of action. Arylalkylamine toxins are noncompetitive NMDAR antagonists in the mammalian central nervous system (CNS). The synthetic toxins argiotoxin-636 (Arg-636), Joro spider toxin (JSTX-3), alpha-agatoxin-489 and -505 (Agel-489 and Agel-505) and philanthotoxin-433 (delta-PhTX) were found in the present study to have no effect on the induction of LTP in the Schaffer collateral-CA1 pyramidal cell pathway in rat hippocampal slices maintained in vitro. Arylalkylamine toxins represent a class of potent NMDAR antagonists that fail to affect hippocampal LTP, and thus provide novel structural leads for the development of NMDAR antagonists that do not impair cognition.

Construction and characterisation of Salmonella typhimurium aroA simultaneously expressing the five pertussis toxin subunits

Pertussis toxin (PT) is a major protective antigen of Bordetella pertussis, the causative agent of whooping cough. Current vaccines against whooping cough are administered parenterally, and generate a systemic immune response. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunisation with live vaccine strains of Salmonella spp. We have individually expressed the five PT subunits in Salmonella typhimurium aroA and subsequently expressed a synthetic pertussis toxin operon (pDP16), via the use of a temperature inducible expression system. In S. typhimurium aroA containing individual subunit expression plasmids the S5 subunit was found to be completely processed, while all other subunits were partially processed. In S. typhimurium aroA (pDP16) S5 was completely processed, S1 was partially processed, whilst S2, S3 and S4 remained unprocessed. Induction of the synthetic PT operon decreased the in vitro invasiveness of S. typhimurium aroA, compared to vector-only and plasmid-less strains. Following oral immunisation of Balb c mice with S. typhimurium aroA (pDP16), statistically significant IgG ( p < 0.05) specific to PT was detected in the serum of mice. Nonetheless, despite a statistically significant anti-PT serum antibody response in immunised mice, vaccination with S. typhimurium aroA (pDP16) failed to protect mice from virulent challenge.

Las neurotoxinas naturales como herramientas farmacológicas para el estudio del sistema nervioso central

Natural venoms produced by different species of animals are very useful to distinguish between the different types and subtypes of ionic channels and neurotransmitters receptors involved in the information processing in the nervous system. Natural venoms permit distinction between the different types and subtypes of Na+, K+ and Ca2+ channels. These differences are based on their structure, physical and chemical characteristics and function with regard to ionic transport and the neurotransmitters release. The use of different molecular biology techniques makes it possible to develop new synthetic toxins, by means of which new perspectives appear for the study and treatment of different neurological disease.

Design, synthesis, and biological evaluation of spider toxin (argiotoxin-636) analogs as NMDA receptor antagonists.

Twelve synthetic spider toxin analogs were prepared in an effort to better understand the structure-activity relationships of the polyamine portion of argiotoxin-636 (Arg-636), a noncompetitive NMDA receptor (NMDAR) antagonist. The 1,13-diamino-4,8-diazatridecane portion of the side chain of Arg-636 was systematically modified in an effort to further our knowledge of the structural requirements for the alkyl linker spacing between the amine nitrogens. Systematic isosteric replacement of each of the amine nitrogens in the polyamine moiety with either oxygen or carbon provided a series of compounds which were evaluated in vitro for NMDAR antagonist activity. One-half of the heteroatoms found in Arg-636 were removed to provide analogs which maintained in vitro potency below 1 microM. However, these simplified analogs produced similar or more pronounced effects on the cardiovascular system than Arg-636 in vivo. In this set of analogs, a minimum of three basic nitrogens in the side chain was required for maximum potency as NMDAR antagonists. Isosteric nitrogen substitutions in the polyamine chain reduced the in vitro potency of these analogs. An analog binding-conformation model was proposed to rationalize the inactivity of these isosterically substituted analogs.

Chick cerebellar purkinje cells express ω-conotoxin GVIA-sensitive rather than funnel-web spider toxin-sensitive calcium channels

Voltage-gated calcium channels form a complex family of distinct molecular entities which participate in multiple neuronal functions. In cerebellar Purkinje cells these channels contribute to the characteristic electrophysiological pattern of complex spikes, first described in birds 7 and later in mammals. 9 A specific calcium channel, the P-type channel, 10 has been shown to mediate the majority of the voltage-gated calcium flux in mammalian Purkinje cells. 13, 25 P-type channels play an essential role in synaptic transmission of mammalian cerebellum. 15, 16, 20, 22 It is unclear whether the P-type calcium channel is present in birds. Studies in chick synaptosomal preparations show that the pharmacological profile of calcium channels is complex 1, 12 and suggest a minimal expression of the P-type channel in avian central nervous system. In the present work, we studied voltage-gated calcium channels in dissociated chick cerebellar Purkinje cells to examine the presence of different calcium channel types. Purkinje cells were used because, in mammals, they express predominantly P-type channels and because the morphology of these cells is thought to be phylogenetically conserved. 21 We found that ω-conotoxin GVIA ω-CgTx GVIA):, a specific antagonist of N-type calcium channel, rather than the synthetic funnel-web spider toxin (sFTX), a P-type channel antagonist, blocks the majority of the barium current flowing through calcium channels in chick Purkinje neurons.

Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin.

The effects of pH, temperature and polypeptide concentration on the solution structure and side chain interactions of ShK toxin, a potassium-channel-blocking polypeptide from the sea anemone Stichodactyla helianthus, have been investigated by means of one-dimensional and two-dimensional 1H-NMR spectroscopy. Resonance assignments have been obtained for most protons in the molecule, and for the alpha and beta carbon atoms. The lack of concentration dependence of the 1H chemical shifts and linewidths indicates that self-association is not significant and cannot account for the sheet-like structure near the N terminus. The structure is stable to high temperature, showing little change even at 353 K. This stability allowed backbone-amide temperature coefficients to be interpreted, and the correlation of these values with hydrogen bonds observed in the structures and with solvent exchange rates is discussed. pKa values have been measured for Asp5, His19 and Tyr23, and the contributions to these pKa values from other residues investigated using the analogues R11Q (denoting substitution of Argll with Gln), R11E, H19K, K22A, Y23A and K30A. These results show that Asp5 (pKa 2.8) makes an electrostatic interaction with Lys30, which may be partially responsible for the importance of these side chains in the folding of synthetic toxin. The phenolic pKa of Tyr23 is reduced to 8.7 in the native toxin, as a result of interactions with the positively charged side chains of Arg11 and to a lesser extent Lys22. Several hydrogen bonds between the Arg11 guanidino group and the Tyr23 phenolic group are found in the solution structures. As these three residues are implicated in the tight binding of ShK toxin to the T-lymphocyte voltage-gated potassium channel Kv1.3, their close interactions should be taken into account in models of binding of this toxin to the pore and vestibule of this and other potassium channels.

Expression of a synthetic pertussis toxin operon in Escherichia coli

Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated bouts of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A B bacterial toxin consisting of five subunits S1–S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized vnto an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli β-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

Voltage-dependent blockage of Ca(2+)-permeable AMPA receptors by joro spider toxin in cultured rat hippocampal neurones.

1. The effect of synthetic joro spider toxin (JSTX-3) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels in cultured rat hippocampal neurones was investigated using the whole-cell patch-clamp technique. 2. A population of cultured neurones had AMPA receptors with strong inward rectification and substantial Ca2+ permeability (type II neurones), whereas most neurones (type I neurones) had slight outward rectification and little Ca2+ permeability. JSTX-3 selectively suppressed the inwardly rectifying and Ca(2+)-permeable AMPA receptors expressed in type II neurones without affecting AMPA receptors in type I neurones. 3. The effect of JSTX-3 on the Ca(2+)-permeable AMPA receptors was use and voltage dependent. In the steady state, current responses induced by ionophoretic applications of kainate (a non-desensitizing agonist of AMPA receptors) were suppressed by the toxin in a dose-dependent manner at negative potentials (IC50 = 56 nM at -60 mV). 4. At the standard membrane potential (-60 mV), recovery from the blockage by JSTX-3 was very slow. Even after washout for more than 7 min, the recovery was only partial. However, the blockage was completely removed immediately after application of a +60 mV voltage pulse for 5 s in conjunction with a single ionophoretic application of kainate.

The use of invertebrate peptide toxins to establish Ca 2+ channel identity of CA3-CA1 neurotransmission in rat hippocampal slices

The relative contribution(s) of different Ca 2+ channel subtypes to synaptic transmission between Schaffer collaterals of hippocampal CA3 pyramidal cells and CA1 pyramidal cell dendrites has been assessed using the synthetic invertebrate peptide toxins ω-conotoxin GVIA to block N-type Ca 2+ channels, ω-agatoxin-IVA to block P-type Ca 2+ channels and ω-conotoxin MVIIC to block N-, P- and Q-type Ca 2+ channels. ω-Agatoxin-IVA, ω-conotoxin GVIA and ω-conotoxin MVIIC all produced dose-dependent inhibitions of the excitatory post-synaptic field potential (fEPSP) recorded from the CA1 region of transverse hippocampal slices. Application of 300 nM ω-conotoxin GVIA generally produced no further inhibition to that observed with 100 nM, resulting in a maximal 50% inhibition of the fEPSP. By contrast, 30 nM ω-agatoxin-IVA reduced the fEPSP slope by only 4.6 ± 11.1% (mean ± S.D., n = 3), suggesting the lack of involvement of classical P-type Ca 2+ channels, whereas 300 nM ω-agatoxin-IVA reduced the fEPSP slope by 85.7 ± 15.3% ( n = 3) at the end of 44 min application. Similar applications of 100 and 300 nM ω-conotoxin MVIIC reduced the fEPSP slope by 30.9 ± 6.6% and 79.7 ± 5.7% respectively. Application of 30 nM ω-agatoxin-IVA together with ω-conotoxin GVIA (300 nM) produced no greater inhibition of the fEPSP than that observed with ω-conotoxin GVIA alone, suggesting that the ω-agatoxin-IVA-sensitive and ω-conotoxin MVIIC-sensitive component presents a pharmacology similar to the reported Q-type Ca 2+ channel. The inhibition produced by ω-conotoxin GVIA and ω-conotoxin MVIIC showed no recovery with prolonged washing (1–2 h) whereas that produced by ω-agatoxin-IVA was slowly reversible. The observation that ω-agatoxin-IVA, which does not effect N-type Ca 2+ channels (Mintz et al. (1992a) Neuron 9, 85), is capable of completely suppressing the fEPSP suggests that, whilst N-type Ca 2+ channels may contribute to normal synaptic transmission at Schaffer collateral-CA1 synapses, they are not capable of supporting transmission when Q-type channels are blocked.

Protection against α-bungarotoxin poisoning by immunization with synthetic toxin peptides

The purpose of the present work was to determine the ability of BgTX peptides, corresponding to the various loops and exposed regions of α-bungarotoxin (BgTX) and representing regions that are recognized by B and/or T cells, to stimulate protective immunity in mice against in vivo challenge with BgTX. The BgTX LD 50 values in non-immune mice or mice that had been immunized with proteins and peptides unrelated to BgTX were: Balb c , 0.128 μg/g; SJL, 0.156 μg/g. Immunization of Balb c and SJL mice with each of the synthetic peptides in its free form afforded considerable protection against BgTX poisoning. Peptides L1 (residues 3–16), L2 (residues 26–41) and C-tail (residues 66–74) of BgTX were the most protective and mice immunized with these peptides survived LD 50 values that were three times higher than control mice. Immunization with an equimolar mixture of the three peptides was even more protective and these mice survived even higher challenge doses of BgTX (4.6-fold higher than LD 50 of controls; i.e. protection index, PI = 4.6). An OVA conjugate carrying all three peptides, when used as an immunogen, conferred extremely high protection ( PI ≥ 18.1) which was almost double the protection obtained by BgTX immunization ( PI = 9.7). Thus, the conjugate of the three peptides should serve as an effective vaccine against BgTX poisoning. Furthermore, these results with BgTX peptides should serve as a prototype for the design and synthesis of peptide vaccines against other members of this large family of toxins which include both long and short neurotoxins as well as cytotoxins.

Epitope repertoire of human CD4+ lines propagated with tetanus toxoid or with synthetic tetanus toxin sequences.

The use of synthetic antigen sequences allows propagation in vitro of T cell lines and clones specific for rare antigens, or for individual epitopes. In the present study we investigated the extent of similarity of the epitope repertoire of CD4+ T cell line specific for the antigen tetanus toxin (TTX), propagated with the complete molecule of tetanus toxoid (TTD), and with the synthetic TTX peptides. We propagated from two healthy subjects CD4+ T cell lines specific for TTD, by cycles of stimulation in vitro with TTD or with pools of overlapping synthetic peptides, 20 residues long and overlapping by five residues, corresponding to all or part of the tetanus toxin (TTX) sequence. One pool corresponded to the complete TTX sequence (peptide pool). Two other pools corresponded to residues 1-305 of the TTX light chain and 476-780 of the TTX heavy chain (peptide minipools). The peptide pool-propagated lines recognized TTD vigorously, at levels comparable with those of the TTD-propagated lines. They recognized several peptides, most of which were also recognized by the TTD-propagated line from the same subject. They also recognized to a low extent a few peptides not recognized by the corresponding TTD-propagated line, which might contain cryptic epitopes. The TTD-propagated lines recognized also several peptides that did not elicit a detectable response by the lines propagated with the complete peptide pool. The peptide minipool propagated lines recognized most of the peptides recognized by the TTD-propagated lines. They also recognized several peptides that did not elicit a measurable response of the TTD-propagated line from the same subject, which might contain cryptic epitopes. Very few peptides recognized by the TTD-propagated line did not evoke a response from the peptide minipool propagated lines. To verify that the response to the TTD molecule of the lines propagated with the peptide pools reflected the response of clones recognizing different epitopes produced upon in vitro processing of the TTD molecule, we propagated from each of the two subjects CD4+ T cell lines by stimulation with individual peptide recognized by the TTD-specific lines of that subject (13 peptide-specific lines from subject #1, and 15 from subject #2). All lines responded to the presence of TTD to an extent comparable to the response induced by the same concentration of the relevant peptide, demonstrating that propagation by synthetic epitope sequences allows expansion of T cell clones specific for epitopes which result from processing of the complete TTD molecule. Therefore, whereas the use of very large pools of synthetic antigen peptides for propagation of antigen specific human CD4+ cell lines might lead to loss of clones recognizing less immunogenic sequence regions, peptide pools comprising a relatively limited number of synthetic sequences allow propagation of the majority of the antigen specific T cell clones. The use of peptide pools, and especially of limited peptide pools, results in propagation of polyclonal T cell lines having a more diverse repertoire than the lines propagated by stimulation with the complete antigen molecule. The T clones propagated by the use of the short peptide sequences, which are not expanded when the complete antigen molecule is used, may recognize poorly processed, cryptic epitopes. This approach may be adopted to propagate and detect minor clonal populations, recognizing less immunogenic parts of the antigen.

Selective inhibition of high voltage-activated L-type and Q-type Ca2+ currents by serotonin in rat melanotrophs.

1. Whole-cell Ca2+ currents (ICa) from cultured rat melanotrophs were identified by their sensitivity to Ca2+ channel blockers, and their modulation by serotonin (5-HT) was studied. All cells displayed high voltage-activated (HVA; > -30 mV) Ca2+ currents. A low voltage-activated (LVA; > -60 mV) Ca2+ current was detected in 92% of the cells. 2. The whole-cell ICa was insensitive to omega-conotoxin GVIA (0.5-1 microM) indicating the absence of N-type Ca2+ channels. 3. At a holding potential (Vh) of -70 mV, the L-type channel blocker nifedipine reduced ICa in a dose-dependent manner with a half-maximal effective concentration (IC50) of 28 nM. The L-type current represented 39% of the total ICa. 4. omega-Agatoxin IVA (omega-Aga IVA) produced a biphasic dose-dependent inhibition of ICa, with IC50 values of 0.4 and 91 nM, indicating the presence of P-type and Q-type Ca2+ channels, which accounted respectively for 16 and 45% of the total ICa. The P-type current was also blocked by synthetic funnel-web spider toxin (sFTX 3.3; 1-10 microM) and was present only in a subpopulation (60-70%) of cells. 5. All cells possessed a Ca2+ current which was resistant to nifedipine (10 microM) and omega-Aga IVA (50 nM). This current was not affected by Ni2+ (40 microM) but was abolished by a low concentration of Cd2+ (10 microM) and by omega-conotoxin MVIIC (1 microM) indicating that it was a Q-type Ca2+ current. 6. 5-HT (10 microM) inhibited the whole-cell ICa in 70% of the cells tested (n = 120) by activating 5-HT1A and 5-HT2C receptors. 5-HT produced either a kinetic slowing of the activation phase (37% of the cells) or a scaling down (14% of the cells) of ICa. In the majority of cells (49%) both types of inhibition were found to coexist. 7. The effects of 5-HT were voltage dependent, rendered irreversible when GTP-gamma-S (30 microM) was present in the pipette solution and abolished by pretreatment of the cells with pertussis toxin (PTX; 150 ng ml-1, 18 h). 8. Low concentrations of omega-Aga IVA (20 nM), which blocked mainly P-type channels, did not reduce the effect of 5-HT on ICa. The scaling down effect of 5-HT on ICa was eliminated in the presence of nifedipine (10 microM) and the kinetic slowing effect of 5-HT persisted after blockade of L- and P-type channels but was abolished by omega-conotoxin MVIIC (1 microM). 9. We conclude that rat melanotrophs possess functional L-, P- and Q-type Ca2+ channels and that 5-HT inhibits selectively L-type and Q-type Ca2+ currents with different modalities. These effects are voltage dependent and mediated by a PTX-sensitive G-protein.

Differential inhibition of Ca 2+ channels in mature rat cerebellar Purkinje cells by sFTX-3.3 and FTX-3.3

Synthetic funnel web spider toxin (sFTX-3.3) is a polyamine amide analogue of FTX, a toxin fraction isolated from the venom of the funnel web spider, Agelenopsis aperta, that blocks P-type Ca 2+ channels. The structures of these polyamine containing compounds are not identical: sFTX-3.3 contains an amide carbonyl oxygen that is absent from the predicted structure of native FTX. Recently, a compound called FTX-3.3 was synthesized with the structure predicted for native FTX. We have compared the effects of polyamine amide sFTX-3.3 and polyamine FTX-3.3, on Ca 2+ channel currents in the soma of mature rat cerebellar Purkinje neurons, in which the predominant Ca 2+ channels are defined as P-type. Differential inhibition by sFTX-3.3 and FTX-3.3 revealed three populations of Ca 2+ channels. One group, mediating ~66% of the current, was blocked by sFTX-3.3 with an IC 50 (concentration producing half maximal inhibition) of 33 nM or by FTX-3.3 with an IC 50 of 55 pM. A second population (5–25% of the total current) was inhibited by sFTX-3.3 with an IC 50 of 33 nM, but was insensitive to FTX-3.3, while a third (10–30%) was blocked by FTX-3.3 with an IC 50 of 125 nM and was resistant to sFTX-3.3. These channels also showed distinctive currentvoltage relationships. Our results suggest that P-type Ca 2+ channels in mature rat cerebellar Purkinje cells may be subdivided according to pharmacological and biophysical properties.

Determination of three-dimensional solution structure of waglerin I, a toxin from Trimeresurus wagleri, using 2D-NMR and molecular dynamics simulation

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By y a combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 ω torsion angle restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.

Cardiovascular and respiratory changes following exposure to a synthetic toxin of

The present study demonstrated cardiorespiratory effects of a synthetic phosphorus-containing ichthyotoxic metabolite elaborated by the marine dinoflagellate Ptychodiscus brevis in anaesthetised cats. The metabolite at a dose of 0.25–1.5 mg/kg i.v., resulted in a dose-dependent fall in blood pressure and such vasodepressor effect was associated with bradycardia. There is initial respiratory apnoea ollowed by increased rate and depth of respiration (hyperapnoea) following the administration of the toxin. The hypotensive response was accompanied by a decrease in aortic baroreceptor activity. The ECG showed atrioventricular conduction block, arrhythmia and depression of S-T segment and T wave which indicated coronary insufficiency. Vasodepressive property of the toxin is presumably muscarinic in nature as atropine counteracted the vasodepression.