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We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

Due to their complexity and diversity, animal venoms represent an extensive source of bioactive compounds such as peptides and proteins. Conventional approaches for their characterization often require large quantities of biological material. Current mass spectrometry (MS) techniques now give access to a wealth of information in a short working time frame with minute amounts of sample. Such MS approaches may now be used for the discovery of novel compounds, and once their structure has been determined they may be synthesized and tested for functional activity. Molecular mass fingerprints of venoms allow the rapid identification of known toxins as well as preliminary structural characterization of new compounds. De novo peptide sequencing by tandem mass spectrometry (MS/MS) offers rapid access to partial or total primary peptide structures. This article, written as a tutorial, also contains new material: molecular mass fingerprint analysis of Orthochirus innesi scorpion venom, and identification of components from bumblebee Bombus lapidarius venom, both collected from one single specimen. The structure of the three major peptides detected in the Bombus venom was fully characterized in one working day by de novo sequencing using an electrospray ionization hybrid quadrupole time-of-flight instrument (ESI-QqTOF) and a matrix-assisted laser desorption ionization time-of-flight instrument (MALDI-LIFT-TOF-TOF). After presenting the MS-based sequence elucidation, perspectives in using MS and MS/MS techniques in toxinology are discussed.

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.

Proteins are essential biomolecules which are frequently involved in major pathological syndromes and are widely used as diagnostic markers or therapeutic agents. The emergence of proteomics will doubtless further increase the significance of proteins both in the clinic and in the life sciences in general. Our main objective is to offer innovative solutions to what we like to call the "post-proteomics era". To achieve our goal, we intend to develop novel approaches and technologies for in vivo metabolic studies of proteins using mass spectrometry (MS), focusing on pharmacokinetics and pharmacodynamics. Using goserelin as a model, we have successfully developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of an intact analogue of luteinizing hormone-releasing hormone (LHRH) in small volumes of rat plasma samples at concentrations ranging from 0.3 to 405.0 microg/l. To this end, a microbore reversed-phase-HPLC system was coupled on-line to a tandem high resolution quadrupole time-of-flight (Q-TOF) instrument fitted with an electrospray ion source and operated in LC-MS/MS mode. External calibration was used and the high resolution was crucial to discard contaminating signals, which would not have been possible with the more conventional triple quadrupole mass spectrometers operated in a static mode. For low sample amounts, calibration curves were constructed corresponding to rat plasma levels of 0.3 to 16.4 microg/l and found to be of third order with a coefficient of determination greater than 0.999. The relative standard deviation was found to be lower than 15%. A lower limit of detection (LLOD) of 0.17 microg/l and a lower limit of quantification (LLOQ) of 0.3 microg/l were determined.

In the Internet community there is a strong demand for platform-independent collaboration software. Java is developed with the major design goals of being a platform-independent, and Internet-oriented programming language. We show how a group of Internet users can share single-user Java applications for synchronous collaboration. Our approach is based on a replicated tool architecture in which each participant runs a copy of the application and the activity of each user is multicast to all the participants in the conference. We have developed a system called Java Collaborative Environment (JCE), on which the Java's Abstract Window Toolkit (AWT) is extended such that mouse and keyboard events are intercepted and distributed among all copies of the shared Java application. In addition we provide an infrastructure and a simple interface for session management and floor control.

The ability to detect biochemical diversity in animal venoms has wide-ranging implications for a diverse array of scientific disciplines. Matrix-assisted laser desorption time-of-flight mass spectrometry (and, for comparative purposes, isoelectric focusing) were used to characterize venoms from a geographically diverse sample of Trimeresurus stejnegeri (n < 229) from Taiwan. Previously unrealised levels of heterogeneity were detected in venom phospholipase A2 isoforms (PLA2) and in whole venom profiles. Geographic variation in venom was primarily between Taiwan and two Pacific islets. Despite the common assumption that venom variation is a product of neutral molecular evolution, statistical testing failed to link venom variation with phylogenetic descent convincingly. Instead, pronounced differences in venom composition may be the product of natural selection for regional diets or of independent founder effects. More data are required on the functional differences between the isoforms to distinguish between these alternatives.

A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.

Although Multimedia desktop conferencing and application sharing among geographically dispersed users are increasingly popular modalities, their spread is inhibited by platform-dependency problems. In this paper, an approach which exploits the use of the Java programming language to accommodate different hardware and window systems is investigated and a prototype is implemented. Our approach is based on replicated tool architecture in which each participant runs a copy of the application and the activity of each user is multicast to all the participants in the conference. The problems associated with this approach such as view synchronization and replicated object management are among the issues addressed in our research. In addition, we are developing standard functions and mechanisms that allow conference participants to seamlessly use the audio and video features available on most PC’s and workstations. Our research on multimedia stream synchronization and adaptation, the incorporation of reliable multicasting and the development of distributed control algorithms are expected to result in increased conference quality, performance and robustness.

With an upgraded version of the Tree and Tabular Combined Notation (TTCN) that encompasses concurrency, it is now possible to foresee a new generation of Abstract Test Suites (ATSs) and Means Of Testing (MOT) for communication protocols. Indeed, the inclusion of concurrency/parallelism allows the complete description of ATSs in TTCN, including the upper tester behavior. This paper introduces a new prototype technology that automates the production of MOT from an ATS completely specified in TTCN. This technology is based on an implementation of the operational semantics of TTCN in Estelle.

Three isomeric lead bis(butylthiolate) and tin bis(butylthiolate) compounds have been prepared and characterized. Their solid state decomposition, and in the case of the lead bis(thiolate) compounds, their thermolyses in suspension in a high boiling hydrocarbon, have been studied. X-ray powder diffraction patterns and scanning electron micrographs of the obtained solid state material are discussed. Volatile co-products of the decomposition have been isolated and characterized.

This paper presents the current status of the joint ISO/CCITT work on formal methods in conformance testing. After a general introduction to the topic and an overview of the ongoing work, the test case generation methods studied are presented. Additionally, some ongoing research is highlighted. Only work that is going on in the immediate environment of the authors is presented here.

The present invention relates to novel mu-conotoxin peptides, biologically active fragments thereof, combinations thereof and /or variants thereof. The invention also relates to their use in pharmaceutical composition for the treatment or prevention of pain, and their use in the preparation of an anesthesic. [La présente invention concerne de nouveaux peptides de type mu-conotoxine, des fragments biologiquement actifs desdits peptides, des combinaisons desdits peptides et/ou des variantes desdits peptides. L'invention concerne également leur emploi dans une préparation pharmaceutique pour le traitement prophylactique ou thérapeutique de la douleur, ainsi que leur emploi dans l'élaboration d'un anesthésique]

International audience; In this paper, we present an overview of the biodiversity of both marine snails of the large genus Conus and their venoms. After a brief survey of Conidae malacology, we focus on the high degree of biodiversity of this genus, its specific biogeography as well as its habitat, and the relatively strict diet of its members. The venom of Conidae species contains a large number of peptides that can interact selectively with key elements of the peripheral and central nervous systems of vertebrates and invertebrates. Emphasis is on summarizing our current knowledge of the specific actions of venom components on ionic channels, receptors and other key elements of cellular communication. The peptides isolated from venoms, called conotoxins, form different families according to both their primary structure and their specific pharmacological targets. Three families encompassing the mu-, muO- and delta-conotoxins target voltage-sensitive sodium channels but with different modes of action or tissue selectivity. Another important class of conotoxins is the omega-conotoxin family which acts on voltage-sensitive calcium channels. The alpha-conotoxin family is represented by several peptides blocking muscular or neuronal nicotinic acetylcholine receptors. Finally, a blocker of potassium channels is presented as well as two conotoxins acting on the N-Methyl-D-Aspartate receptor. Primary structures and cysteine frameworks of all these conotoxins are shown and compared. At the end of the review, we report the contribution of molecular biology to identification of new conotoxins having original pharmacological properties. In conclusion, conotoxins have received increasing attention from physiologists, pharmacologists, biochemists and physicians because of their selectivity as well as their pharmacological and therapeutic potential.