Neurotoxins

were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Centaurea solstitialis (yellow star thistle) has been proven to cause equine nigropallidal encephalomalacia in horses. Over the last fifty years, nigropallidal encephalomalacia has been of interest to human medicine due to the possible connection with Parkinson's disease. Previous studies indicated the presence of neurotoxic nitrogenous compounds in polar extracts of the plant. In order to give a more detailed description of the nitrogen-containing fraction of C. solstitialis, various samples were collected at different development stages. Different aliquots of the same aqueous extract were directly derivatized with ophthaldialdehyde and dansyl chloride and analyzed separately by reversed-phase HPLC. A complete profile of the free nitrogenous fraction of C. solstitialis was given and results obtained with the two derivatization procedures were compared. No particularly high level of free aspartic and glutamic acids, two potent neuroexcitotoxic amino acids, were found in polar extracts of the plant. Tyramine resulted to be the most important biologically active amine present in C. solstitialis (with a mean concentration of 2.0 mg/100 g of dry weight). q

The aim of this study was to investigate the possible involvement of the glutamatergic system in the toxicity of organochalcogens, since this is an important neurotransmitter system for signal transduction and neural function. The results indicated that 100 μM diphenyl diselenide ( ...

Use of an amino-cupric-silver technique for the detection of early and semiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma. NEUROTOXICOL TERATOL 16(6) 545-561, 1994.-A new amino-cupric silver protocol is described for detection of neuronal degeneration. We describe its selectivity in visualizing both early and semiacute degeneration after intracerebrai or systemic administration of a variety of neurotoxicants in rats, and after transient ischemie episodes in gerbils. As early as 5 min after physical trauma, or 15 min following either intrastriatal injections of glutamate analogs or exposure to ischemic episodes, neuronal silver staining was evident at primary sites of trauma (i.g. injection sites) and at hodologically related secondary sites. With intoxication by peripheral injections of trimethyltin (IP) or intracerebral injections of Doxorubicin, reproducible patterns of degeneration are demonstrable after 24 h or after 9-13 days, respectively. The amino-cupric silver method permits simultaneous detection of all neuronal compartments against a clear background. Degeneration in the neuronal cell bodies, dendrites, axons and terminals, as well as the recruitment of new structures in a progressive pathologic process, could be accurately followed. The inclusion of new reagents increased the sensitivity visA -vis previous versions of the cupric-silver method. The advantages and disadvantages of the current method in comparison with other means of neurotoxic assessment are discussed in detail, with special emphasis on its unique ability to discriminate irreversible degenerative phenomena and degeneration of axonal components in cases where the cell body remains apparently intact. The amino-cupric silver method is an especially useful tool for surveying neuronal damage in basic neuroscience investigations and in neuropathologic and neurotoxic assessment. Brain Silver method Neuronal degeneration Excitotoxicity Ischemia Physical trauma Neuropathology Neuroscience Neurotoxicology NMDA Glutamate analogs THE USE OF silver impregnation procedures for the study of degenerative changes in the nervous system has increased significantly during the past few years, as investigators have rediscovered the unique value of these procedures in identifying damaged neuronal elements. Recent studies include the demonstration of axonal projections arising from neurons undergoing transneuronal anterograde degeneration (17,41), the identification of cells undergoing developmental neuronal death (50,66), or the detection of neuronal damage by neurotoxins including colchicine, 6-hydroxydopamine, puromycin, tetanus toxin, capsaicin, and organometallic compounds (4-5,26,35,57,61). The silver methods have also been used to detect the excitotoxic effects of glutamate and its analogues, the action of 3-acetylpyridine and diazepam, as well as the effects of ischemia, drugs of abuse, epilepsy, hypoglycemia,

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

Mushrooms have been used as components of human diet and many ancient documents written in oriental countries have already described the medicinal properties of fungal species. Some mushrooms are known because of their nutritional and therapeutical properties and all over the world some species are known because of their toxicity that causes fatal accidents every year mainly due to misidentification. Many different substances belonging to poisonous mushrooms were already identified and are related with different symptoms and signs. Carcinogenicity, alterations in respiratory and cardiac rates, renal failure, rhabidomyolisis and other effects were observed in toxicity studies with various species including edible and therapeutic ones. Proper identification is important to avoid accidents and toxicity studies are necessary to assure the safe use of mushrooms as food and for medicinal purposes.

h i g h l i g h t s " The neurotoxins BMAA and DAB are deposited in Lesser Flamingo feathers. " Using deuterated BMAA, good recoveries were obtained after SPE. " DAB was found to occur at concentrations up to 8.5 lg g À1 in feathers. " BMAA and DAB co-occur with microcystins and anatoxin-a. " Feather analysis may be useful post-mortem to determine bird cause of death.

The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx 3, inhibited Ca2÷-dependent glutamate release and the increase in cytosolic free Ca 2÷ in response to membrane depolarization. In the present work, we investigated the effect of PhTx 3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx 3 decreased the release of [3H]-ACh induced by tityustoxin and KC1 in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extraceUular Ca 2+. However, in brain PhTx 3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx 3 is coupled to the process of release of ACh in brain and autonomic nervous system.

Neurolathyrism, an upper motor neuron disease, has been thought to be caused by long-term dietary consumption of lathyrus pulse, which contains the toxin b-N-oxalyl-L-a,b-diaminopropionic acid. Earlier behavioural studies employing oral feeding of lathyrus pulse to animals has been conducted without evaluating the biochemical toxicity potential. In the present investigation the effect of dietary feeding of 10%, 50% and 80% lathyrus pulse to rats and guinea pigs for 3 months on neurobehavioural parameters, including locomotor activity, inclined plain test and neurotoxicological parameters such as neurotransmitter receptor binding, Ca 2Y influx and membrane fluidity, was investigated. Exposure of 50% low and high toxin lathyrus to rats did not cause any significant change in locomotor activity, whereas guinea pigs at the same dosage regimen of high toxin lathyrus showed significant lowering of inclined plain test scores. Furthermore, studies of neuroreceptor binding in rats fed 50% low and high toxin lathyrus showed significant changes in glutamate, dopamine and muscarinic receptors, whereas the benzodiazepine receptor elicited no change. Guinea pigs, on the other hand, fed 50% and 80% lathyrus in the diet showed significant changes in glutamate, dopamine, muscarinic and benzodiazepine receptors. Interestingly, significant elevation in intracellular calcium with a concomitant increase in membrane fluidity was observed in rats (50% low and high toxin) and guinea pigs (50% and 80%) fed a lathyrus diet. These results indicate that although both species (rats and guinea pigs) are susceptible to neurochemical changes on exposure to lathyrus, locomotor changes are only noticed in guinea pigs. Thus, guinea pigs may be more prone to lathyrus toxicity and may serve as a sensitive animal model compared with rats.

Cisplatin causes apoptosis of dorsal root ganglia (DRG) neurons. The amount of platinum binding to DNA correlates with cisplatin toxicity in cancer cells 1 . Cisplatin binds neuronal DNA more than a neuron-like dividing cell line (PC12); 10-fold at 24 h and 24-fold greater at 72 h. ...

The neurotoxicity of methamphetamine to monoaminergic neurons was examined. Neurotoxicity was assessed by quantitative autoradiography using radioligands specific for binding to norepinephrine, dopamine, and serotonin uptake sites. High-dose administration of methamphetamine led to decreases in binding to uptake sites for the three monoamines. Norepinephrine binding sites were decreased in certain amygdaloid nuclei and in the dorsomedial hypothalamic nucleus. Serotonin binding sites were reduced in widespread brain areas, while dopamine binding sites were reduced in the caudate putamen, olfactory tubercle, and nucleus accumbens. The decreases in binding site density for the three monoamines are limited to terminal field areas; cell body areas are not affected. Our results indicate that methamphetamine is neurotoxic to serotonin, dopamine, and norepinephrine neurons. The neurotoxicity to norepinephrine neurons is in selected brain areas. Neurotoxicity, Serotonin, Dopamine, Norepinephrine o 1992 Wiley-Liss, Inc.

nor contamination by vacuolar and mitochondrial membranes, with no significant difference between ves-Sensitivity of ATP Hydrolysis to Inhibitors icles purified by the older column method and the Inhibition c (%) newer gradient method. In summary, we have demonstrated that yeast secre-Conditions a Isolation b Vanadate Azide Bafilomycin tory vesicles can be isolated by a simple two-step sucrose gradient procedure yielding a preparation compa-Standard Column 99.3 { 0.3 0.8 { 0.4 1.5 { 0.5 Gradient 99.3 { 0.3 1.1 { 0.5 2.1 { 0.8 rable to that previously obtained by size-exclusion Transport Column 87.7 { 2.4 3.5 { 2.5 3.7 { 1.8 chromatography (1) and can be accomplished in sig-Gradient 86.1 { 1.4 3.5 { 1.4 7.8 { 4.4 nificantly less time. Furthermore, fluorescence quenching results confirm that the secretory vesicles are well a ATP hydrolysis was measured either at 30ЊC in 50 mM Mes/Tris, sealed and suited to transport assays. The improved pH 5.7, mM Na 2 ATP, 10 mM MgCl 2 , 5 mM phosphoenolpyruvate, and 50 mg/ml pyruvate kinase (standard) or at 23ЊC in 0.6 M sorbitol, 0.1 isolation method should be useful to investigators in-M KCl, 20 mM Hepes/KOH, pH 6.7, 3 mM Na 2 ATP, and 8 mM MgCl 2 terested in studying other yeast transport systems or (transport). in expressing heterologous transporters in yeast. b Secretory vesicles wre isolated either by the column method (Ref. 1) or by the method described in the text (gradient).

The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 × 10-3 M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. 45Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H2O2-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m2 surface area with its calcium gradient in the 10−3 M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.

Regioselective and univocal Suzuki cross-coupling reactions performed on halopyridinyl boronic acids provide a flexible and versatile route to a multigram scale synthesis of 2,2′-dichloro-3,4′-bipyridine 14, which allows couplings with excess pyridin-3-yl boronic acid to give a new and efficient two-step rapid synthesis of nemertelline, the quaterpyridine neurotoxin isolated from a Hoplonemertine sea worm.

Clinicians in the emergency department are often confronted with coma patients due to poisoning. A systematic general approach involving early consultation with a neurologist is of paramount importance. A high index of suspicion, a systematic first assessment already in the prehospital phase and early stabilisation of vital functions are the essential first steps. Specific antidotes like hypertonic glucose and thiamine are part of a "coma cocktail". The opiate antagonist naloxone should be used only

We have shown previously that aberrant hippocampal (HPC) output underlies the dopamine (DA) dysfunction observed in the methylazoxymethanol acetate (MAM) developmental model of schizophrenia in the rodent. This alteration of HPC activity was proposed to result from a reduction in parvalbumin (PV)-expressing GABAergic interneurons and consequent destabilization of the output of pyramidal neurons, as well as disrupted activation across a broad neural network. In vivo extracellular recordings were performed in the ventral tegmental area (VTA) and ventral HPC of saline-(SAL) and MAM-treated animals. A novel benzodiazepine-positive allosteric modulator (PAM), selective for the a5 subunit of the GABA A receptor, SH-053-2 0 F-R-CH3, was tested for its effects on the output of the HPC, leading to dopamine system hyperactivity in MAM-treated animals. In addition, the effect of SH-053-2 0 F-R-CH3 on the hyperactive locomotor response to amphetamine in MAM animals was examined. We demonstrate that treatment with the a5GABA A R PAM reduced the number of spontaneously active DA neurons in the VTA of MAM animals to levels observed in SAL rats, both when administered systemically and when directly infused into the ventral HPC. Moreover, HPC neurons in both SAL and MAM animals showed diminished cortical-evoked responses following a5GABA A R PAM treatment. In addition, the increased locomotor response to amphetamine observed in MAM rats was reduced following a5GABA A R treatment. This study supports a novel treatment of schizophrenia that targets abnormal HPC output, which in turn normalizes dopaminergic neuronal activity.

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS spiders / neurotoxins / peptides / ion channels / structure / pharmacology * Correspondence and reprints.

Pha1b is a potent toxin obtained from the spider Phoneutria nigriventer that blocks neuronal voltage-sensitive Ca 2þ channels. This study compared the antiallodynic effects of Pha1b, u-conotoxin MVIIA and morphine in mice and their side effects in rats. Mechanical allodynia was measured in mice receiving single intrathecal administration of Pha1b, u-conotoxin MVIIA or morphine before or after the incisional plantar procedure. The effect of the treatments on cardiovascular profile and global neurological were evaluated in rats. The expression of pro or anti-inflammatory cytokines of human polymorph mononuclear cells was also evaluated.

BACKGROUND: Cacopsylla melanoneura (Förster) is a vector of Candidatus Phytoplasma mali, the causal agent of one of the most serious diseases in European apple orchards, apple proliferation (AP). The aim of this study was to evaluate the toxicity of five insecticides from different chemical groups (carbamates, chitin inhibitors, insect growth regulators, organophosphates and pyrethroids) towards overwintered adults and young stages of C. melanoneura under semi-field conditions. RESULTS: The organophosphate chlorpyrifos and the pyrethroid etofenprox caused higher mortality of overwintered adults than diflubenzuron, fenoxycarb and abamectin, with mortality values exceeding 90%. All the pesticides tested reduced the number of progeny per female and, except for abamectin, were highly effective against young stages, with a long-lasting effect.

Clostridium is a genus of sporulating and anaerobic Gram-positive, rod-shaped bacteria that includes more than 150 species. These bacteria are widely distributed in the environment and in anaerobic regions of the intestines of several animals, where they are typically found as spores, which are resistant to physical and chemical stresses and can persist for long periods of time until favourable conditions enable germination 1,2 . Under appropriate environmental conditions (such as humidity, nutrients and the absence of oxygen), spores germinate into vegetative cells; conversely, exposure to oxygen, as well as water and nutrient deprivation, trigger sporulation. Several clostridia, including Clostridium difficile, Clostridium perfringens and Clostridium sordelli, are pathogenic, owing to the release of protein toxins, but only a few species are neurotoxigenic. For example, Clostridium tetani produces tetanus neurotoxin, which blocks neurotransmitter release in spinal cord interneurons and causes the spastic paralysis of tetanus 3 . In addition, six phylogenetically distinct clostridia produce more than 40 different botulinum neurotoxins (BoNTs) (BOX 1). BoNTs consist of three primary domains: two of these domains enable binding to nerve terminals and translocation of the toxin into the neuronal cytosol, and the third domain comprises a metalloprotease that inhibits the release of neurotransmitter by peripheral nerve terminals (BOX 2), which causes the flaccid paralysis and autonomic dysfunctions that are typical of botulism 2,4 . The neurospecificity and toxic potency of BoNTs make them the most powerful known toxins, and they are potential bioterrorism weapons 5,6 . By contrast, their absolute neurospecificity has enabled BoNTs to be used as effective therapeutic agents for human diseases that Neurotransmitter An endogenous chemical that transmits signals across a synapse from a neuron to a postsynaptic cell.

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-a-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin ~ binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-a-benzoate.

Phospholipases A 2 (PLA 2 ) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA 2 from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA 2 s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of $1 mM. In addition, 4nerolidylcatechol inhibited representatives of groups I and III of PLA 2 s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA 2 activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA 2 s studied.

We obtained a neurotoxic fraction (AcTx) from star fruit (Averrhoa carambola) and studied its effects on GABAergic and glutamatergic transmission systems. AcTx had no effect on GABA/glutamate uptake or release, or on glutamate binding. However, it specifically inhibited GABA binding in a concentration-dependent manner (IC 50 = 0.89 mM). Video-electroencephalogram recordings demonstrated that following cortical administration of AcTx, animals showed behavioral changes, including tonic-clonic seizures, evolving into status epilepticus, accompanied by cortical epileptiform activity. Chemical characterization of AcTx showed that this compound is a nonproteic molecule with a molecular weight less than 500, differing from oxalic acid. This neurotoxic fraction of star fruit may be considered a new tool for neurochemical and neuroethological research. #

Parkinson's disease (PD) is one of the most important neurodegenerative worldwide disorders. It is characterized by a selective and progressive degeneration of dopaminergic neurons, causing a series of symptoms which might ultimately induce programmed cell death. The potential cytoprotective effects of one of the commercial extracts of Anemopaegma mirandum (Catuaba), a Brazilian tree, on Rotenone-induced apoptosis in human neuroblastomas SH-SY5Y cells was demonstrated. The cell viability, analysis of cellular morphology, nuclei morphology and ultra structural research were done by MTT-tetrazole (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay, phase contrast microscopy, stained with Hoechst 33258 and electron microscopy transmission, respectively. Three different concentrations of Catuaba extract were used (0.312, 0.625 and 1.250 mg/mL). These extracts promoted an increase of 22.3 ± 3.6%, 22.0 ± 2.1% and 15.8± 0.7% on the cell viability. Notable changes in the cellular morphology, condensation of the cell body, nuclear fragmentation and condensation into discrete dense chromatin clumps were observed when the cells were treated with 300 nM Rotenone for 48 h.

The purpose of this study was to investigate the efficacy and persistence of the hypohidrosis induced by iontophoresis of botulinum toxin type A in patients with palmar hyperhidrosis. Eight patients with severe palmar hyperhidrosis participated in this study. Iontophoresis of botulinum toxin was applied to the patients' dominant (right) hand and the other hand was the control treated with normal saline. Gravimetry and iodine-starch tests were performed to evaluate the rate of sweating. The evaluation sessions were at baseline, 2 and 4 days, 1, 2, 3, 4, 8 and 12 weeks after treatment. The patients were also asked to fill out the Dermatology Life Quality Index questionnaire at 2, 4, 8 and 12 weeks after treatment. The mean sweating rate significantly reduced at the first, second, third and fourth week and the second and third month (P < 0.05) after the treatment in the right hand, and the third and fourth week (P < 0.05) after treatment in the left hand. Iontophoresis of botulinum toxin rapidly induced a quick reduction of sweating rate for at least 3 months with no pain, muscle weakness or other side-effects.

Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two a-helices connected by a b-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

The NMR structure of a new toxin, butantoxin (BuTX), which is present in the venoms of the three Brazilian scorpions Tityus serrulatus, Tityus bahiensis, and Tityus stigmurus, has been investigated. This toxin was shown to reversibly block the Shaker B potassium channels (K d ϳ ϳ 660 nM) and inhibit the proliferation of T-cells and the interleukin-2 production of antigen-stimulated T-helper cells. BuTX is a 40 amino acid basic protein stabilized by the four disulfide bridges: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36, and Cys20-Cys38. The latter three are conserved among all members of the short-chain scorpion toxin family, while the first is unique to BuTX. The three-dimensional structure of BuTX was determined using 1 H-NMR spectroscopy. NOESY, phase sensitive COSY (PH-COSY), and amide hydrogen exchange data were used to generate constraints for molecular modeling calculations. Distance geometry and simulated annealing calculations were performed to generate a family of 49 structures free of constraint violations. The secondary structure of BuTX consists of a short 2 1 2 turn ␣-helix (Glu15-Phe23) and a ␤-sheet. The ␤-sheet is composed of two well-defined antiparallel strands (Gly29-Met32 and Lys35-Cys38) connected by a type-I ␤-turn (Asn33-Asn34). Residues Cys5-Ala9 form a quasi-third strand of the ␤-sheet. The N-terminal C2-C5 disulfide bridge unique to this toxin does not appear to confer stability to the protein.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in Caenorhabditis elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2 and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C-and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C-and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.

The safe use of grasspea (Lathyrus sativus) and allied species (L. cicera, L. clymenum and L. ochrus) requires a better understanding of the factors that are involved in the development of neurolathyrism. A suitable animal model is needed. The nutritional quality, seed chemical composition, the role of malnutrition, synergistic action of antinutritional factors, the toxicity of both seed and forage to animals, metabolism and tissue distribution of the toxic amino acid beta-N-oxalyl-alpha,beta-L-diaminopropionic acid (ODAP) in mammals are reviewed. Malnutrition is not necessary for the development of neurolathyrism, however, the supply of sulfur amino acids by Lathyrus spp. is limited by the combined action of several antinutritional factors and the low inherent levels in the seeds. Metabolism or excretion of ODAP and clearance from the central nervous system appear to function well under normal circumstances, while species differences exist. Interruptions to these processes and excessive concurrent demands for reduced sulfur amino acids are likely to be conducive to the onset of neurotoxicity.

The aim of this work was to develop an analytical method capable of determining the presence of anisatin in star anise. This neurotoxin may induce severe side effects such as epileptic convulsions. It is therefore of prime importance to have rapid and accurate analytical methods able to detect and quantify anisatin in samples that are purportedly edible star anise. The sample preparation combined an automated accelerated solvent extraction with a solid-supported liquid-liquid purification step on EXtrelut ® . Samples were analysed on a porous graphitic carbon HPLC column and quantified by tandem mass spectrometry operating in the negative ionisation mode. The quantification range of anisatin was between 0.2 and 8 mg kg −1 . The applicability of this validated method was demonstrated by the analysis of several Illicium species and star anise samples purchased on the Swiss market. High levels of anisatin were measured in Illicium lanceolatum, I. majus and I. anisatum, which may cause health concerns if they are misidentified or mixed with edible Illicium verum.

In 2008, a group of clinicians, scientists, engineers, and industry representatives met to discuss advances in the application of engineering technologies to the diagnosis and treatment of patients with epilepsy. The presentations also provided a guide for further technological development, specifically in the evaluation of patients for epilepsy surgery, seizure onset detection and seizure prediction, intracranial treatment systems, and extracranial treatment systems. This article summarizes the discussions and demonstrates that cross-disciplinary interactions can catalyze collaborations between physicians and engineers to address and solve many of the pressing unmet needs in epilepsy. Near-infrared spectroscopy (NIRS) is a relatively new technique that measures hemodynamic changes associated with neural activity during brain activation [1]. Cerebral activation induces blood flow fluctuations, leading to changes in oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) concentrations. HbO and HbR present different absorption coefficients for near-infrared light. Simultaneous use of two different wavelengths between 680 and 1000 Fig. 12. Illustration of intracranial EEG-based surgical planning. Blue, electrodes; orange, right-hand activations; gray, left-hand activations; red, lesion.

Background Azzalure â (Galderma SA), a newly approved European botulinum neurotoxin type A (BoNT-A), is derived from Dysport TM (Ipsen Ltd.), which has a 20-year history of product consistency and has been widely used for various aesthetic and therapeutic applications. Azzalure â and Dysport TM are collectively referred to as BoNT-A (Speywood Unit) after the unit of their activity, and are distinct from other commercial BoNT-A preparations.

Synapses are specialised structures where interneuronal communication takes place. Not only brain function is absolutely dependent on synaptic activity, but also most of our organs are intimately controlled by synaptic activity. Synapses re therefore an ideal target to act upon and poisonous species have evolved fascinating neurotoxins capable of shutting down neuronal communication by blocking or activating essential components of the synapse. By hijacking key proteins of the communication machinery, neurotoxins are therefore extremely valuable tools that have, in turn, greatly helped our understanding of synaptic biology. Moreover, analysis and understanding of the molecular strategy used by certain neurotoxins has allowed the design of entirely new classes of drugs acting on specific targets with high selectivity and efficacy. This chapter will discuss the different classes of marine neurotoxins, their effects on neurotransmitter release and how they act to incapacitate key steps...

Voltage-gated sodium, calcium, and potassium channels generate electrical signals required for action potential generation and conduction and are the molecular targets for a broad range of potent neurotoxins. These channels are built on a common structural motif containing six transmembrane segments and a pore loop. Their pores are formed by the S5/S6 segments and the pore loop between them and are gated by bending of the S6 segments at a hinge glycine or proline residue. The voltage sensor domain consists of the S1 to S4 segments, with positively charged residues in the S4 segment serving as gating charges. The diversity of toxin action on these channels is illustrated by sodium channels, which are the molecular targets for toxins that act at five or more distinct receptor sites on the channel protein. Both hydrophilic low molecular weight toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Hydrophobic alkaloid toxins and related lipid-soluble toxins act at intramembrane sites and alter voltage-dependent gating of sodium channels via an allosteric mechanism. In contrast, polypeptide toxins alter channel gating by voltage-sensor trapping through binding to extracellular receptor sites, and this toxin interaction has now been modeled at the atomic level for a -scorpion toxin. The voltage sensor trapping mechanism may be a common mode of action for polypeptide gating modifier β toxins acting on all of the voltage gated ion channels.

Criminal behaviors have become a phenomenon that disturbs the communities. The objective of this study is to review the literature about neurotoxicity and criminal behavior.Criminalbehavior implies the use of violence against others. We think that the theories explaining criminal behaviors have their limitations because they put focus mainly on social aspects. From our findings and others, we have reached to some assumptions that neurotoxicity induces changes in brain chemistry that, in turn, changes the perception and lowers the tolerance to external violent stimuli leading to criminal behaviors. In this study, we put focus on the exposure to heavy metals such as lead (pb) due to their impact on nervous system.We have shown the experience of authors in this aspect and how the prevalence of crime is associated with the wide spread of lead (pb).As a conclusion, we think that the environmental theory of violent actions can explain the increasing trend of crime and violence.

In this paper a methodology is presented for the purification of toxins acting on neuronal potassium channels. These neurotoxins are ideal tools for studying the physiological functioning of ion channels, for classifying them and for exploring them in several neuronal regions. Separation procedures leading to the identification of neurotoxins almost always include gel filtration chromatography as well as other chromatographic techniques such as ion exchange and reversed phase chromatography. The use of a Superdex 30 prep grade gel filtration column, which has the advantage of superior resolution in comparison with traditionally used gel filtration columns such as a Sephadex G-50 column, is reported here. The biological assay incorporates the use of Xenopus laevis oocytes, which express potassium channels. Screening of the collected venom fractions was performed by means of the whole-cell voltage clamp technique. The combination of these techniques represents a fast and efficient identification procedure in the search for new and selective neurotoxins for cloned channels and receptors.

Neurotoxins represent unique chemical tools, providing a means to 1) gain insight into cellular mechanisms of apopotosis and necrosis, 2) achieve a morphological template for studies otherwise unattainable, 3) specifically produce a singular phenotype of denervation, and 4) provide the starting point to delve into processes and mechanisms of nerve regeneration and sprouting. There are many other notable uses of neurotoxins in neuroscience research, and ever more being discovered each year. The objective of this review paper is to highlight the broad areas of neuroscience in which neurotoxins and neurotoxicity mechanism come into play. This shifts the focus away from neurotoxins per se, and onto the major problems under study today. Neurotoxins broadly defined are used to explore neurodegenerative disorders, psychiatric disorders and substance use disorders. Neurotoxic mechanisms relating to protein aggregates are indigenous to Alzheimer disease, Parkinson's disease. NeuroAIDS is a disorder in which microglia and macrophages have enormous import. The gap between the immune system and nervous system has been bridged, as neuroinflammation is now considered to be part of the neurodegenerative process. Related mechanisms now arise in the process of neurogenesis. Accordingly, the entire spectrum of neuroscience is within the purview of neurotoxins and neurotoxicity mechanisms. Highlights on discoveries in the areas noted, and on selective neurotoxins, are included, mainly from the past 2 to 3 years. FP Graham Publishing Co., www.NeurotoxicityResearch.com damage and chromosomal aberrations in cultured Chinese hamster lung fibroblast cells. Mutat. Res. 474, 25-33. Kadiu I, JG Glanzer, J Kipnis, HE Gendelman and MP Thomas (2005) Mononuclear phagocytes in the pathogenesis of neurodegenerative diseases. Neurotox. Res. 8, 25-50. Kalivas PW, KK Szumlinski and P Worley (2004) Homer2 gene deletion in mice produces a phenotype similar to chronic cocaine treated rats. Neurotox. Res. 6, 385-387. Kaul M and SA Lipton (2005) Experimental and potential future therapeutic approaches for HIV-1 associated dementia targeting receptors for chemokines, glutamate and erythropoietin. Neurotox. Res. 8, 167-186. Kawashima H, Y Iida, Y Kitamura and H Saji (2004) Binding of 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium ion (HPP + ), a metabolite of haloperidol, to synthetic melanin: implications for the dopaminergic neurotoxicity of HPP + . Neurotox. Res. 6, 535-542. Kheradpezhouh M, S Shavali and M Ebadi (2003) Salsolinol causing parkinsonism activates endoplasmic reticulum-stress signaling pathways in human dopaminergic SK-N-SH cells. Neurosignals 12, 315-324. Kim WK, X Avarez and K Williams (2005) The role of monocytes and perivascular macrophages in HIV and SIV neuropathogenesis: information from non-human primate models. Neurotox. Res. 8, 107-115. Kinney GG, M Burno, UC Campbell, LM Hernandez, D Rodriguez, LJ Bristow and PJ Conn (2003) Metabotropic glutamate subtype 5 receptors (mGluR5) modulate locomotor activity and sensorimotor gating in rodents. J. Pharm. Exp. Ther. 306, 116-123. Kiss P, A Tamas, A Lubics, M Szalai, L Szalontay, I Lengvari and D Reglodi (2005) Development of neurological reflexes and motor coordination in rats neonatally treated with monosodium glutamate. Neurotox. Res. 8, 235-244. Kemmmerling U, P Muñoz, MA Carrasco and C Hidalgo (2005) Redox activation of ryanodine receptors induces calcium signals and CREB AND ERK phosphorylation in hippocampal and N2a neurons. Neurotox. Res. 8, 336. Klawitter V, P Morales, P Huaiquín, D Bustamante and M Herrera-Marschitz (2005) Effects of perinatal asphyxia on cell survival, neuronal phenotype and neurite growth evaluated with organotypic triple cultures. Neurotox. Res. 8, 334. Klein DF and J Stewart (2004) Genes and environment: nosology and psychiatry. Neurotox. Res. 6, 11-16. Klivenyi P, D Siwek, G Gardian, L Yang, A Starkov, C Cleren, RJ Ferrante, NW Kowall, A Abeliovich and MF Beal (2006) Mice lacking α-synuclein are resistant to mitochondrial toxins. Neurobiol. Dis. 21, 541-548. Kostrzewa RM (1995) Dopamine receptor supersensitivity. Neurosci. Biobehav. Rev. 19, 1-17. Kostrzewa RM, JP Kostrzewa and R Brus (2003) Dopamine receptor supersensitivity: an outcome and index of neurotoxicity. Neurotox. Res. 5, 111-118. Kostrzewa RM, JP Kostrzewa, P Nowak, RA Kostrzewa and R Brus (2004) Dopamine D 2 agonist priming in intact and dopaminelesioned rats. Neurotox. Res. 6, 457-462. Kostrzewa JP and RM Kostrzewa (2005) p-Chloroamphetamine enhances dopamine release (striatal in vivo microdialysates) after 5-HT lesion of Parkinsonian rats. Neurotox. Res. 8, 321. Kostrzewa RM, JP Kostrzewa and R Brus (2005) Serotonin systems mediate and modulate dopamine in a Parkinsonian rodent model.