Attentional bias to pictures of fear-relevant animals in a dot probe task

Attentional bias to fear-relevant animals was assessed in 69 participants not preselected on self-reported anxiety with the use of a dot probe task showing pictures of snakes, spiders, mushrooms, and flowers. Probes that replaced the fear-relevant stimuli (snakes and spiders) were found faster than probes that replaced the non-fear-relevant stimuli, indicating an attentional bias in the entire sample. The bias was not correlated with self-reported state or trait anxiety or with general fearfulness. Participants reporting higher levels of spider fear showed an enhanced bias to spiders, but the bias remained significant in low scorers. The bias to snake pictures was not related to snake fear and was significant in high and low scorers. These results indicate preferential processing of fear-relevant stimuli in an unselected sample.

Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Of snakes and flowers: Does preferential detection of pictures of fear-relevant animals in visual search reflect on fear-relevance?

Previous research in visual search indicates that animal fear-relevant deviants, snakes/spiders, are found faster among non fear-relevant backgrounds, flowers/mushrooms, than vice versa. Moreover, deviant absence was indicated faster among snakes/spiders and detection time for flower/mushroom deviants, but not for snake/spider deviants, increased in larger arrays. The current research indicates that the latter 2 results do not reflect on fear-relevance, but are found only with flower/mushroom controls. These findings may reflect on factors such as background homogeneity, deviant homogeneity, or background-deviant similarity. The current research removes contradictions between previous studies that used animal and social fear-relevant stimuli and indicates that apparent search advantages for fear-relevant deviants seem likely to reflect on delayed attentional disengagement from fear-relevance on control trials.

SCORPION2: A database for structure-function analysis of scorpion toxins

Scorpion toxins are important experimental tools for characterization of vast array of ion channels and serve as scaffolds for drug design. General public database entries contain limited annotation whereby rich structure-function information from mutation studies is typically not available. SCORPION2 contains more than 800 records of native and mutant toxin sequences enriched with binding affinity and toxicity information, 624 three-dimensional structures and some 500 references. SCORPION2 has a set of search and prediction tools that allow users to extract and perform specific queries: text searches of scorpion toxin records, sequence similarity search, extraction of sequences, visualization of scorpion toxin structures, analysis of toxic activity, and functional annotation of previously uncharacterized scorpion toxins. The SCORPION2 database is available at http://sdmc.i2r.a-star.edu.sg/scorpion/. (c) 2006 Elsevier Ltd. All rights reserved.

Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Toxin insights into nicotinic acetylcholine receptors

Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. in the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin Iml. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR. (c) 2006 Elsevier Inc. All rights reserved.

Cloning and characterization of natriuretic peptides from the venom glands of Australian elapids

The venom from Australian elapid snakes contains a complex mixture of polypeptide toxins that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Included in these toxin families are the recently described venom natriuretic peptides, which display similar structure and vasoactive functions to mammalian natriuretic peptides. This paper describes the identification and detailed comparative analysis of the cDNA transcripts coding for the mature natriuretic peptide from a total of nine Australian elapid snake species. Multiple isoforms were identified in a number of species and represent the first description of a natriuretic peptide from the venom gland for most of these snakes. Two distinct natriuretic peptide isoforms were selected from the common brown snake (Pseudonaja textilis), PtNP-a, and the mulga (Pseudechis australis), PaNP-c, for recombinant protein expression and functional analysis. Only one of these peptides, PtNP-a, displayed cGMP stimulation indicative of normal natriuretic peptide activity. Interestingly, both recombinant peptides demonstrated a dose-dependent inhibition of angiotensin converting enzyme (ACE) activity, which is predictive of the vasoactive effects of the toxin. The natriuretic peptides, however, did not possess any coagulopathic activity, nor did they inhibit or potentiate thrombin, adenosine diphosphate or arachidonic acid induced platelet aggregation. The data presented in this study represent a significant resource for understanding the role of various natriuretic peptides isoforms during the envenomation process by Australian elapid snakes. (c) 2006 Published by Elsevier Masson SAS.

N-type calcium channel blockers: Novel therapeutics for the treatment of pain

Highly selective Cav2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (ω-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Cav2.2 calcium channel have met with varied success. This review focuses on the properties of the Cav2.2 calcium channel in different pain states, the action of ω-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Cav2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

The stability of P in coral reef fishes

The constancy of phenotypic variation and covariation is an assumption that underlies most recent investigations of past selective regimes and attempts to predict future responses to selection. Few studies have tested this assumption of constancy despite good reasons to expect that the pattern of phenotypic variation and covariation may vary in space and time. We compared phenotypic variance-covariance matrices (P) estimated for Populations of six species of distantly related coral reef fishes sampled at two locations on Australia's Great Barrier Reef separated by more than 1000 km. The intraspecific similarity between these matrices was estimated using two methods: matrix correlation and common principal component analysis. Although there was no evidence of equality between pairs of P, both statistical approaches indicated a high degree of similarity in morphology between the two populations for each species. In general, the hierarchical decomposition of the variance-covariance structure of these populations indicated that all principal components of phenotypic variance-covariance were shared but that they differed in the degree of variation associated with each of these components. The consistency of this pattern is remarkable given the diversity of morphologies and life histories encompassed by these species. Although some phenotypic instability was indicated, these results were consistent with a generally conserved pattern of multivariate selection between populations.

Modulation of insect Cav channels by peptidic spider toxins

Insects have a much smaller repertoire of voltage-gated calcium (Ca-v) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate Ca(v)1, Ca(v)2, and Ca(v)3 subtypes, although its basal inventory is expanded by alternative splicing and editing of Ca-v channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect Ca-v channels, since severe loss-of-function mutations in genes encoding the pore-forming a, subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect Ca-v channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect Ca-v channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect Ca-v channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect Ca-v channels. This review focuses on peptidic spider toxins that specifically target insect Ca-v channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests. (c) 2006 Elsevier Ltd. All rights reserved.

Organohalogen contaminants in wildlife from the Yangtze River Delta : Development of methods and assessments of legacy and emerging persistent organic pollutants

Rapid economic development has occurred during the past few decades in China with the Yangtze River Delta (YRD) area as one of the most progressive areas. The urbanization, industrialization, agricultural and aquaculture activities result in extensive production and application of chemicals. Organohalogen contaminants (OHCs) have been widely used as i.e. pesticides, flame retardants and plasticizers. They are persistent, bioaccumulative and pose a potential threat to ecosystem and human health. However, limited research has been conducted in the YRD with respect to chemicals environmental exposure. The main objective of this thesis is to investigate the contamination level, distribution pattern and sources of OHCs in the YRD. Wildlife from different habitats are used to indicate the environmental pollution situation, and evaluate selected matrices for use in long term biomonitoring to determine the environmental stress the contamination may cause. In addition, a method is developed for dicofol analysis. Moreover, a specific effort is made to introduce statistic power analysis to assist in optimal sampling design. The thesis results show extensive contamination of OHCs in wildlife in the YRD. The occurrences of high concentrations of chlorinated paraffins (CPs) are reported in wildlife, in particular in terrestrial species, (i.e. short-tailed mamushi snake and peregrine falcon). Impurities and byproducts of pentachlorophenol products, i.e. polychlorinated diphenyl ethers (PCDEs) and hydroxylated polychlorinated diphenyl ethers (OH-PCDEs) are identified and reported for the first time in eggs from black-crowned night heron and whiskered tern. High concentrations of octachlorodibenzo-p-dioxin (OCDD) are determined in these samples. The toxic equivalents (TEQs) of polychlorinated dibenzo-p-dioxin (PCDDs) and polychlorinated dibenzofurans (PCDFs) are at mean levels of 300 and 520 pg TEQ g-1lw (WHO2005 TEQ) in eggs from the two bird species, respectively. This is two orders of magnitude higher than European Union (EU) regulation limit in chicken eggs. Also, a novel pattern of polychlorinated biphenyls (PCBs) with octa- to decaCBs, contributing to as much as 20% of total PCBs therein, are reported in birds. The legacy POPs shows a common characteristic with relatively high level of organochlorine pesticides (i.e. DDT, hexacyclohexanes (HCHs) and Mirex), indicating historic applications. In contrast, rather low concentrations are shown of industrial chemicals such as PCBs and polybrominated diphenyl ethers (PBDEs). A refined and improved analytical method is developed to separate dicofol from its major decomposition compound, 4,4’-dichlorobenzophenone. Hence dicofol is possible to assess as such. Statistic power analysis demonstrates that sampling of sedentary species should be consistently spread over a larger area to monitor temporal trends of contaminants in a robust manner. The results presented in this thesis show high CPs and OCDD concentrations in wildlife. The levels and patterns of OHCs in YRD differ from other well studied areas of the world. This is likely due to the extensive production and use of chemicals in the YRD. The results strongly signal the need of research biomonitoring programs that meet the current situation of the YRD. Such programs will contribute to the management of chemicals and environment in YRD, with the potential to grow into the human health sector, and to expand to China as a whole.

Identification and evaluation of agents isolated from traditionally used herbs against Ophiophagus hannah venom

The aim of this study was firstly to identify active molecules in herbs, that are traditionally used for the treatment of snake bite, such as Curcuma antinaia, Curcuma contravenenum, Andrographis paniculata, and Tanacetum parthenium; secondly to test similar structurally related molecules and finally to prepare and evaluate an efficient formulation against Ophiophagus hannah venom intoxification. Three labdane based compounds, including labdane dialdehyde, labdane lactone, and labdane trialdehyde and two lactones including 14-deoxy-11,12-didehydroandrographolide and parthenolide were isolated by column chromatography and characterised. Using the isolated rat phrenic nerve-hemidiaphragm preparation, the antagonistic effect of crude extracts, isolated compounds and prepared formulations were measured in vitro on the inhibition of the neuromuscular transmission. Inhibition on muscle contraction, produced by the 5 μg/mL venom, was reversed by test agents in organ bath preparations. A labdane trialdehyde, isolated from C. contravenenum, was identified as the best antagonising agent in the low micromolar range. Tests on formulations of the most potent C. contravenenum extract showed, that the suppository with witepsol H15 was an effective medicine against O. hannah venom. This study elucidated the active compounds, accounting for the antivenin activity of traditionally used herbs and suggested the most suitable formulation, which may help to develop potent medicines for the treatment of snake bite in the future.

Editorial

Full text: Welcome to issue 1 of 2012 and a belated Happy New Year to all CLAE readers and BCLA members. The hardest job I had for this issue was to decide which papers to include and which papers to hang on to and save for issue 2 of 2012. At the end of December when I was choosing the content for this issue there were additional 5 papers that could have been included. The final choice came down to various factors; such as first come first served – i.e. which papers had been ready and waiting the longest; secondly which papers had been submitted the earliest; are there similar papers so that it may be beneficial to publish them alongside this paper; and also was the content something which needed to be out there quicker than other papers as it was a current hot topic? But it should be noted that once papers are proofed and deemed ready by authors they are published in the epub version and put online for others to see in their final version. An epub version is given a DOI number (digital object identifier) so that it can be cited by other authors. Apart from being on line the only other difference is that an epub version is essentially waiting to be assigned to a particular issue. So those papers that are being held off for issue 2 of this year are actually already available for you to read (and cite) on line. In this issue there is a paper related to the cost of different contact lens replacement schedules – this may be a topic that is debated more in the future since as a society we are thinking more ‘green’ and all trying to help by reducing our carbon footprint, whether that be by recycling or using less in the first place. A timely review paper on the management of allergic eye disease may help us to better manage those patients we see in the spring with pollen allergies. We have two papers looking at different aspects in keratoconus patients, another looking at a modified fluoret strip and its application in measuring tear break up time and a review paper on corneal erosions. Another interesting paper comes from Professor Harminder Dua and his team. Professor Dua is the UK Royal College of Ophthalmologists’ president and has been interested in corneal anatomy and physiology for much of his research career. Finally, an unusual case of an ocular injury related to a snake bite. Overall I would say there is enough to sink your ‘fangs’ into! Finally, it gives me great pleasure in announcing the newest person to join our Editorial Board, Dr Florence Malet. Dr Malet is an Ophthalmologist and since September 2000 she has been at the Bordeaux Hospital in France developing the Contact Lens Unit of in the Ophthalmology University Department. She is ex-president of the French Contact Lens Society and president of the European Contact Lens Society of Ophthalmologists.

In-vitro and in-vivo antivenin activity of 2-[2-(5,5,8a-trimethyl-2- methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde against Ophiophagus hannah venom

Objectives Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan-Paya-Ngoo-Tua-Mia, is commonly used in the North-Eastern part of Thailand as a 'snakebite antidote'. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom. Methods The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X-ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve-hemidiaphragms and in vivo in mice. Key findings The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene] -succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve-hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 μg/ml venom, was reversed by 2-16 μg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. Conclusions The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source. © 2010 Royal Pharmaceutical Society of Great Britain.