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Conservation of sea snakes is virtually nonexistent in Asia, and its role in human-snake interactions in terms of catch, trade, and snakebites as an occupational hazard is mostly unexplored. We collected data on sea snake landings from the Gulf of Thailand, a hotspot for sea snake harvest by squid fishers operating out of the ports of Song Doc and Khanh Hoi, Ca Mau Province, Vietnam. The data were collected during documentation of the steps of the trading process and through interviewers with participants in the trade. Squid vessels return to ports once per lunar synodic cycle and fishers sell snakes to merchants who sort, package, and ship the snakes to various destinations in Vietnam and China for human consumption and as a source of traditional remedies. Annually, 82 t, roughly equal to 225,500 individuals, of live sea snakes are brought to ports. To our knowledge, this rate of harvest constitutes one of the largest venomous snake and marine reptile harvest activities in the world today. Lapemis curtus and Hydrophis cyanocinctus constituted about 85% of the snake biomass, and Acalyptophis peronii, Aipysurus eydouxii, Hydrophis atriceps, H. belcheri, H. lamberti, and H. ornatus made up the remainder. Our results establish a quantitative baseline for characteristics of catch, trade, and uses of sea snakes. Other key observations include the timing of the trade to the lunar cycle, a decline of sea snakes harvested over the study period (approximately 30% decline in mass over 4 years), and the treatment of sea snake bites with rhinoceros horn. Emerging markets in Southeast Asia drive the harvest of venomous sea snakes in the Gulf of Thailand and sea snake bites present a potentially lethal occupational hazard. We call for implementation of monitoring programs to further address the conservation implications of this large-scale marine reptile exploitation., (© 2014 Society for Conservation Biology.)

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca(2+)-activated K(+) channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BKCa channel. Calculation of an electrostatic surface map of the BKCa gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BKCa channel activation gating.

Autophagy is a catabolic pathway in which the cell sequesters cytoplasmic material, including long-lived proteins, lipids and organelles, in specialized double-membrane vesicles, called autophagosomes. Subsequently, autophagosomes communicate with the lysosomal compartment and acquire acidic hydrolases for final cargo degradation. This process of partial self-eating secures the survival of eukaryotic cells during starvation periods and is critically regulated by mTORC1 (mammalian target of rapamycin complex 1). Under nutrient-poor conditions, inhibited mTORC1 permits localized PtdIns(3)P production at particular membranes that contribute to autophagosome formation. Members of the human WIPI (WD-repeat protein interacting with phosphoinositides) family fulfil an essential role as PtdIns(3)P effectors at the initiation step of autophagosome formation. In the present article, we discuss the role of human WIPIs in autophagy, and the identification of evolutionarily conserved amino acids of WIPI-1 that confer PtdIns(3)P binding downstream of mTORC1 inhibition. We also discuss the PtdIns(3)P effector function of WIPIs in the context of longevity and autophagy-related human diseases, such as cancer and neurodegeneration.

Cryptophane-C is composed of two nonequivalent cyclotribenzylene caps, one of which contains methoxy group substituents on the phenyl rings. The two caps are connected by three OCH(2)CH(2)O linkers in an anti arrangement. Host-guest complexes of cryptophane-C with dichloromethane and chloroform in solution were investigated in detail by nuclear magnetic resonance techniques and density functional theory (DFT) calculations. Variable temperature proton and carbon-13 spectra show a variety of dynamic processes, such as guest exchange and host conformational transitions. The guest exchange was studied quantitatively by exchange spectroscopy measurements or by line-shape analysis. The conformational preferences of the guest-containing host were interpreted through cross-relaxation measurements, providing evidence of the gauche+2 and gauche-2 conformations of the linkers. In addition, the mobility of the chloroform guest inside the cavity was studied by carbon-13 relaxation experiments. Combining different types of evidence led to a detailed picture of molecular recognition, interpreted in terms of conformational selection., (Copyright © 2012 John Wiley & Sons, Ltd.)

Cryptophane-D is composed of two nonequivalent cyclotribenzylene caps bound together by three OCH2CH2O bridges in a syn arrangement. Host-guest complexes with chloroform and dichloromethane were investigated in solution by NMR spectroscopy. Variable temperature NMR (1)H and (13)C spectra showed effects of chemical exchange between the free and bound guest and of conformational exchange for the host, strongly and specifically affected by guest binding. We found in particular that the carbon-13 chemical shifts for the linkers connecting the two cyclotribenzylene units are very informative. The NMR results were supported by DFT calculations. The guest exchange was also studied quantitatively, either by EXSY measurements (for chloroform as guest) or by line-shape analysis (for dichloromethane as guest). In the case of chloroform guest, we also investigated cross-relaxation between the guest and host protons, as well as carbon-13 longitudinal relaxation and heteronuclear NOE at three different fields. The results were interpreted in terms of orientation and dynamics of the guest inside the host cavity. Putting together various types of evidence resulted in remarkably detailed insight into the process of molecular recognition of the two guests by cryptophane-D host.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the alpha-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an alpha-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

Complexation equilibria and kinetics of exchange of chloroform and dichloromethane molecules between the cavity of cryptophane-E and bulk solution were investigated using NMR methods. Using one-dimensional magnetization transfer (1D-EXSY type sequence), chemical exchange rates were measured in different temperature ranges, limited by the equilibrium constant values of the complexes and the boiling points of the guest substances. From the kinetic data, activation energies were calculated using the Arrhenius equation. From the temperature-dependence of the association constant data, the enthalpy and entropy of complexation were estimated and compared with values for similar complexes of other cryptophanes., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Unlabelled: For women who desire pregnancy or who wish to retain their uterus, myomectomy is the standard approach for the treatment of fibroids. Abdominal myomectomy seems to be the best choice when there are large subserosal or intramural fibroids (> 5-7 cm), or submucosal fibroids > 3 cm or when multiple fibroids (> 3) are to be removed. When submucosal myomas are present or multiple fibroids are to be removed, opening the uterine cavity during the surgical procedure is more likely to happen. There is lack of published evidence about whether there is any difference in perioperative morbidity and management of those cases where the uterine cavity is opened during the surgical procedure compared with those where the uterine cavity remains closed., Methods: We undertook a retrospective review of 423 abdominal myomectomies via either an opened or closed uterine cavity. As a primary outcome we assessed the overall perioperative morbidity rate and as a secondary outcome we compared the necessity of pre and postoperative transfusions, intraoperative bleeding, febrile morbidity, unintended surgical interventions, life-threatening events, need for relaparotomies and duration of hospital stay between the opened and non opened uterine cavity groups., Results: The overall perioperative morbidity rate was significantly higher in those cases where the uterine cavity was opened during surgery; however the difference was caused only by the increased risk of intraoperative bleeding. All the other variables, such as febrile morbidity, number of relaparotomies, unintended surgical procedures and life-threatening events did not differ between the two groups., Conclusion: Although there is an increased risk of intraoperative bleeding it seems that entering the uterine cavity during abdominal myomectomy can be considered as safe a procedure as in those cases where the uterine cavity remains closed.

Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca2+-activated K+ channels with high affinity and specificity. Automated solid-phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N--(D-biotin-6-amidocaproate)-L-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx-LC-biotin exhibits high affinity (KD = 26 nM) and a slow dissociation rate (koff = 5.4 x 10(-4) s(-1)) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx-LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx-LC-biotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx-LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patch-like surface distribution of BK channel protein. The results support the feasibility of using IbTx-LC-biotin and similar biotin-tagged K+ channel toxins for diverse applications in cellular neurobiology. .

The Hylobatidae (gibbons) are among the most endangered primates and their evolutionary history and systematics remain largely unresolved. We have investigated the species-level phylogenetic relationships among hylobatids using 1257 bases representing all species and an expanded data set of up to 2243 bases for select species from the mitochondrial ND3-ND4 region. Sequences were obtained from 34 individuals originating from all 12 recognized extant gibbon species. These data strongly support each of the four previously recognized clades or genera of gibbons, Nomascus, Bunopithecus, Symphalangus, and Hylobates, as monophyletic groups. Among these clades, there is some support for either Bunopithecus or Nomascus as the most basal, while in all analyses Hylobates appears to be the most recently derived. Within Nomascus, Nomascus sp. cf. nasutus is the most basal, followed by N. concolor, and then a clade of N. leucogenys and N. gabriellae. Within Hylobates, H. pileatus is the most basal, while H. moloch and H. klossii clearly, and H. agilis and H. muelleri likely form two more derived monophyletic clades. The segregation of H. klossii from other Hylobates species is not supported by this study. The present data are (1) consistent with the division of Hylobatidae into four distinct clades, (2) provide the first genetic evidence for all the species relationships within Nomascus, and (3) call for a revision of the current relationships among the species within Hylobates. We propose a phylogenetic tree as a working hypothesis against which intergeneric and interspecific relationships can be tested with additional genetic, morphological, and behavioral data.

Snake alpha-neutotoxins of Elapidae venoms are grouped into two structural classes, short-chain and long-chain alpha-neutotoxins. While these two classes share many chemical and biological characteristics, there are also distinct dissimilarities between them, including their binding site on the nicotinic acetylcholine receptor (nAChR), specificity among species of Chordata, and the associated pharmacological effects. In the present study we test the hypothesis that structural motifs that evolved to confer natural resistance against conspecific long-chain alpha-neurotoxins in Elapidae snakes also interfere with the biological action of short-chain alpha-neurotoxins. We expressed functional nAChRs that contains segments or single residues of the Elapidae nAChR ligand binding domain and tested the effect of short-chain alpha-neurotoxin erabutoxin-a (ETX-a) from the Erabu sea snake Laticauda semifasciata on the acetylcholine-induced currents as measured by two-microelectrode voltage clamp. Our results show that the Elapidae nAChR alpha subunit segment T(154)-L(208) ligand binding domain has an inhibitory effect on the pharmacological action of ETX-a. This effect is primarily attributed to the presence of glycosylation at position N(189). If the glycosylation is removed from the T(154)-L(208) segment, the nAChR will be inhibited, however, to a lesser extent than seen in the mouse. This effect correlates with the variations in alpha-neurotoxin sensitivity of different species and, importantly, reflects the evolutionary conservation of the binding site on the nAChR polypeptide backbone per se. Phylogenetic analysis of alpha-neurotoxin resistance suggests that alpha-neurotoxin-resistant nAChR evolved first, which permitted the evolution of snake venom alpha-neurotoxins. A model describing alpha-neurotoxin resistance in Elapidae snakes is presented.

Evolutionary success requires that animal venoms are targeted against phylogenetically conserved molecular structures of fundamental physiological processes. Species producing venoms must be resistant to their action. Venoms of Elapidae snakes (e.g., cobras, kraits) contain alpha-neurotoxins, represented by alpha-bungarotoxin (alpha-BTX) targeted against the nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction. The model which presumes that cobras (Naja spp., Elapidae) have lost their binding site for conspecific alpha-neurotoxins because of the unique amino acid substitutions in their nAChR polypeptide backbone per se is incompatible with the evolutionary theory that (1) the molecular motifs forming the alpha-neurotoxin target site on the nAChR are fundamental for receptor structure and/or function, and (2) the alpha-neurotoxin target site is conserved among Chordata lineages. To test the hypothesis that the alpha-neurotoxin binding site is conserved in Elapidae snakes and to identify the mechanism of resistance against conspecific alpha-neurotoxins, we cloned the ligand binding domain of the Egyptian cobra (Naja haje) nAChR alpha subunit. When expressed as part of a functional Naja/mouse chimeric nAChR in Xenopus oocytes, this domain confers resistance against alpha-BTX but does not alter responses induced by the natural ligand acetylcholine. Further mutational analysis of the Naja/mouse nAChR demonstrated that an N-glycosylation signal in the ligand binding domain that is unique to N. haje is responsible for alpha-BTX resistance. However, when the N-glycosylation signal is eliminated, the nAChR containing the N. haje sequence is inhibited by alpha-BTX with a potency that is comparable to that in mammals. We conclude that the binding site for conspecific alpha-neurotoxin in Elapidae snakes is conserved in the nAChR ligand binding domain polypeptide backbone per se. This conclusion supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. Such conservation also calls for a revision of the present model of the alpha-BTX binding site. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.

Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.