Cloud-point extraction and preconcentration of cyanobacterial toxins (microcystins) from natural waters using a cationic surfactant

A new cloud-point extraction and preconcentration method, using a cationic, surfactant, Aliquat-336 (tricaprylyl-methy;ammonium chloride), his-been developed for the determination of cyanobacterial toxins, microcystins, in natural waters. Sodium sulfate was used to induce phase separation at 25 degreesC. The phase behavior of Aliquat-336 with respect to concentration of Na2SO4 was studied. The cloud-point system revealed a very high phase volume ratio compared to other established systems of nonionic, anionic, and cationic surfactants: At pH 6-7, it showed an outstanding selectivity in ahalyte extraction for anionic species. Only MC-LR and MC-YR, which are known to be predominantly anionic, were extracted (with averaged recoveries of 113.9 +/- 9% and 87.1 +/- 7%, respectively). MC-RR, which is likely to be amphoteric at the above pH range, was. not cle tectable in.the extract. Coupled to HPLC/UV separation and detection, the cloud-point extraction method (with 2.5 mM Aliquat-336 and 75 mM Na2SO4 at 25 degreesC) offered detection limits of 150 +/- 7 and 470 +/- 72 pg/mL for MC-LR and MC-YR, respectively, in 25 mL of deionized water. Repeatability of the method was 7.6% for MC-LR and 7.3% for MC-YR: The cloud-point extraction process can be. completed within 10-15 min with no cleanup steps required. Applicability of the new method to the determination of microcystins in real samples was demonstrated using natural surface waters, collected from a local river and a local duck pond spiked with realistic. concentrations of microcystins. Effects of salinity and organic matter (TOC) content in the water sample on the extraction efficiency were also studied.

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.

Dietary selectivity for the toxic cyanobacterium Lyngbya majuscula and resultant growth rates in two species of opisthobranch mollusc

Trophodynamics of blooms of the toxic marine cyanobacterium Lyngkya majuscula were investigated to determine dietary specificity in two putative grazers: the opisthobranch molluscs, Stylocheilus striatus and Bursatella leachii. S. striatus is associated with L. majuscula blooms and is known to sequester L. majuscula metabolites. The dietary specificity and toxicodynamics of B. leachii in relation to L. majuscula is less well documented. In this study we found diet history had no significant effect upon dietary selectivity of S. striatus when offered a range of plant species. However, L. majuscula chemotype may alter S. striatus' selectivity for this cyanobacterium. Daily biomass increases between small and large size groups of both species were recorded in no-choice consumption trials using L. majuscula. Both S. striatus and B. leachii preferentially consumed L. majuscula containing lyngbyatoxin-a. Increase in mass over a 10-day period in B. leachii (915%) was significantly greater than S. striatus (150%), yet S. striatus consumed greater quantities of L. majuscula (g day(-1)) and thus had a lower conversion efficiency (0.038) than B. leachii (0.081) based on sea hare weight per gram of L. majuscula consumed day(-1). Our findings suggest that growth rates and conversion efficiencies may be influenced by sea hare maximum growth potential, acquisition of secondary metabolites or diet type. (C) 2005 Elsevier B.V. All rights reserved.

High selectivity microporous silica membranes for lactic acid dehydration

Lactic acid (LA) has significant market potential for many industries including food, cosmetics, pharmaceuticals, medical and biodegradable materials. Production of LA usually begins with the fermentation of glucose but subsequent stages for the enrichment of lactic acid are complex and energy intensive and could be minimised using water selective membrane technology. In this work, we trialled a highly selective hydrostable carbonised template molecular sieve silica (CTMSS) membrane for the dehydration of a 15 vol% aqueous lactic acid solution with 0.1 vol% glucose. CTMSS membrane films were developed by dip-coating ceramic substrates with silica sols made using the acid catalysed sol-gel process. Permeation was performed by feeding LA/glucose solution to the membrane cell at 18°C in a standard pervaporation setup. The membrane showed selective transport of water from the aqueous feed to the permeate while glucose was not detected. CTMSS membrane permeate flux stabilised at 0.2 kg.m-2.hr-1 in 3.9 hours, and reduced LA to lower than 0.2 vol%. Flux through the CTMSS micropores was activated, displaying increased initial flux to 1.58 kg.m-2.hr-1 at 60°C. To enrich a 1 l.min-1 stream to 85% LA in a single stage, a minimum membrane area of 324 m2 would be required at 18°C. Increased operating temperature to 80°C significantly reduced this area to 24 m2 but LA levels in the permeate stream increased to 0.5 vol%. The highly selective CTMSS membrane technology is an ideal candidate for LA purification. CTMSS membrane systems operate stably in aqueous systems leading to potential cost reductions in LA processing for future markets.

Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists

1 Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2 We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3 The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4 The average pIC(50) Values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC(50) values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5 The V/C ratio calculated as antilog [pIC(50)V-pIC(50)C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6 In rat small mesenteric arteries the pIC(50) values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 mu M) contraction in the rat arteries the pIC(50) values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7 In conclusion in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Spider toxins: A new group of potassium channel modulators

Spider toxins that target potassium channels constitute a new class of pharmacological tools that can be used to probe the structure and function of these channels at the molecular level. The limited studies performed to date indicate that these peptide toxins may facilitate the analysis of K+ channels that have proved insensitive to peptide inhibitors isolated from other animal sources. Thus far, two classes of K+ channel-selective spider toxins have been isolated, sequenced, and pharmacologically characterised - the hanatoxins (HaTx) from Grammastola spatulata and heteropodatoxins (HpTx) from Heteropoda venatoria. The hanatoxins block Kv2.1 and Kv4.2 voltage-gated K+ channels. In Kv2.1 K+ channels this occurs as a consequence of a depolarising shift in the voltage dependence of activation and not by occlusion of the channel pore. These toxins show minimal sequence homology with other peptide inhibitors of K+ channels, but they do share some homology with other ion channel toxins from spiders, particularly with regard to the spacing between cysteine residues. We have recently isolated three K+ channel antagonists from the venom of the Australian funnel-web spider Hadronyche versuta; at least two of these toxins are likely to constitute a new class of spider toxins active on K+ channels as they are approximately twice as large as HaTx and HpTx.

Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication, In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC50 values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nM and a maximally effective concentration caused 91% block, [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in similar to 80% of neurons, with an IC50 value of 1.4 nM and 46% maximal block of the total current, The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the cu-conotoxins PnIA or PnIB, and by mecamylamine. H-1 NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and II of PnIA and PnIB influence potency and determine selectivity among alpha 7 and other nAChR subtypes, including alpha 3 beta 2 and alpha 3 beta 4, Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.

Quantitative analysis of vascular to cardiac selectivity of L- and T-type voltage-operated calcium channel antagonists in human tissues

1. Classical L-type voltage-operated calcium channel (VOCC) antagonists dilate blood vessels, depress myocardial contractility and slow cardiac conduction. 2. We compared four L-type VOCC antagonists and a novel tetralol derivative, mibefradil, reportedly 10-fold more selective for T- (transient) over L-type VOCC in two in vitro assays of human tissue, namely isolated small arteries from the aortic vasa vasorum in a myograph and right atrial trabeculae muscle under isometric force conditions. 3. In arteries contracted with K+ (62 mmol/L), the relaxation pIC(50) values for the VOCC antagonists felodipine, nifedipine, amlodipine, verapamil and mibefradil were 8.30, 7.78, 6.64, 6.26 and 6.22, respectively. In atrial trabeculae, the pIC(50) values to inhibit the inotropic response to a submaximal concentration of isoprenaline (6 nmol/L) for felodipine, nifedipine, verapamil, amlodipine and mibefradil were 7.21, 6.95, 6.91, 5.94 and 4.61, respectively. 4. Taking the anti-log (pIC(50) vessel - pIC(50) atrium) the vascular relaxation to cardiac depression potency ratios for mibefradil, felodipine, nifedipine, amlodipine and verapamil were 41, 12, 7, 5 and 0.22, respectively. 5. We conclude that, in human tissue assays, perhaps T- over L-type VOCC selectivity confers the most favourable vascular selectivity on mibefradil. Alternatively, splice variants of L-type VOCC in the vasculature (CaV1.2b) may be more sensitive to mibefradil than the splice variants in the heart (CaV1.2a).

Molecular evolution and phylogeny of elapid snake venom three-finger toxins

Animal venom components are of considerable interest to researchers across a wide variety of disciplines, including molecular biology, biochemistry, medicine, and evolutionary genetics. The three-finger family of snake venom peptides is a particularly interesting and biochemically complex group of venom peptides, because they are encoded by a large multigene family and display a diverse array of functional activities. In addition, understanding how this complex and highly varied multigene family evolved is an interesting question to researchers investigating the biochemical diversity of these peptides and their impact on human health. Therefore, the purpose of our study was to investigate the long-term evolutionary patterns exhibited by these snake venom toxins to understand the mechanisms by which they diversified into a large, biochemically diverse, multigene family. Our results show a much greater diversity of family members than was previously known, including a number of subfamilies that did not fall within any previously identified groups with characterized activities. In addition, we found that the long-term evolutionary processes that gave rise to the diversity of three-finger toxins are consistent with the birth-and-death model of multigene family evolution. It is anticipated that this three-finger toxin toolkit will prove to be useful in providing a clearer picture of the diversity of investigational ligands or potential therapeutics available within this important family.

alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT(3) chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.