http://www.mdpi.com/journal/toxins Latest open access articles published in Toxins at http://www.mdpi.com/journal/toxins http://www.mdpi.com/2072-6651/4/6/390 Cyanobacteria (“blue-green algae”) are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems. http://www.mdpi.com/2072-6651/4/6/390 Fri, 25 May 2012 00:00:00 CEST Toxins 2012-05-25 4 6 Article 390 404 2072-6651 Effects of Cyanobacterial Lipopolysaccharides from Microcystis on Glutathione-Based Detoxification Pathways in the Zebrafish (Danio rerio) Embryo 2012-05-25 doi: 10.3390/toxins4060390 Asha Jaja-Chimedza Miroslav Gantar Gregory D. Mayer Patrick D. L. Gibbs John P. Berry http://www.mdpi.com/2072-6651/4/5/373 On the market since 1996, genetically modified plants expressing an insecticidal toxin (Cry toxin stemmed from Bacillus thuringiensis) target several lepidopteran and coleopteran pests. In this study, we assessed the impact of two varieties of Bt maize producing different toxins (Cry1Ab or Cry1Fa, respectively) on the biology of a storage pest: Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The Indianmeal moths were susceptible to both toxins but showed an escape behavior only from Cry1Fa. The weight of females issued from larvae reared on Cry1Ab increased with increasing toxin concentration, but adults of both sexes reared on Cry1Fa had decreased weight. Both toxins increased development time from egg to adult regardless of sex and had no impact on the male adult lifespan. Finally, we recorded a time lag between metamorphosis from the non-Bt and the Bt diets, which increased proportionally to Cry concentration in the Bt diet. http://www.mdpi.com/2072-6651/4/5/373 Thu, 24 May 2012 00:00:00 CEST Toxins 2012-05-24 4 5 Article 373 389 2072-6651 Effects of Two Varieties of Bacillus thuringiensis Maize on the Biology of Plodia interpunctella 2012-05-24 doi: 10.3390/toxins4050373 Aiko Gryspeirt Jean-Claude Grégoire http://www.mdpi.com/2072-6651/4/5/364 Ochratoxin A (OTA) is a mycotoxin commonly present in cereals, grapes, coffee, spices, and cocoa. Even though the main objective of the food and feed chain processors and distributors is to avoid the extended contamination of plant-derived foods and animal feeds with mycotoxins, until now, complete OTA removal from foods and feedstuffs is not feasible. Prevention through pre-harvest management is the best method for controlling mycotoxin contamination. However, in the case that the contamination occurs after this stage, the hazards associated with OTA must be managed through post-harvest strategies. Due to the increasing number of fungal strains resistant to chemical fungicides and the impact of these pesticides on the environment and human health, maximum levels of chemical residues have been regulated in many products. Alternative methods are necessary to substitute or complement treatments with fungicides to control fungi under field or storage conditions. Yeasts are considered one of the most potent biocontrol agents due to their biology and non-toxic properties. Epiphytic yeasts are the major component of the microbial community on the surface of grape berries and they are evolutionarily adapted to this ecological niche. Nowadays, several yeast species included in different genera are considered as potential biocontrol agents to control both, growth of ochratoxigenic Aspergillus species and OTA accumulation. http://www.mdpi.com/2072-6651/4/5/364 Mon, 14 May 2012 00:00:00 CEST Toxins 2012-05-14 4 5 Review 364 372 2072-6651 Control of Ochratoxin A Production in Grapes 2012-05-14 doi: 10.3390/toxins4050364 María Lorena Ponsone María Laura Chiotta Juan Manuel Palazzini Mariana Combina Sofía Chulze http://www.mdpi.com/2072-6651/4/5/353 The aim of the present study was to evaluate the effect of long-term cyanidin 3-O-β-D-glucoside (C3G) and/or Ochratoxin A (OTA)-exposure on dimethylarginine dimethylamino hydrolase/nitric oxide synthase (DDAH/NOS) pathway in rats. The experiments were performed in rats supplemented with C3G (1 g/kg feed), OTA (200 ppb), and OTA + C3G. After 4 weeks of daily treatment, liver and kidneys were processed for eNOS, iNOS and DDAH-1 Western blotting, nitrite levels evaluation and DDAH activity determination. Results show that OTA is able to induce iNOS both in kidney and liver, whereas OTA is able to induce eNOS and DDAH-1 overexpression and DDAH activation only in kidney, resulting in increased nitrite levels. In kidney of OTA + C3G fed rats, iNOS, eNOS and DDAH-1 expression were less pronounced compared with those observed in the OTA-treated group. Coherent with the decreased iNOS, eNOS and DDAH-1 expression a decrease in nitrite levels and DDAH activity was observed in the OTA + C3G group. Results demonstrate that C3G is able to counteract the deleterious effects of chronic consumption of OTA and also suggest a possible involvement of iNOS-eNOS-DDAH impairment in OTA nephrocarcinogenity. http://www.mdpi.com/2072-6651/4/5/353 Tue, 08 May 2012 00:00:00 CEST Toxins 2012-05-08 4 5 Article 353 363 2072-6651 Dimethylarginine Dimethylaminohydrolase/Nitric Oxide Synthase Pathway in Liver and Kidney: Protective Effect of Cyanidin 3-O-β-D-Glucoside on Ochratoxin-A Toxicity 2012-05-08 doi: 10.3390/toxins4050353 Valeria Sorrenti Claudia Di Giacomo Rosaria Acquaviva Matteo Bognanno Ester Grilli Nicolantonio D’Orazio Fabio Galvano http://www.mdpi.com/2072-6651/4/5/339 A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC50 values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78–101%; king scallop, 98–114%), DTX-1 (king scallop, 79–102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS. http://www.mdpi.com/2072-6651/4/5/339 Mon, 30 Apr 2012 00:00:00 CEST Toxins 2012-04-30 4 5 Article 339 352 2072-6651 Single Laboratory Validation of A Ready-to-Use Phosphatase Inhibition Assay for Detection of Okadaic Acid Toxins 2012-04-30 doi: 10.3390/toxins4050339 Henry G. F. Smienk Dolores Calvo Pedro Razquin Elena Domínguez Luis Mata http://www.mdpi.com/2072-6651/4/5/323 A divalent cation-independent lectin—HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAcβ1-4GlcNAcβ1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4–12 and temperatures lower than 60 °C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner. http://www.mdpi.com/2072-6651/4/5/323 Mon, 30 Apr 2012 00:00:00 CEST Toxins 2012-04-30 4 5 Article 323 338 2072-6651 Cytotoxicity and Glycan-Binding Properties of an 18 kDa Lectin Isolated from the Marine Sponge Halichondria okadai 2012-04-30 doi: 10.3390/toxins4050323 Ryo Matsumoto Yuki Fujii Sarkar M. A. Kawsar Robert A. Kanaly Hidetaro Yasumitsu Yasuhiro Koide Imtiaj Hasan Chihiro Iwahara Yukiko Ogawa Chang Hun Im Shigeki Sugawara Masahiro Hosono Kazuo Nitta Jiharu Hamako Taei Matsui Yasuhiro Ozeki http://www.mdpi.com/2072-6651/4/5/296 Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyomma sp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely available for download (right click with the mouse) at links http://exon.niaid.nih.gov/transcriptome/RGD/RGD-Peps-WEB.xlsx (web version) and http://exon.niaid.nih.gov/transcriptome/RGD/RGD-sialogenins.zip (stand alone version). http://www.mdpi.com/2072-6651/4/5/296 Thu, 26 Apr 2012 00:00:00 CEST Toxins 2012-04-26 4 5 Review 296 322 2072-6651 Disintegrins from Hematophagous Sources 2012-04-26 doi: 10.3390/toxins4050296 Teresa C. F. Assumpcao José M. C. Ribeiro Ivo M. B. Francischetti http://www.mdpi.com/2072-6651/4/4/281 It is recognized that mycotoxins can cause a variety of adverse health effects in animals, including altered gastrointestinal barrier function. It is the aim of the present study to determine whether mycotoxin-contaminated diets can alter the oral bioavailability of the antibiotics doxycycline and paromomycin in pigs, and whether a mycotoxin adsorbing agent included into diets interacts with those antibiotics. Experiments were conducted with pigs utilizing diets that contained blank feed, mycotoxin-contaminated feed (T-2 toxin or deoxynivalenol), mycotoxin-contaminated feed supplemented with a glucomannan mycotoxin binder, or blank feed supplemented with mycotoxin binder. Diets with T-2 toxin and binder or deoxynivalenol and binder induced increased plasma concentrations of doxycycline administered as single bolus in pigs compared to diets containing blank feed. These results suggest that complex interactions may occur between mycotoxins, mycotoxin binders, and antibiotics which could alter antibiotic bioavailability. This could have consequences for animal toxicity, withdrawal time for oral antibiotics, or public health. http://www.mdpi.com/2072-6651/4/4/281 Tue, 24 Apr 2012 00:00:00 CEST Toxins 2012-04-24 4 4 Article 281 295 2072-6651 Influence of Mycotoxins and a Mycotoxin Adsorbing Agent on the Oral Bioavailability of Commonly Used Antibiotics in Pigs 2012-04-24 doi: 10.3390/toxins4040281 Joline Goossens Virginie Vandenbroucke Frank Pasmans Siegrid De Baere Mathias Devreese Ann Osselaere Elin Verbrugghe Freddy Haesebrouck Sarah De Saeger Mia Eeckhout Kris Audenaert Geert Haesaert Patrick De Backer Siska Croubels http://www.mdpi.com/2072-6651/4/4/267 Ochratoxin A (OTA) is a mycotoxin that enhances renal tumor formation in the outer medulla of male rat kidney. Direct DNA damage and subsequent mutagenicity may contribute to these processes. In this study we have determined whether OTA in the absence or presence of activated rat liver microsomes (RLM) or redox-active transition metals (Fe(III) or Cu(II)) causes promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. In addition, we have assessed the mutagenicity of the hydroquinone metabolite (OTHQ) of OTA in the absence or presence of cysteine without added cofactors. Our results show that oxidation of OTA, either by RLM or by transition metal ions, activates OTA to a directly genotoxic mutagen(s). The Fe(III)/OTA system was the most potent mutagen in our experimental system, causing a 32-fold increase in mutant fraction (MF) above the spontaneous control MF. The Cu(II)/OTA system caused a 9-fold increase in MF, while a 6–10-fold increase in MF was observed for OTA in the presence of RLM. The OTHQ metabolite is also mutagenic, especially in the presence of cysteine, in which a 6-fold increase in MF was observed. Our data provide further insight into OTA bioactivation that may account for its in vivo mutagenicity in male rat kidney. http://www.mdpi.com/2072-6651/4/4/267 Mon, 16 Apr 2012 00:00:00 CEST Toxins 2012-04-16 4 4 Article 267 280 2072-6651 Mutagenicity of Ochratoxin A and Its Hydroquinone Metabolite in the SupF Gene of the Mutation Reporter Plasmid Ps189 2012-04-16 doi: 10.3390/toxins4040267 Steven A. Akman Marissa Adams Doug Case Gyungse Park Richard A. Manderville http://www.mdpi.com/2072-6651/4/4/244 The safety of food and feed depends to a great deal on quality control. Numerous compounds and organisms may contaminate food and feed commodities and thus pose a health risk for consumers. The compound of interest in this review is ochratoxin A (OTA), a secondary metabolite of the fungi Aspergillus and Penicillium. Due to its adverse health effects, detection and quantification are of utmost importance. Quality control of food and feed requires extraction and analysis, including TLC, HPLC, MS, and immunochemical methods. Each of these methods has its advantages and disadvantages. However, with regard to costs and rapidity, immunochemical methods have gained much interest in the last decade. In this review an introduction to immunochemistry and assay design will be given to elucidate the principles. Further, the application of the various formats to the detection and quantification of ochratoxin will be described, including the use of commercially available kits. http://www.mdpi.com/2072-6651/4/4/244 Fri, 13 Apr 2012 00:00:00 CEST Toxins 2012-04-13 4 4 Review 244 266 2072-6651 Immunochemical Methods for Ochratoxin A Detection: A Review 2012-04-13 doi: 10.3390/toxins4040244 Eline P. Meulenberg http://www.mdpi.com/2072-6651/4/4/228 Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects’ vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology. http://www.mdpi.com/2072-6651/4/4/228 Tue, 10 Apr 2012 00:00:00 CEST Toxins 2012-04-10 4 4 Review 228 243 2072-6651 Plant Insecticidal Toxins in Ecological Networks 2012-04-10 doi: 10.3390/toxins4040228 Sébastien Ibanez Christiane Gallet Laurence Després http://www.mdpi.com/2072-6651/4/3/191 Over 10,000 arthropod species are currently considered to be pest organisms. They are estimated to contribute to the destruction of ~14% of the world’s annual crop production and transmit many pathogens. Presently, arthropod pests of agricultural and health significance are controlled predominantly through the use of chemical insecticides. Unfortunately, the widespread use of these agrochemicals has resulted in genetic selection pressure that has led to the development of insecticide-resistant arthropods, as well as concerns over human health and the environment. Bioinsecticides represent a new generation of insecticides that utilise organisms or their derivatives (e.g., transgenic plants, recombinant baculoviruses, toxin-fusion proteins and peptidomimetics) and show promise as environmentally-friendly alternatives to conventional agrochemicals. Spider-venom peptides are now being investigated as potential sources of bioinsecticides. With an estimated 100,000 species, spiders are one of the most successful arthropod predators. Their venom has proven to be a rich source of hyperstable insecticidal mini-proteins that cause insect paralysis or lethality through the modulation of ion channels, receptors and enzymes. Many newly characterized insecticidal spider toxins target novel sites in insects. Here we review the structure and pharmacology of these toxins and discuss the potential of this vast peptide library for the discovery of novel bioinsecticides. http://www.mdpi.com/2072-6651/4/3/191 Thu, 22 Mar 2012 00:00:00 CET Toxins 2012-03-22 4 3 Review 191 227 2072-6651 Spider-Venom Peptides as Bioinsecticides 2012-03-22 doi: 10.3390/toxins4030191 Monique J. Windley Volker Herzig Sławomir A. Dziemborowicz Margaret C. Hardy Glenn F. King Graham M. Nicholson http://www.mdpi.com/2072-6651/4/3/157 Shiga toxins and ricin are well characterized similar toxins belonging to quite different biological kingdoms. Plant and bacteria have evolved the ability to produce these powerful toxins in parallel, while humans have evolved a defense system that recognizes molecular patterns common to foreign molecules through specific receptors expressed on the surface of the main actors of innate immunity, namely monocytes and neutrophils. The interactions between these toxins and neutrophils have been widely described and have stimulated intense debate. This paper is aimed at reviewing the topic, focusing particularly on implications for the pathogenesis and diagnosis of hemolytic uremic syndrome. http://www.mdpi.com/2072-6651/4/3/157 Thu, 01 Mar 2012 00:00:00 CET Toxins 2012-03-01 4 3 Review 157 190 2072-6651 The Interactions of Human Neutrophils with Shiga Toxins and Related Plant Toxins: Danger or Safety? 2012-03-01 doi: 10.3390/toxins4030157 Maurizio Brigotti http://www.mdpi.com/2072-6651/4/2/139 Cyclotides are plant mini-proteins whose natural function is thought to be to protect plants from pest or pathogens, particularly insect pests. They are approximately 30 amino acids in size and are characterized by a cyclic peptide backbone and a cystine knot arrangement of three conserved disulfide bonds. This article provides an overview of the reported pesticidal or toxic activities of cyclotides, discusses a possible common mechanism of action involving disruption of biological membranes in pest species, and describes methods that can be used to produce cyclotides for potential applications as novel pesticidal agents. http://www.mdpi.com/2072-6651/4/2/139 Wed, 15 Feb 2012 00:00:00 CET Toxins 2012-02-15 4 2 Review 139 156 2072-6651 Host-Defense Activities of Cyclotides 2012-02-15 doi: 10.3390/toxins4020139 David J. Craik http://www.mdpi.com/2072-6651/4/2/110 During the development of selective peptides against highly homologous targets, a reliable tool is sought that can predict information on both mechanisms of binding and relative affinities. These tools must first be tested on known profiles before application on novel therapeutic candidates. We therefore present a comparative docking protocol in HADDOCK using critical motifs, and use it to “predict” the various selectivity profiles of several major αKTX scorpion toxin families versus Kv1.1, Kv1.2 and Kv1.3. By correlating results across toxins of similar profiles, a comprehensive set of functional residues can be identified. Reasonable models of channel-toxin interactions can be then drawn that are consistent with known affinity and mutagenesis. Without biological information on the interaction, HADDOCK reproduces mechanisms underlying the universal binding of αKTX-2 toxins, and Kv1.3 selectivity of αKTX-3 toxins. The addition of constraints encouraging the critical lysine insertion confirms these findings, and gives analogous explanations for other families, including models of partial pore-block in αKTX-6. While qualitatively informative, the HADDOCK scoring function is not yet sufficient for accurate affinity-ranking. False minima in low-affinity complexes often resemble true binding in high-affinity complexes, despite steric/conformational penalties apparent from visual inspection. This contamination significantly complicates energetic analysis, although it is usually possible to obtain correct ranking via careful interpretation of binding-well characteristics and elimination of false positives. Aside from adaptations to the broader potassium channel family, we suggest that this strategy of comparative docking can be extended to other channels of interest with known structure, especially in cases where a critical motif exists to improve docking effectiveness. http://www.mdpi.com/2072-6651/4/2/110 Mon, 06 Feb 2012 00:00:00 CET Toxins 2012-02-06 4 2 Article 110 138 2072-6651 Developing a Comparative Docking Protocol for the Prediction of Peptide Selectivity Profiles: Investigation of Potassium Channel Toxins 2012-02-06 doi: 10.3390/toxins4020110 Po-Chia Chen Serdar Kuyucak http://www.mdpi.com/2072-6651/4/2/98 The ability of a cyclodextrin-polyurethane polymer to remove ochratoxin A from aqueous solutions was examined by batch rebinding assays. The results from the aqueous binding studies were fit to two parameter models to gain insight into the interaction of ochratoxin A with the nanosponge material. The ochratoxin A sorption data fit well to the heterogeneous Freundlich isotherm model. The polymer was less effective at binding ochratoxin A in high pH buffer (9.5) under conditions where ochratoxin A exists predominantly in the dianionic state. Batch rebinding assays in red wine indicate the polymer is able to remove significant levels of ochratoxin A from spiked solutions between 1–10 μg·L−1. These results suggest cyclodextrin nanosponge materials are suitable to reduce levels of ochratoxin A from spiked aqueous solutions and red wine samples. http://www.mdpi.com/2072-6651/4/2/98 Mon, 06 Feb 2012 00:00:00 CET Toxins 2012-02-06 4 2 Communication 98 109 2072-6651 Sorption of Ochratoxin A from Aqueous Solutions Using β-Cyclodextrin-Polyurethane Polymer 2012-02-06 doi: 10.3390/toxins4020098 Michael Appell Michael A. Jackson http://www.mdpi.com/2072-6651/4/2/83 Bacillus anthracis, the agent of anthrax, has gained virulence through its exotoxins produced by vegetative bacilli and is composed of three components forming lethal toxin (LT) and edema toxin (ET). So far, little is known about the effects of these toxins on the eukaryotic cytoskeleton. Here, we provide an overview on the general effects of toxin upon the cytoskeleton architecture. Thus, we shall discuss how anthrax toxins interact with their receptors and may disrupt the interface between extracellular matrix and the cytoskeleton. We then analyze what toxin molecular effects on cytoskeleton have been described, before discussing how the cytoskeleton may help the pathogen to corrupt general cell processes such as phagocytosis or vascular integrity. http://www.mdpi.com/2072-6651/4/2/83 Mon, 06 Feb 2012 00:00:00 CET Toxins 2012-02-06 4 2 Review 83 97 2072-6651 Cytoskeleton as an Emerging Target of Anthrax Toxins 2012-02-06 doi: 10.3390/toxins4020083 Yannick Trescos Jean-Nicolas Tournier http://www.mdpi.com/2072-6651/4/2/68 Twelve yeast strains isolated from the surface of Italian typical dry-cured hams, belonging to D. hansenii, D. maramus, C. famata, C. zeylanoides and H. burtonii species, and previously selected for their ability to grow in dry-cured ham-like substrates, were screened for antagonistic activity against a toxigenic strain of P. nordicum and inhibition of ochratoxin A (OTA) biosynthesis. On average, yeast inhibitory activity was lowered by increasing fungal inoculum and enhanced by NaCl presence. In the assay conditions, H. burtonii and C. zeylanoides were the most effective, both in inhibiting P. nordicum growth and OTA production. D. hansenii was the species with the lowest inhibitory activity, especially in the absence of salt. OTA production dropped from the range < LOD − 5000 ppb in P. nordicum control plates to the range < LOD − 200 ppb in yeast-added plates. OTA production increased in the presence of NaCl in P. nordicum control plates, while salt enhanced inhibition against OTA production in yeast-added plates. http://www.mdpi.com/2072-6651/4/2/68 Wed, 01 Feb 2012 00:00:00 CET Toxins 2012-02-01 4 2 Article 68 82 2072-6651 Biocontrol of Penicillium nordicum Growth and Ochratoxin A Production by Native Yeasts of Dry Cured Ham 2012-02-01 doi: 10.3390/toxins4020068 Roberta Virgili Nicoletta Simoncini Tania Toscani Marco Camardo Leggieri Silvia Formenti Paola Battilani http://www.mdpi.com/2072-6651/4/2/55 Recently, ureases were included in the arsenal of plant defense proteins, alongside many other proteins with biotechnological potential such as insecticides. Isoforms of Canavalia ensiformis urease (canatoxin—CNTX and jack bean urease—JBURE-I) are toxic to insects of different orders. This toxicity is due in part to the release of a 10 kDa peptide from the native protein, by cathepsin-like enzymes present in the insect digestive tract. The entomotoxic peptide, Jaburetox-2Ec, exhibits potent insecticidal activity against several insects, including many resistant to the native ureases. JBURE-I and Jaburetox-2Ec cause major alterations of post-feeding physiological processes in insects, which contribute to, or can be the cause of, their entomotoxic effect. An overview of the current knowledge on plant urease processing and mechanisms of action in insects is presented in this review. http://www.mdpi.com/2072-6651/4/2/55 Wed, 01 Feb 2012 00:00:00 CET Toxins 2012-02-01 4 2 Review 55 67 2072-6651 Plant Ureases and Related Peptides: Understanding Their Entomotoxic Properties 2012-02-01 doi: 10.3390/toxins4020055 Fernanda Stanisçuaski Célia R. Carlini http://www.mdpi.com/2072-6651/4/2/42 A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP) and tested for ochratoxin A (OTA) production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%), while A. carbonarius was the major species during 2007 (46.6%). All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES), as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7%) isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46) in both years, but also some strains of A. tubingensis (11) and A. japonicus (6) produced lower amounts of OTA. http://www.mdpi.com/2072-6651/4/2/42 Mon, 30 Jan 2012 00:00:00 CET Toxins 2012-01-30 4 2 Article 42 54 2072-6651 Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure 2012-01-30 doi: 10.3390/toxins4020042 Davide Spadaro Subban Patharajan Alessia Lorè Angelo Garibaldi Maria Lodovica Gullino http://www.mdpi.com/2072-6651/4/1/28 Lethal toxin (LT) of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8) is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb) with toxin-neutralising (TN) activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection. http://www.mdpi.com/2072-6651/4/1/28 Thu, 19 Jan 2012 00:00:00 CET Toxins 2012-01-19 4 1 Article 28 41 2072-6651 Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies 2012-01-19 doi: 10.3390/toxins4010028 Gail Whiting Michael Baker Sjoerd Rijpkema http://www.mdpi.com/2072-6651/4/1/15 Throughout the last decade, efforts to identify and develop effective inhibitors of the ricin toxin have focused on targeting its N-glycosidase activity. Alternatively, molecules disrupting intracellular trafficking have been shown to block ricin toxicity. Several research teams have recently developed high-throughput phenotypic screens for small molecules acting on the intracellular targets required for entry of ricin into cells. These screens have identified inhibitory compounds that can protect cells, and sometimes even animals against ricin. We review these newly discovered cellular inhibitors of ricin intoxication, discuss the advantages and drawbacks of chemical-genetics approaches, and address the issues to be resolved so that the therapeutic development of these small-molecule compounds can progress. http://www.mdpi.com/2072-6651/4/1/15 Fri, 06 Jan 2012 00:00:00 CET Toxins 2012-01-06 4 1 Review 15 27 2072-6651 Inhibitors of the Cellular Trafficking of Ricin 2012-01-06 doi: 10.3390/toxins4010015 Julien Barbier Céline Bouclier Ludger Johannes Daniel Gillet http://www.mdpi.com/2072-6651/4/1/1 Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD50 value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD50 values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure. http://www.mdpi.com/2072-6651/4/1/1 Fri, 30 Dec 2011 00:00:00 CET Toxins 2011-12-30 4 1 Article 1 14 2072-6651 Investigations into the Toxicology of Spirolides, a Group of Marine Phycotoxins 2011-12-30 doi: 10.3390/toxins4010001 Rex Munday Michael A. Quilliam Patricia LeBlanc Nancy Lewis Pamela Gallant Sandra A. Sperker H. Stephen Ewart Shawna L. MacKinnon http://www.mdpi.com/2072-6651/3/12/1569 Up to 60 species of fungi in the Botryosphaeriaceae family, genera Cadophora, Cryptovalsa, Cylindrocarpon, Diatrype, Diatrypella, Eutypa, Eutypella, Fomitiporella, Fomitiporia, Inocutis, Phaeoacremonium and Phaeomoniella have been isolated from decline-affected grapevines all around the World. The main grapevine trunk diseases of mature vines are Eutypa dieback, the esca complex and cankers caused by the Botryospheriaceae, while in young vines the main diseases are Petri and black foot diseases. To understand the mechanism of these decline-associated diseases and the symptoms associated with them, the toxins produced by the pathogens involved in these diseases were isolated and characterised chemically and biologically. So far the toxins of only a small number of these decline fungi have been studied. This paper presents an overview of the toxins produced by the most serious of these vine wood pathogens: Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and some taxa in the Botryosphaeriaceae family, and examines how these toxins produce decline symptoms. The chemical structure of these metabolites and in some cases their vivotoxin nature are also discussed. http://www.mdpi.com/2072-6651/3/12/1569 Tue, 20 Dec 2011 00:00:00 CET Toxins 2011-12-20 3 12 Review 1569 1605 2072-6651 Phytotoxins Produced by Fungi Associated with Grapevine Trunk Diseases 2011-12-20 doi: 10.3390/toxins3121569 Anna Andolfi Laura Mugnai Jordi Luque Giuseppe Surico Alessio Cimmino Antonio Evidente http://www.mdpi.com/2072-6651/3/12/1554 The trichothecenes produced by solid and liquid cultures of Fusarium sporotrichioides were evaluated with high performance liquid chromatography—tandem mass spectrometry (LC-MS/MS). Along with the expected T-2 toxin HT-2 toxin and neosolaniol, two additional compounds were detected, which had ions 162 m/z higher than those in the mass spectra of T-2 toxin or HT-2 toxin. Fragmentation behavior of these two compounds was similar to that of T-2 toxin and HT-2 toxin. Based on LC-MS/MS behavior, it is proposed that the two compounds are T-2 toxin 3-O-glucoside and HT-2 toxin 3-O-glucoside. Production of the two glucosides was measured in kernels from wheat and oat inoculated with F. sporotrichiodes, as well as in cultures grown in liquid media and on cracked corn or rice. Production of glucosides in wheat and oats suggest that they may also be present in naturally contaminated cereals. http://www.mdpi.com/2072-6651/3/12/1554 Tue, 20 Dec 2011 00:00:00 CET Toxins 2011-12-20 3 12 Article 1554 1568 2072-6651 Observation of T-2 Toxin and HT-2 Toxin Glucosides from Fusarium sporotrichioides by Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS/MS) 2011-12-20 doi: 10.3390/toxins3121554 Mark Busman Stephen M. Poling Chris M. Maragos http://www.mdpi.com/2072-6651/3/12/1518 Fungal toxins, such as those produced by members of the order Hypocreales, have widespread effects on cereal crops, resulting in yield losses and the potential for severe disease and mortality in humans and livestock. Among the most toxic are the trichothecenes. Trichothecenes have various detrimental effects on eukaryotic cells including an interference with protein production and the disruption of nucleic acid synthesis. However, these toxins can have a wide range of toxicity depending on the system. Major differences in the phytotoxicity and cytotoxicity of these mycotoxins are observed for individual members of the class, and variations in toxicity are observed among different species for each individual compound. Furthermore, while diverse toxicological effects are observed throughout the whole cellular system upon trichothecene exposure, the mechanism of toxicity is not well understood. In order to comprehend how these toxins interact with the cell, we must first have an advanced understanding of their structure and dynamics. The structural analysis of trichothecenes was a subject of major interest in the 1980s, and primarily focused on crystallographic and solution-state Nuclear Magnetic Resonance (NMR) spectroscopic studies. Recent advances in structural determination through solution- and solid-state NMR, as well as computation based molecular modeling is leading to a resurgent interest in the structure of these and other mycotoxins, with the focus shifting in the direction of structural dynamics. The purpose of this work is to first provide a brief overview of the structural data available on trichothecenes and a characterization of the methods commonly employed to obtain such information. A summary of the current understanding of the relationship between structure and known function of these compounds is also presented. Finally, a prospectus on the application of new emerging structural methods on these and other related systems is discussed. http://www.mdpi.com/2072-6651/3/12/1518 Mon, 19 Dec 2011 00:00:00 CET Toxins 2011-12-19 3 12 Review 1518 1553 2072-6651 Current and Future Experimental Strategies for Structural Analysis of Trichothecene Mycotoxins—A Prospectus 2011-12-19 doi: 10.3390/toxins3121518 Roxanne A. Shank Nora A. Foroud Paul Hazendonk François Eudes Barbara A. Blackwell http://www.mdpi.com/2072-6651/3/12/1502 PA1b (Pea Albumin 1, subunit b) is a peptide extract from pea seeds showing significant insecticidal activity against certain insects, such as cereal weevils (genus Sitophilus), the mosquitoes Culex pipiens and Aedes aegyptii, and certain species of aphids. PA1b has great potential for use on an industrial scale and for use in organic farming: it is extracted from a common plant; it is a peptide (and therefore suitable for transgenic applications); it can withstand many steps of extraction and purification without losing its activity; and it is present in a seed regularly consumed by humans and mammals without any known toxicity or allergenicity. The potential of this peptide to limit pest damage has stimulated research concerning its host range, its mechanism of action, its three-dimensional structure, the natural diversity of PA1b and its structure–function relationships. http://www.mdpi.com/2072-6651/3/12/1502 Thu, 08 Dec 2011 00:00:00 CET Toxins 2011-12-08 3 12 Review 1502 1517 2072-6651 Pea Albumin 1 Subunit b (PA1b), a Promising Bioinsecticide of Plant Origin 2011-12-08 doi: 10.3390/toxins3121502 Frédéric Gressent Pedro Da Silva Vanessa Eyraud Lamis Karaki Corinne Royer http://www.mdpi.com/2072-6651/3/12/1484 Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes. http://www.mdpi.com/2072-6651/3/12/1484 Fri, 02 Dec 2011 00:00:00 CET Toxins 2011-12-02 3 12 Article 1484 1501 2072-6651 Trichothecene Mycotoxins Inhibit Mitochondrial Translation—Implication for the Mechanism of Toxicity 2011-12-02 doi: 10.3390/toxins3121484 Mohamed Anwar Bin-Umer John E. McLaughlin Debaleena Basu Susan McCormick Nilgun E. Tumer http://www.mdpi.com/2072-6651/3/11/1453 Fungicide application is a key factor in the control of mycotoxin contamination in the harvested wheat grain. However, the practical results are often disappointing. In 2000–2004, 2006–2008 and 2007 and 2008, three experiments were made to test the efficacy of fungicide control on Fusarium Head Blight (FHB) in wheat and to find ways to improve control of the disease and toxin contamination. In a testing system we have used for 20 years, tebuconazole and tebuconazole + prothioconazole fungicides regularly reduced symptoms by about 80% with a correlating reduction in toxin contamination. Averages across the years normally show a correlation of r = 0.90 or higher. The stability differences (measured by the stability index) between the poorest and the best fungicides are about 10 or more times, differing slightly in mycotoxin accumulation, FHB index (severity) and Fusarium damaged kernels (FDK). The weak fungicides, like carbendazim, were effective only when no epidemic occurred or epidemic severity was at a very low level. Similar fungicide effects were seen on wheat cultivars which varied in FHB resistance. In this study, we found three fold differences in susceptibility to FHB between highly susceptible and moderately resistant cultivars when treated with fungicides. In the moderately resistant cultivars, about 50% of the fungicide treatments lowered the DON level below the regulatory limit. In the most susceptible cultivars, all fungicides failed to reduce mycotoxin levels low enough for grain acceptance, in spite of the fact that disease was significantly reduced. The results correlated well with the results of the large-scale field tests of fungicide application at the time of natural infection. The Turbo FloodJet nozzle reduced FHB incidence and DON contamination when compared to the TeeJet XR nozzle. Overall, the data suggest that significant decreases in FHB incidence and deoxynivalenol contamination in field situations are possible with proper fungicide applications. Additionally, small plot tests can be used to evaluate the quality of the field disease and toxin production. http://www.mdpi.com/2072-6651/3/11/1453 Wed, 16 Nov 2011 00:00:00 CET Toxins 2011-11-16 3 11 Article 1453 1483 2072-6651 Role of Fungicides, Application of Nozzle Types, and the Resistance Level of Wheat Varieties in the Control of Fusarium Head Blight and Deoxynivalenol 2011-11-16 doi: 10.3390/toxins3111453 Ákos Mesterházy Beáta Tóth Monika Varga Tibor Bartók Ágnes Szabó-Hevér László Farády Szabolcs Lehoczki-Krsjak http://www.mdpi.com/2072-6651/3/11/1433 B. anthracis is a bioweapon of primary importance and its pathogenicity depends on its lethal and edema toxins, which belong to the A-B model of bacterial toxins, and on its capsule. These toxins are secreted early in the course of the anthrax disease and for this reason antibiotics must be administered early, in addition to other limitations. Antibodies (Abs) may however neutralize those toxins and target this capsule to improve anthrax treatment, and many Abs have been developed in that perspective. These Abs act at various steps of the cell intoxication and their mechanisms of action are detailed in the present review, presented in correlation with structural and functional data. The potential for clinical application is discussed for Abs targeting each step of entry, with four of these molecules already advancing to clinical trials. Paradoxically, certain Abs may also enhance the lethal toxin activity and this aspect will also be presented. The unique paradigm of Abs neutralizing anthrax toxins thus exemplifies how they may act to neutralize A-B toxins and, more generally, be active against infectious diseases. http://www.mdpi.com/2072-6651/3/11/1433 Wed, 16 Nov 2011 00:00:00 CET Toxins 2011-11-16 3 11 Review 1433 1452 2072-6651 Antibodies against Anthrax: Mechanisms of Action and Clinical Applications 2011-11-16 doi: 10.3390/toxins3111433 Jeffrey W. Froude II Philippe Thullier Thibaut Pelat http://www.mdpi.com/2072-6651/3/11/1420 In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation. http://www.mdpi.com/2072-6651/3/11/1420 Mon, 14 Nov 2011 00:00:00 CET Toxins 2011-11-14 3 11 Review 1420 1432 2072-6651 Pharmacological Aspects of Vipera xantina palestinae Venom 2011-11-14 doi: 10.3390/toxins3111420 Tatjana Momic Franziska T. Arlinghaus Hadar Arien-Zakay Jeoshua Katzhendler Johannes A. Eble Cezary Marcinkiewicz Philip Lazarovici http://www.mdpi.com/2072-6651/3/11/1405 Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. http://www.mdpi.com/2072-6651/3/11/1405 Fri, 11 Nov 2011 00:00:00 CET Toxins 2011-11-11 3 11 Article 1405 1419 2072-6651 Llama-Derived Single Domain Antibodies Specific for Abrus Agglutinin 2011-11-11 doi: 10.3390/toxins3111405 Ellen R. Goldman George P. Anderson Dan Zabetakis Scott Walper Jinny L. Liu Rachael Bernstein Alena Calm James P. Carney Thomas W. O’Brien Jennifer L. Walker Eric A. E. Garber http://www.mdpi.com/2072-6651/3/11/1393 Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M1 muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M1 receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M5 subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M1 receptor. An M5 receptor construct (M5-E175Y184E474), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent Ki = 24 nM; apparent Ki for M1 = 0.5 nM). After screening for different domains and residues, we found a specific residue (P179 to L in M5) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M5-EL179YE, apparent Ki = 0.5 nM). Mutation of P179 to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors. http://www.mdpi.com/2072-6651/3/11/1393 Fri, 11 Nov 2011 00:00:00 CET Toxins 2011-11-11 3 11 Article 1393 1404 2072-6651 Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein 2011-11-11 doi: 10.3390/toxins3111393 Sergio Rondinelli Katja Näreoja Johnny Näsman http://www.mdpi.com/2072-6651/3/11/1373 The toxin ricin has long been understood to have potential for criminal activity and there has been concern that it might be used as a mass-scale weapon on a military basis for at least two decades. Currently, the focus has extended to encompass terrorist activities using ricin to disrupt every day activities on a smaller scale. Whichever scenario is considered, there are features in common which need to be understood; these include the knowledge of the toxicity from ricin poisoning by the likely routes, methods for the detection of ricin in relevant materials and approaches to making an early diagnosis of ricin poisoning, in order to take therapeutic steps to mitigate the toxicity. This article will review the current situation regarding each of these stages in our collective understanding of ricin and how to defend against its use by an aggressor. http://www.mdpi.com/2072-6651/3/11/1373 Fri, 04 Nov 2011 00:00:00 CET Toxins 2011-11-04 3 11 Review 1373 1392 2072-6651 Understanding Ricin from a Defensive Viewpoint 2011-11-04 doi: 10.3390/toxins3111373 Gareth D. Griffiths http://www.mdpi.com/2072-6651/3/10/1332 Accidental and intended Ricinus communis intoxications in humans and animals have been known for centuries but the causative agent remained elusive until 1888 when Stillmark attributed the toxicity to the lectin ricin. Ricinus communis is grown worldwide on an industrial scale for the production of castor oil. As by-product in castor oil production ricin is mass produced above 1 million tons per year. On the basis of its availability, toxicity, ease of preparation and the current lack of medical countermeasures, ricin has gained attention as potential biological warfare agent. The seeds also contain the less toxic, but highly homologous Ricinus communis agglutinin and the alkaloid ricinine, and especially the latter can be used to track intoxications. After oil extraction and detoxification, the defatted press cake is used as organic fertilizer and as low-value feed. In this context there have been sporadic reports from different countries describing animal intoxications after uptake of obviously insufficiently detoxified fertilizer. Observations in Germany over several years, however, have led us to speculate that the detoxification process is not always performed thoroughly and controlled, calling for international regulations which clearly state a ricin threshold in fertilizer. In this review we summarize knowledge on intended and unintended poisoning with ricin or castor seeds both in humans and animals, with a particular emphasis on intoxications due to improperly detoxified castor bean meal and forensic analysis. http://www.mdpi.com/2072-6651/3/10/1332 Mon, 24 Oct 2011 00:00:00 CEST Toxins 2011-10-24 3 10 Review 1332 1372 2072-6651 Ricinus communis Intoxications in Human and Veterinary Medicine—A Summary of Real Cases 2011-10-24 doi: 10.3390/toxins3101332 Sylvia Worbs Kernt Köhler Diana Pauly Marc-André Avondet Martin Schaer Martin B. Dorner Brigitte G. Dorner http://www.mdpi.com/2072-6651/3/10/1310 The structure of T-2 toxin in the solid-state is limited to X-ray crystallographic studies, which lack sufficient resolution to provide direct evidence for hydrogen-bonding interactions. Furthermore, its solution-structure, despite extensive Nuclear Magnetic Resonance (NMR) studies, has provided little insight into its hydrogen-bonding behavior, thus far. Hydrogen-bonding interactions are often an important part of biological activity. In order to study these interactions, the structure of T-2 toxin was compared in both the solution- and solid-state using NMR Spectroscopy. It was determined that the solution- and solid-state structure differ dramatically, as indicated by differences in their carbon chemical shifts, these observations are further supported by solution proton spectral parameters and exchange behavior. The slow chemical exchange process and cross-relaxation dynamics with water observed between the hydroxyl hydrogen on C-3 and water supports the existence of a preferential hydrogen bonding interaction on the opposite side of the molecule from the epoxide ring, which is known to be essential for trichothecene toxicity. This result implies that these hydrogen-bonding interactions could play an important role in the biological function of T-2 toxin and posits towards a possible interaction for the trichothecene class of toxins and the ribosome. These findings clearly illustrate the importance of utilizing solid-state NMR for the study of biological compounds, and suggest that a more detailed study of this whole class of toxins, namely trichothecenes, should be pursued using this methodology. http://www.mdpi.com/2072-6651/3/10/1310 Fri, 21 Oct 2011 00:00:00 CEST Toxins 2011-10-21 3 10 Article 1310 1331 2072-6651 Hydrogen-Bonding Interactions in T-2 Toxin Studied Using Solution and Solid-State NMR 2011-10-21 doi: 10.3390/toxins3101310 Praveen Chaudhary Roxanne A. Shank Tony Montina James T. Goettel Nora A. Foroud Paul Hazendonk François Eudes http://www.mdpi.com/2072-6651/3/10/1294 The Fusarium graminearum species complex (FGSC) is a group of mycotoxigenic fungi that are the primary cause of Fusarium head blight (FHB) of wheat worldwide. The distribution, frequency of occurrence, and genetic diversity of FGSC species in cereal crops in South America is not well understood compared to some regions of Asia, Europe and North America. Therefore, we examined the frequency and genetic diversity of a collection of 183 FGSC isolates recovered from wheat grown during multiple growing seasons and across a large area of eastern Argentina, a major wheat producing region in South America. Sequence analysis of the translation elongation factor 1−α and β-tubulin genes as well as Amplified Fragment Length Polymorphism (AFLP) analyses indicated that all isolates were the FGSC species F. graminearum sensu stricto. AFLP analysis resolved at least 11 subgroups, and all the isolates represented different AFLP haplotypes. AFLP profile and geographic origin were not correlated. Previously obtained trichothecene production profiles of the isolates revealed that the 15-acetyldeoxynivalenol chemotype was slightly more frequent than the 3-acetyldeoxynivalenol chemotype among the isolates. These data extend the current understanding of FGSC diversity and provide further evidence that F. graminearum sensu stricto is the predominant cause of FHB in the temperate main wheat-growing area of Argentina. Moreover, two isolates of F. crookwellense and four of F. pseudograminearum were also recovered from wheat samples and sequenced. The results also suggest that, although F. graminearum sensu stricto was the only FGSC species recovered in this study, the high level of genetic diversity within this species should be considered in plant breeding efforts and development of other disease management strategies aimed at reducing FHB. http://www.mdpi.com/2072-6651/3/10/1294 Thu, 20 Oct 2011 00:00:00 CEST Toxins 2011-10-20 3 10 Article 1294 1309 2072-6651 Genetic Diversity in Fusarium graminearum from a Major Wheat-Producing Region of Argentina 2011-10-20 doi: 10.3390/toxins3101294 Cora Lilia Alvarez Stefania Somma Robert H. Proctor Gaetano Stea Giuseppina Mulè Antonio F. Logrieco Virginia Fernandez Pinto Antonio Moretti http://www.mdpi.com/2072-6651/3/10/1278 Systemic anthrax disease is characterized by vascular leakage pathologies. We previously reported that anthrax lethal toxin (LT) induces human endothelial barrier dysfunction in a cell death-independent manner with actin stress fiber formation and disruption of adherens junctions (AJs). In the present study, we further characterize the molecular changes in the AJ complex and investigate whether AJ structure and barrier function can be preserved by modulating key cytoskeletal signaling pathways. Here, we show that LT reduces total VE-cadherin protein and gene expression but the expression of the key linker protein beta-catenin remained unchanged. The changes in VE-cadherin expression correlated temporally with the appearance of actin stress fibers and a two-fold increase in phosphorylation of the stress fiber-associated protein myosin light chain (p-MLC) and cleavage of Rho-associated kinase-1 (ROCK-1). Co-treatment with ROCK inhibitors (H-1152 and Y27632), but not an inhibitor of MLC kinase (ML-7), blocked LT-induced p-MLC enhancement and stress fiber formation. This was accompanied by the restoration of VE-cadherin expression and membrane localization, and attenuation of the LT-induced increase in monolayer permeability to albumin. Together, these findings suggest the ROCK pathway may be a relevant target for countering LT-mediated endothelial barrier dysfunction. http://www.mdpi.com/2072-6651/3/10/1278 Thu, 20 Oct 2011 00:00:00 CEST Toxins 2011-10-20 3 10 Article 1278 1293 2072-6651 Anthrax Lethal Toxin-Mediated Disruption of Endothelial VE-Cadherin Is Attenuated by Inhibition of the Rho-Associated Kinase Pathway 2011-10-20 doi: 10.3390/toxins3101278 Jason M. Warfel Felice D’Agnillo http://www.mdpi.com/2072-6651/3/10/1263 Ribosome-inactivating (ribotoxic) xenobiotics are capable of using cleavage and modification to damage 28S ribosomal RNA, which leads to translational arrest. The blockage of global protein synthesis predisposes rapidly dividing tissues, including gut epithelia, to damage from various pathogenic processes, including epithelial inflammation and carcinogenesis. In particular, mucosal exposure to ribotoxic stress triggers integrated processes that are important for barrier regulation and re-constitution to maintain gut homeostasis. In the present study, various experimental models of the mucosal barrier were evaluated for their response to acute and chronic exposure to ribotoxic agents. Specifically, this review focuses on the regulation of epithelial junctions, epithelial transporting systems, epithelial cytotoxicity, and compensatory responses to mucosal insults. The primary aim is to characterize the mechanisms associated with the intestinal epithelial responses induced by ribotoxic stress and to discuss the implications of ribotoxic stressors as chemical modulators of mucosa-associated diseases such as ulcerative colitis and epithelial cancers. http://www.mdpi.com/2072-6651/3/10/1263 Thu, 20 Oct 2011 00:00:00 CEST Toxins 2011-10-20 3 10 Review 1263 1277 2072-6651 Mucosal Injuries due to Ribosome-Inactivating Stress and the Compensatory Responses of the Intestinal Epithelial Barrier 2011-10-20 doi: 10.3390/toxins3101263 Yuseok Moon http://www.mdpi.com/2072-6651/3/10/1249 A total of 155 puffers caught from two of Thailand’s seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000–2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992–1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication. http://www.mdpi.com/2072-6651/3/10/1249 Tue, 18 Oct 2011 00:00:00 CEST Toxins 2011-10-18 3 10 Article 1249 1262 2072-6651 Toxic Marine Puffer Fish in Thailand Seas and Tetrodotoxin They Contained 2011-10-18 doi: 10.3390/toxins3101249 Monrat Chulanetra Nitat Sookrung Potjanee Srimanote Nitaya Indrawattana Jeeraphong Thanongsaksrikul Yuwaporn Sakolvaree Manas Chongsa-Nguan Hisao Kurazono Wanpen Chaicumpa http://www.mdpi.com/2072-6651/3/10/1233 Ricin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. Development of these lead compounds into potent drug candidates is a challenging prospect for numerous reasons, including poor solubility of pterins, the large and highly polar secondary binding pocket of RTA, as well as the enzyme’s near perfect catalytic efficiency and tight binding affinity for its natural substrate, the eukaryotic ribosome. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10−4 M range, leaving significant room for improvement. This review highlights the variety of techniques routinely employed in structure-based drug design projects, as well as the challenges faced in the design of RTA inhibitors. http://www.mdpi.com/2072-6651/3/10/1233 Thu, 13 Oct 2011 00:00:00 CEST Toxins 2011-10-13 3 10 Review 1233 1248 2072-6651 Structure-Based Design of Ricin Inhibitors 2011-10-13 doi: 10.3390/toxins3101233 Karl Jasheway Jeffrey Pruet Eric V. Anslyn Jon D. Robertus http://www.mdpi.com/2072-6651/3/9/1220 Trichoderma brevicompactum IBT 40841 produces trichodermin, a trichothecene-type toxin that shares most of the steps of its biosynthesis with harzianum A, another trichothecene produced by several Trichoderma species. The first specific step in the trichothecene biosynthesis is carried out by a terpene cylcase, trichodiene synthase, that catalyzes the conversion of farnesyl pyrophosphate to trichodiene and that is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin production, but also in an increase in tyrosol and hydroxytyrosol production, two antioxidant compounds that may play a regulatory role in trichothecene biosynthesis, and also in a higher expression of three trichothecene genes, tri4, tri6 and tri10, and of the erg1 gene, which participates in the synthesis of triterpenes. The effect of tri5 overexpression on tomato seedling disease response was also studied. http://www.mdpi.com/2072-6651/3/9/1220 Fri, 23 Sep 2011 00:00:00 CEST Toxins 2011-09-23 3 9 Article 1220 1232 2072-6651 Overexpression of the Trichoderma brevicompactum tri5 Gene: Effect on the Expression of the Trichodermin Biosynthetic Genes and on Tomato Seedlings 2011-09-23 doi: 10.3390/toxins3091220 Anamariela Tijerino Rosa Hermosa Rosa E. Cardoza Javier Moraga Monica G. Malmierca Josefina Aleu Isidro G. Collado Enrique Monte Santiago Gutierrez http://www.mdpi.com/2072-6651/3/9/1203 Ricin is a protein toxin classified as a bioterror agent, for which there are no known treatment options available after intoxication. It is composed of an enzymatically active A-chain connected by a disulfide bond to a cell binding B-chain. After internalization by endocytosis, ricin is transported retrogradely to the Golgi and ER, from where the ricin A-chain is translocated to the cytosol where it inhibits protein synthesis and thus induces cell death. We have identified cytoplasmic phospholipase A2 (PLA2) as an important factor in ricin retrograde transport. Inhibition of PLA2 protects against ricin challenge, however the toxin can still be endocytosed and transported to the Golgi. Interestingly, ricin transport from the Golgi to the ER is strongly impaired in response to PLA2 inhibition. Confocal microscopy analysis shows that ricin is still colocalized with the trans-Golgi marker TGN46 in the presence of PLA2 inhibitor, but less is colocalized with the cis-Golgi marker GM130. We propose that PLA2 inhibition results in impaired ricin transport through the Golgi stack, thus preventing it from reaching the ER. Consequently, ricin cannot be translocated to the cytosol to exert its toxic action. http://www.mdpi.com/2072-6651/3/9/1203 Fri, 23 Sep 2011 00:00:00 CEST Toxins 2011-09-23 3 9 Article 1203 1219 2072-6651 Role of Phospholipase A2 in Retrograde Transport of Ricin 2011-09-23 doi: 10.3390/toxins3091203 Tove Irene Klokk Anne Berit Dyve Lingelem Anne-Grethe Myrann Kirsten Sandvig http://www.mdpi.com/2072-6651/3/9/1185 Outbreaks of Bacillus anthracis in the US and Europe over the past 10 years have emphasized the health threat this lethal bacteria poses even for developed parts of the world. In contrast to cutaneous anthrax, inhalational disease in the US during the 2001 outbreaks and the newly identified injectional drug use form of disease in the UK and Germany have been associated with relatively high mortality rates. One notable aspect of these cases has been the difficulty in supporting patients once shock has developed. Anthrax bacilli produce several different components which likely contribute to this shock. Growing evidence indicates that both major anthrax toxins may produce substantial cardiovascular dysfunction. Lethal toxin (LT) can alter peripheral vascular function; it also has direct myocardial depressant effects. Edema toxin (ET) may have even more pronounced peripheral vascular effects than LT, including the ability to interfere with the actions of conventional vasopressors. Additionally, ET also appears capable of interfering with renal sodium and water retention. Importantly, the two toxins exert their actions via quite different mechanisms and therefore have the potential to worsen shock and outcome in an additive fashion. Finally, both toxins have the ability to inhibit host defense and microbial clearance, possibly contributing to the very high bacterial loads noted in patients dying with anthrax. This last point is clinically relevant since emerging data has begun to implicate other bacterial components such as anthrax cell wall in the shock and organ injury observed with infection. Taken together, accumulating evidence regarding the potential contribution of LT and ET to anthrax-associated shock supports efforts to develop adjunctive therapies that target both toxins in patients with progressive shock. http://www.mdpi.com/2072-6651/3/9/1185 Tue, 20 Sep 2011 00:00:00 CEST Toxins 2011-09-20 3 9 Review 1185 1202 2072-6651 The Potential Contributions of Lethal and Edema Toxins to the Pathogenesis of Anthrax Associated Shock 2011-09-20 doi: 10.3390/toxins3091185 Caitlin W. Hicks Xizhong Cui Daniel A. Sweeney Yan Li Amisha Barochia Peter Q. Eichacker http://www.mdpi.com/2072-6651/3/9/1163 Ricin toxin (RT) is derived from castor beans, produced by the plant Ricinus communis. RT and its toxic A chain (RTA) have been used therapeutically to arm ligands that target disease-causing cells. In most cases these ligands are cell-binding monoclonal antibodies (MAbs). These ligand-toxin conjugates or immunotoxins (ITs) have shown success in clinical trials [1]. Ricin is also of concern in biodefense and has been classified by the CDC as a Class B biothreat. Virtually all reports of RT poisoning have been due to ingestion of castor beans, since they grow abundantly throughout the world and are readily available. RT is easily purified and stable, and is not difficult to weaponize. RT must be considered during any “white powder” incident and there have been documented cases of its use in espionage [2,3]. The clinical syndrome resulting from ricin intoxication is dependent upon the route of exposure. Countermeasures to prevent ricin poisoning are being developed and their use will depend upon whether military or civilian populations are at risk of exposure. In this review we will discuss ricin toxin, its cellular mode of action, the clinical syndromes that occur following exposure and the development of pre- and post-exposure approaches to prevent of intoxication. http://www.mdpi.com/2072-6651/3/9/1163 Thu, 15 Sep 2011 00:00:00 CEST Toxins 2011-09-15 3 9 Article 1163 1184 2072-6651 Passive and Active Vaccination Strategies to Prevent Ricin Poisoning 2011-09-15 doi: 10.3390/toxins3091163 Seth H. Pincus Joan E. Smallshaw Kejing Song Jody Berry Ellen S. Vitetta http://www.mdpi.com/2072-6651/3/9/1146 Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrhea in humans and animals. Heat-stable (STa) and heat-labile (LT) enterotoxins produced by ETEC disrupt fluid homeostasis in host small intestinal epithelial cells and cause fluid and electrolyte hyper-secretion that leads to diarrhea. ETEC strains producing STa or LT are sufficiently virulent to cause diarrhea, therefore STa and LT antigens must be included in ETEC vaccines. However, potent toxicity and poor immunogenicity (of STa) prevent them from being directly applied as vaccine components. While LT toxoids, especially LT(R192G), being used in vaccine development, STa toxoids have not been included. A recent study (IAI, 78:316-325) demonstrated porcine-type STa toxoids [pSTa(P12F) and pSTa(A13Q)] elicited protective anti-STa antibodies after being fused to a porcine-type LT toxoid [pLT(R192G)]. In this study, we substituted the 8th, 9th, 16th, or the 17th amino acid of a human-type STa (hSTa) and generated 28 modified STa peptides. We tested each STa peptide for toxicity and structure integrity, and found nearly all modified STa proteins showed structure alteration and toxicity reduction. Based on structure similarity and toxic activity, three modified STa peptides: STa(E8A), STa(T16Q) and STa(G17S), were selected to construct LT192-STa-toxoid fusions. Constructed fusions were used to immunize mice, and immunized mice developed anti-STa antibodies. Results from this study provide useful information in developing toxoid vaccines against ETEC diarrhea. http://www.mdpi.com/2072-6651/3/9/1146 Thu, 15 Sep 2011 00:00:00 CEST Toxins 2011-09-15 3 9 Article 1146 1162 2072-6651 Modified Heat-Stable Toxins (hSTa) of Enterotoxigenic Escherichia coli Lose Toxicity but Display Antigenicity after Being Genetically Fused to Heat-Labile Toxoid LT(R192G) 2011-09-15 doi: 10.3390/toxins3091146 Mei Liu Chengxian Zhang Kristy Mateo James P. Nataro Donald C. Robertson Weiping Zhang http://www.mdpi.com/2072-6651/3/9/1131 The entry of ricin toxin into macrophages and certain other cell types in the spleen and liver results in toxin-induced inflammation, tissue damage and organ failure. It has been proposed that uptake of ricin into macrophages is facilitated by the mannose receptor (MR; CD206), a C-type lectin known to recognize the oligosaccharide side chains on ricin’s A (RTA) and B (RTB) subunits. In this study, we confirmed that the MR does indeed promote ricin binding, uptake and killing of monocytes in vitro. To assess the role of MR in the pathogenesis of ricin in vivo, MR knockout (MR−/−) mice were challenged with the equivalent of 2.5× or 5× LD50 of ricin by intraperitoneal injection. We found that MR−/− mice were significantly more susceptible to toxin-induced death than their age-matched, wild-type control counterparts. These data are consistent with a role for the MR in scavenging and degradation of ricin, not facilitating its uptake and toxicity in vivo. http://www.mdpi.com/2072-6651/3/9/1131 Fri, 09 Sep 2011 00:00:00 CEST Toxins 2011-09-09 3 9 Article 1131 1145 2072-6651 Role of the Mannose Receptor (CD206) in Innate Immunity to Ricin Toxin 2011-09-09 doi: 10.3390/toxins3091131 Emily Gage Maria O. Hernandez Joanne M. O’Hara Elizabeth A. McCarthy Nicholas J. Mantis http://www.mdpi.com/2072-6651/3/9/1111 A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx. http://www.mdpi.com/2072-6651/3/9/1111 Wed, 07 Sep 2011 00:00:00 CEST Toxins 2011-09-07 3 9 Article 1111 1130 2072-6651 Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung 2011-09-07 doi: 10.3390/toxins3091111 Eric K. Dumas Philip M. Cox Charles O’Connor Fullenwider Melissa Nguyen Michael Centola Mark Barton Frank Igor Dozmorov Judith A. James A. Darise Farris http://www.mdpi.com/2072-6651/3/9/1089 Pseudomonas syringae is a phytopathogenic bacterium present in a wide variety of host plants where it causes diseases with economic impact. The symptoms produced by Pseudomonas syringae include chlorosis and necrosis of plant tissues, which are caused, in part, by antimetabolite toxins. This category of toxins, which includes tabtoxin, phaseolotoxin and mangotoxin, is produced by different pathovars of Pseudomonas syringae. These toxins are small peptidic molecules that target enzymes of amino acids’ biosynthetic pathways, inhibiting their activity and interfering in the general nitrogen metabolism. A general overview of the toxins’ chemistry, biosynthesis, activity, virulence and potential applications will be reviewed in this work. http://www.mdpi.com/2072-6651/3/9/1089 Wed, 31 Aug 2011 00:00:00 CEST Toxins 2011-08-31 3 9 Review 1089 1110 2072-6651 Chemical and Metabolic Aspects of Antimetabolite Toxins Produced by Pseudomonas syringae Pathovars 2011-08-31 doi: 10.3390/toxins3091089 Eva Arrebola Francisco M. Cazorla Alejandro Perez-García Antonio de Vicente http://www.mdpi.com/2072-6651/3/9/1065 Inhibition of AB5-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin’s pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB5 complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor’s activity. http://www.mdpi.com/2072-6651/3/9/1065 Thu, 25 Aug 2011 00:00:00 CEST Toxins 2011-08-25 3 9 Article 1065 1088 2072-6651 Impact of the Nature and Size of the Polymeric Backbone on the Ability of Heterobifunctional Ligands to Mediate Shiga Toxin and Serum Amyloid P Component Ternary Complex Formation 2011-08-25 doi: 10.3390/toxins3091065 Pavel I. Kitov Eugenia Paszkiewicz Joanna M. Sadowska Zhicheng Deng Marya Ahmed Ravin Narain Thomas P. Griener George L. Mulvey Glen D. Armstrong David R. Bundle http://www.mdpi.com/2072-6651/3/8/1038 Some of the most potent phytotoxins are synthesized by microbes. A few of these share molecular target sites with some synthetic herbicides, but many microbial toxins have unique target sites with potential for exploitation by the herbicide industry. Compounds from both non-pathogenic and pathogenic microbes are discussed. Microbial phytotoxins with modes of action the same as those of commercial herbicides and those with novel modes of action of action are covered. Examples of the compounds discussed are tentoxin, AAL-toxin, auscaulitoxin aglycone, hydantocidin, thaxtomin, and tabtoxin. http://www.mdpi.com/2072-6651/3/8/1038 Mon, 22 Aug 2011 00:00:00 CEST Toxins 2011-08-22 3 8 Review 1038 1064 2072-6651 Modes of Action of Microbially-Produced Phytotoxins 2011-08-22 doi: 10.3390/toxins3081038 Stephen O. Duke Franck E. Dayan http://www.mdpi.com/2072-6651/3/8/1020 The Fusarium graminearum species complex (Fg complex) consists of phylogenetically distinct species some of which cannot be discriminated based on their morphology. Their chemotypes and geographic distributions are dramatically different, and these highlight the challenges that Fusarium head blight (FHB) poses to plant disease specialists and plant breeders, thereby requiring that quarantine officials employ molecular diagnostic tools in their active surveillance programs. Molecular marker technologies play essential roles in species identification of the Fg complex, and they are being used widely to assess the genetic diversity of the clade. The utility, applicability and limitations of molecular methods for assessing the population structure and genetic diversity within the Fg complex are discussed with suitable examples. Knowledge gained from these studies will provide a baseline for monitoring changes in FHB pathogen diversity and mycotoxin potential over time, both of which are critical to the ultimate control and elimination of this economically devastating disease. http://www.mdpi.com/2072-6651/3/8/1020 Fri, 19 Aug 2011 00:00:00 CEST Toxins 2011-08-19 3 8 Review 1020 1037 2072-6651 Population Structure and Genetic Diversity of the Fusarium graminearum Species Complex 2011-08-19 doi: 10.3390/toxins3081020 Jian-Hua Wang Mbacke Ndoye Jing-Bo Zhang He-Ping Li Yu-Cai Liao http://www.mdpi.com/2072-6651/3/8/1004 Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. It not only causes natural infection in humans but also poses a great threat as an emerging bioterror agent. The lethality of anthrax is primarily attributed to the two major virulence factors: toxins and capsule. An extensive effort has been made to generate therapeutically useful monoclonal antibodies to each of the virulence components: protective antigen (PA), lethal factor (LF) and edema factor (EF), and the capsule of B. anthracis. This review summarizes the current status of anti-anthrax mAb development and argues for the potential therapeutic advantage of a cocktail of mAbs that recognize different epitopes or different virulence factors. http://www.mdpi.com/2072-6651/3/8/1004 Mon, 15 Aug 2011 00:00:00 CEST Toxins 2011-08-15 3 8 Review 1004 1019 2072-6651 Monoclonal Antibody Therapies against Anthrax 2011-08-15 doi: 10.3390/toxins3081004 Zhaochun Chen Mahtab Moayeri Robert Purcell http://www.mdpi.com/2072-6651/3/8/991 The Malayan pit viper, Calloselasma rhodostoma, produces a potent venom toxin, rhodocytin (aggretin) which causes platelet aggregation. Rhodocytin is a ligand for the receptor CLEC-2 on the surface of platelets. The interaction of these two molecules initiates a signaling pathway which results in platelet activation and aggregation. We have previously solved the crystal structures of CLEC-2 and of rhodocytin, and have proposed models by which tetrameric rhodocytin may interact with either two monomers of CLEC-2, or with one or two copies of dimeric CLEC-2. In the current study we use a range of approaches to analyze the molecular interfaces and dynamics involved in the models of the interaction of rhodocytin with either one or two copies of dimeric CLEC-2, and their implications for clustering of CLEC-2 on the platelet surface. http://www.mdpi.com/2072-6651/3/8/991 Wed, 10 Aug 2011 00:00:00 CEST Toxins 2011-08-10 3 8 Article 991 1003 2072-6651 Molecular Analysis of the Interaction of the Snake Venom Rhodocytin with the Platelet Receptor CLEC-2 2011-08-10 doi: 10.3390/toxins3080991 Aleksandra A. Watson Christopher A. O’Callaghan http://www.mdpi.com/2072-6651/3/8/979 The primary immunogenic component of the currently approved anthrax vaccine is the protective antigen (PA) unit of the binary toxin system. PA-specific antibodies neutralize anthrax toxins and protect against infection. Recent research has determined that in humans, only antibodies specific for particular determinants are capable of effecting toxin neutralization, and that the neutralizing epitopes recognized by these antibodies are distributed throughout the PA monomer. The mechanisms by which the majority of these epitopes effect neutralization remain unknown. In this report we investigate the process by which a human monoclonal antibody specific for the amino-terminal domain of PA neutralizes lethal toxin in an in vitro assay of cytotoxicity, and find that it neutralizes LT by blocking the requisite cleavage of the amino-terminal 20 kD portion of the molecule (PA20) from the remainder of the PA monomer. We also demonstrate that the epitope recognized by this human monoclonal does not encompass the 166RKKR169 furin recognition sequence in domain 1 of PA. http://www.mdpi.com/2072-6651/3/8/979 Tue, 09 Aug 2011 00:00:00 CEST Toxins 2011-08-09 3 8 Article 979 990 2072-6651 Mechanism of Lethal Toxin Neutralization by a Human Monoclonal Antibody Specific for the PA20 Region of Bacillus anthracis Protective Antigen 2011-08-09 doi: 10.3390/toxins3080979 Donald Reason Justine Liberato Jinying Sun Jessica Camacho Jianhui Zhou http://www.mdpi.com/2072-6651/3/8/968 Deoxynivalenol (DON), one of the trichothecene mycotoxins, is a worldwide contaminant of wheat and barley, especially when infected by Fusarium graminearum, the causative agent of an epidemic wheat disease called Fusarium Head Blight. Because of the high risk of DON ingestion and the possibility of frequent exposure, it is important to develop a rapid and highly sensitive method for easy identification and quantification of DON in grain samples. In this study, we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect DON in wheat. We conjugated 3-O-Hemisuccinyl-DON (3HS-DON) to Bovine serum albumin (BSA) and Ovalbumin (OVA), and obtained DON-specific mice antisera. The indirect competitive ELISA revealed that the optimal concentration of mice serum and the coated antigen was 1/1600 and 1/1500, respectively. The antiserum cross-reacted with the trichothecenes 3-acetyl-DON and T-2 toxin, reaching about 55.2% and 6.3%, respectively, as compared with DON. Results showed that the assay could be performed satisfactorily using an extraction buffer containing less than 15% methanol. Recovery from DON was 82–93% in grains. The linear detection range of DON in grains was between 0.01 and 100 μg/mL. http://www.mdpi.com/2072-6651/3/8/968 Thu, 04 Aug 2011 00:00:00 CEST Toxins 2011-08-04 3 8 Article 968 978 2072-6651 Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol (DON) 2011-08-04 doi: 10.3390/toxins3080968 Fang Ji Hua Li Jianhong Xu Jianrong Shi http://www.mdpi.com/2072-6651/3/8/949 Maize is frequently infected by the Fusarium species producing mycotoxins. Numerous investigations have focused on grain maize, but little is known about the Fusarium species in the entire plant used for silage. Furthermore, mycotoxins persist during the ensiling process and thus endanger feed safety. In the current study, we analyzed 20 Swiss silage maize samples from growers’ fields for the incidence of Fusarium species and mycotoxins. The species spectrum was analyzed morphologically and mycotoxins were measured by LC-MS/MS. A pre-harvest visual disease rating showed few disease symptoms. In contrast, the infection rate of two-thirds of the harvest samples ranged from 25 to 75% and twelve different Fusarium species were isolated. The prevailing species were F. sporotrichioides, F. verticillioides and F. graminearum. No infection specificity for certain plant parts was observed. The trichothecene deoxynivalenol (DON) was found in each sample (ranging from 780 to 2990 µg kg−1). Other toxins detected in descending order were zearalenone, further trichothecenes (nivalenol, HT-2 and T-2 toxin, acetylated DON) and fumonisins. A generalized linear regression model containing the three cropping factors harvest date, pre-precrop and seed treatment was established, to explain DON contamination of silage maize. Based on these findings, we suggest a European-wide survey on silage maize. http://www.mdpi.com/2072-6651/3/8/949 Thu, 04 Aug 2011 00:00:00 CEST Toxins 2011-08-04 3 8 Article 949 967 2072-6651 Incidence of Fusarium Species and Mycotoxins in Silage Maize 2011-08-04 doi: 10.3390/toxins3080949 Sonja Eckard Felix E. Wettstein Hans-Rudolf Forrer Susanne Vogelgsang http://www.mdpi.com/2072-6651/3/8/932 Aflatoxins are carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Previous studies found that repeated serial mycelial transfer or treatment of A. parasiticus with 5-azacytidine produced colonies with a fluffy phenotype and inability to produce aflatoxins. To understand how these treatments affect expression of genes involved in aflatoxin production and development, we carried out expressed sequence tag (EST)-based microarray assays to identify genes in treated clones that are differentially expressed compared to the wild-type. Expression of 183 genes was significantly dysregulated. Of these, 38 had at least two-fold or lower expression compared to the untreated control and only two had two-fold or higher expression. The most frequent change was downregulation of genes predicted to encode membrane-bound proteins. Based on this result we hypothesize that the treatments cause changes in the structure of cellular and organelle membranes that prevent normal development and aflatoxin biosynthesis. http://www.mdpi.com/2072-6651/3/8/932 Tue, 02 Aug 2011 00:00:00 CEST Toxins 2011-08-02 3 8 Article 932 948 2072-6651 Expression Profiling of Non-Aflatoxigenic Aspergillus parasiticus Mutants Obtained by 5-Azacytosine Treatment or Serial Mycelial Transfer 2011-08-02 doi: 10.3390/toxins3080932 Jeffrey R. Wilkinson Shubha P. Kale Deepak Bhatnagar Jiujiang Yu Kenneth C. Ehrlich http://www.mdpi.com/2072-6651/3/7/920 Key impediments to increased corn yield and quality in the southeastern US coastal plain region are damage by ear-feeding insects and aflatoxin contamination caused by infection of Aspergillus flavus. Key ear-feeding insects are corn earworm, Helicoverpa zea, fall armyworm, Spodoptera frugiperda, maize weevil, Sitophilus zeamais, and brown stink bug, Euschistus servus. In 2006 and 2007, aflatoxin contamination and insect damage were sampled before harvest in three 0.4-hectare corn fields using a grid sampling method. The feeding damage by each of ear/kernel-feeding insects (i.e., corn earworm/fall armyworm damage on the silk/cob, and discoloration of corn kernels by stink bugs), and maize weevil population were assessed at each grid point with five ears. The spatial distribution pattern of aflatoxin contamination was also assessed using the corn samples collected at each sampling point. Aflatoxin level was correlated to the number of maize weevils and stink bug-discolored kernels, but not closely correlated to either husk coverage or corn earworm damage. Contour maps of the maize weevil populations, stink bug-damaged kernels, and aflatoxin levels exhibited an aggregated distribution pattern with a strong edge effect on all three parameters. The separation of silk- and cob-feeding insects from kernel-feeding insects, as well as chewing (i.e., the corn earworm and maize weevil) and piercing-sucking insects (i.e., the stink bugs) and their damage in relation to aflatoxin accumulation is economically important. Both theoretic and applied ramifications of this study were discussed by proposing a hypothesis on the underlying mechanisms of the aggregated distribution patterns and strong edge effect of insect damage and aflatoxin contamination, and by discussing possible management tactics for aflatoxin reduction by proper management of kernel-feeding insects. Future directions on basic and applied research related to aflatoxin contamination are also discussed. http://www.mdpi.com/2072-6651/3/7/920 Thu, 21 Jul 2011 00:00:00 CEST Toxins 2011-07-21 3 7 Article 920 931 2072-6651 Spatial Patterns of Aflatoxin Levels in Relation to Ear-Feeding Insect Damage in Pre-Harvest Corn 2011-07-21 doi: 10.3390/toxins3070920 Xinzhi Ni Jeffrey P. Wilson G. David Buntin Baozhu Guo Matthew D. Krakowsky R. Dewey Lee Ted E. Cottrell Brian T. Scully Alisa Huffaker Eric A. Schmelz http://www.mdpi.com/2072-6651/3/7/911 The uremic syndrome is attributed to the progressive retention of a large number of compounds which, under normal conditions, are excreted by healthy kidneys. The compounds are called uremic toxins when they interact negatively with biological functions. The present review focuses on a specific class of molecules, namely the family of protein-bound uremic toxins. Recent experimental studies have shown that protein-bound toxins are involved not only in the progression of chronic kidney disease (CKD), but also in the generation and aggravation of cardiovascular disease. Two protein-bound uremic retention solutes, namely indoxyl sulfate and p-cresyl sulfate, have been shown to play a prominent role. However, although these two molecules belong to the same class of molecules, exert toxic effects on the cardiovascular system in experimental animals, and accumulate in the serum of patients with CKD they may have different clinical impacts in terms of cardiovascular disease and other complications. The principal aim of this review is to evaluate the effect of p-cresyl sulfate and indoxyl sulfate retention on CKD patient outcomes, based on recent clinical studies. http://www.mdpi.com/2072-6651/3/7/911 Wed, 20 Jul 2011 00:00:00 CEST Toxins 2011-07-20 3 7 Article 911 919 2072-6651 Protein-Bound Uremic Toxins: New Insight from Clinical Studies 2011-07-20 doi: 10.3390/toxins3070911 Sophie Liabeuf Tilman B. Drüeke Ziad A. Massy http://www.mdpi.com/2072-6651/3/7/900 A novel non-hemorrhagic basic metalloprotease, rubelase, was isolated from the venom of Crotalus ruber ruber. Rubelase hydrolyzes succinyl-L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA), a specific substrate for elastase, and the hydrolytic activity was inhibited by chelating agents. It also hydrolyzes collagen and fibrinogen. However, hemorrhagic activity was not observed. By ESI/Q-TOF and MALDI/TOF mass spectrometry combined with Edman sequencing procedure, the molecular mass of rubelase was determined to be 23,266 Da. Although its primary structure was similar to rubelysin (HT-2), a hemorrhagic metalloprotease isolated from the same snake venom, the circumstances surrounding putative zinc binding domain HEXXHXXGXXH were found to be different when the three-dimensional computer models of both metalloproteases were compared. The cytotoxic effects of rubelase and rubelysin on cultured endothelial and smooth muscle cells were also different, indicating that the substitution of several amino acid residues causes the changes of active-site conformation and cell preference. http://www.mdpi.com/2072-6651/3/7/900 Tue, 19 Jul 2011 00:00:00 CEST Toxins 2011-07-19 3 7 Article 900 910 2072-6651 Isolation and Biochemical Characterization of Rubelase, a Non-Hemorrhagic Elastase from Crotalus ruber ruber (Red Rattlesnake) Venom 2011-07-19 doi: 10.3390/toxins3070900 Yumiko Komori Kaname Sakai Katsuyoshi Masuda Toshiaki Nikai http://www.mdpi.com/2072-6651/3/7/884 Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric Gi/o proteins, resulting in the receptors being uncoupled from the Gi/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gαi/o proteins by the A-protomer (Gi/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (Gi/o protein-independent action). http://www.mdpi.com/2072-6651/3/7/884 Fri, 15 Jul 2011 00:00:00 CEST Toxins 2011-07-15 3 7 Article 884 899 2072-6651 Gi/o Protein-Dependent and -Independent Actions of Pertussis Toxin (PTX) 2011-07-15 doi: 10.3390/toxins3070884 Supachoke Mangmool Hitoshi Kurose http://www.mdpi.com/2072-6651/3/7/848 Immunotoxins and antibody-drug conjugates are protein-based drugs combining a target-specific binding domain with a cytotoxic domain. Such compounds are potentially therapeutic against diseases including cancer, and several clinical trials have shown encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit conjugates’ therapeutic use, including inefficient cellular uptake, low cytotoxicity, and off-target effects. During the preparation of immunoconjugates by chemical synthesis, the choice of the hinge component joining the two building blocks is of paramount importance: the conjugate must remain stable in vivo but must afford efficient release of the toxic moiety when the target is reached. Vast efforts have been made, and the present article reviews strategies employed in developing immunoconjugates, focusing on the evolution of chemical linkers. http://www.mdpi.com/2072-6651/3/7/848 Thu, 14 Jul 2011 00:00:00 CEST Toxins 2011-07-14 3 7 Review 848 883 2072-6651 Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components 2011-07-14 doi: 10.3390/toxins3070848 Franco Dosio Paola Brusa Luigi Cattel http://www.mdpi.com/2072-6651/3/7/834 The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER. http://www.mdpi.com/2072-6651/3/7/834 Tue, 05 Jul 2011 00:00:00 CEST Toxins 2011-07-05 3 7 Article 834 847 2072-6651 Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking 2011-07-05 doi: 10.3390/toxins3070834 Björn Becker Manfred J. Schmitt http://www.mdpi.com/2072-6651/3/7/815 1H NMR spectroscopy of urine has been applied to exploring metabolomic differences between people diagnosed with Balkan endemic nephropathy (BEN), and treated by haemodialysis, and those without overt renal disease in Romania and Bulgaria. Convenience sampling was made from patients receiving haemodialysis in hospital and healthy controls in their village. Principal component analysis clustered healthy controls from both countries together. Bulgarian BEN patients clustered separately from controls, though in the same space. However, Romanian BEN patients not only also clustered away from controls but also clustered separately from the BEN patients in Bulgaria. Notably, the urinary metabolomic data of two people sampled as Romanian controls clustered within the Romanian BEN group. One of these had been suspected of incipient symptoms of BEN at the time of selection as a ‘healthy’ control. This implies, at first sight, that metabolomic analysis can be predictive of impending morbidity before conventional criteria can diagnose BEN. Separate clustering of BEN patients from Romania and Bulgaria could indicate difference in aetiology of this particular silent renal atrophy in different geographic foci across the Balkans. http://www.mdpi.com/2072-6651/3/7/815 Mon, 04 Jul 2011 00:00:00 CEST Toxins 2011-07-04 3 7 Article 815 833 2072-6651 Comparative 1H NMR Metabolomic Urinalysis of People Diagnosed with Balkan Endemic Nephropathy, and Healthy Subjects, in Romania and Bulgaria: A Pilot Study 2011-07-04 doi: 10.3390/toxins3070815 Peter Mantle Mirela Modalca Andrew Nicholls Calin Tatu Diana Tatu Draga Toncheva http://www.mdpi.com/2072-6651/3/7/802 As the world’s population grows, access to a safe food supply will continue to be a global priority. In recent years, the world has experienced an increase in mycotoxin contamination of grains due to climatic and agronomic changes that encourage fungal growth during cultivation. A number of the molds that are plant pathogens produce trichothecene mycotoxins, which are known to cause serious human and animal toxicoses. This review covers the types of trichothecenes, their complexity, and proposed biosynthetic pathways of trichothecenes. http://www.mdpi.com/2072-6651/3/7/802 Fri, 01 Jul 2011 00:00:00 CEST Toxins 2011-07-01 3 7 Review 802 814 2072-6651 Trichothecenes: From Simple to Complex Mycotoxins 2011-07-01 doi: 10.3390/toxins3070802 Susan P. McCormick April M. Stanley Nicholas A. Stover Nancy J. Alexander http://www.mdpi.com/2072-6651/3/7/787 Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans). The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin) and Ricinus communis agglutinin l (RCA). These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes. http://www.mdpi.com/2072-6651/3/7/787 Thu, 30 Jun 2011 00:00:00 CEST Toxins 2011-06-30 3 7 Article 787 801 2072-6651 Ricin Trafficking in Plant and Mammalian Cells 2011-06-30 doi: 10.3390/toxins3070787 J. Michael Lord Robert A. Spooner http://www.mdpi.com/2072-6651/3/7/766 Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and through storage. Previous studies have highlighted the constitutive production of proteins involved in maize kernel resistance against A. flavus’ infection. However, little is known about induced resistance nor about defense gene expression and regulation in kernels. In this study, maize oligonucleotide arrays and a pair of closely-related maize lines varying in aflatoxin accumulation were used to reveal the gene expression network in imbibed mature kernels in response to A. flavus’ challenge. Inoculated kernels were incubated 72 h via the laboratory-based Kernel Screening Assay (KSA), which highlights kernel responses to fungal challenge. Gene expression profiling detected 6955 genes in resistant and 6565 genes in susceptible controls; 214 genes induced in resistant and 2159 genes induced in susceptible inoculated kernels. Defense related and regulation related genes were identified in both treatments. Comparisons between the resistant and susceptible lines indicate differences in the gene expression network which may enhance our understanding of the maize-A. flavus interaction. http://www.mdpi.com/2072-6651/3/7/766 Wed, 29 Jun 2011 00:00:00 CEST Toxins 2011-06-29 3 7 Article 766 786 2072-6651 Transcriptional Profiles Uncover Aspergillus flavus-Induced Resistance in Maize Kernels 2011-06-29 doi: 10.3390/toxins3070766 Meng Luo Robert L. Brown Zhi-Yuan Chen Abebe Menkir Jiujiang Yu Deepak Bhatnagar http://www.mdpi.com/2072-6651/3/7/754 A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline. http://www.mdpi.com/2072-6651/3/7/754 Fri, 24 Jun 2011 00:00:00 CEST Toxins 2011-06-24 3 7 Article 754 765 2072-6651 A Public Platform for the Verification of the Phenotypic Effect of Candidate Genes for Resistance to Aflatoxin Accumulation and Aspergillus flavus Infection in Maize 2011-06-24 doi: 10.3390/toxins3070754 Marilyn L. Warburton William Paul Williams Leigh Hawkins Susan Bridges Cathy Gresham Jonathan Harper Seval Ozkan J. Erik Mylroie Xueyan Shan http://www.mdpi.com/2072-6651/3/7/737 Aspergillus flavus and A. parasiticus infect peanut seeds and produce aflatoxins, which are associated with various diseases in domestic animals and humans throughout the world. The most cost-effective strategy to minimize aflatoxin contamination involves the development of peanut cultivars that are resistant to fungal infection and/or aflatoxin production. To identify peanut Aspergillus-interactive and peanut Aspergillus-resistance genes, we carried out a large scale peanut Expressed Sequence Tag (EST) project which we used to construct a peanut glass slide oligonucleotide microarray. The fabricated microarray represents over 40% of the protein coding genes in the peanut genome. For expression profiling, resistant and susceptible peanut cultivars were infected with a mixture of Aspergillus flavus and parasiticus spores. The subsequent microarray analysis identified 62 genes in resistant cultivars that were up-expressed in response to Aspergillus infection. In addition, we identified 22 putative Aspergillus-resistance genes that were constitutively up-expressed in the resistant cultivar in comparison to the susceptible cultivar. Some of these genes were homologous to peanut, corn, and soybean genes that were previously shown to confer resistance to fungal infection. This study is a first step towards a comprehensive genome-scale platform for developing Aspergillus-resistant peanut cultivars through targeted marker-assisted breeding and genetic engineering. http://www.mdpi.com/2072-6651/3/7/737 Fri, 24 Jun 2011 00:00:00 CEST Toxins 2011-06-24 3 7 Article 737 753 2072-6651 Gene Expression Profiling and Identification of Resistance Genes to Aspergillus flavus Infection in Peanut through EST and Microarray Strategies 2011-06-24 doi: 10.3390/toxins3070737 Baozhu Guo Natalie D. Fedorova Xiaoping Chen Chun-Hua Wan Wei Wang William C. Nierman Deepak Bhatnagar Jiujiang Yu http://www.mdpi.com/2072-6651/3/6/721 Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals’ heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins’ direct impact on the heart. http://www.mdpi.com/2072-6651/3/6/721 Thu, 23 Jun 2011 00:00:00 CEST Toxins 2011-06-23 3 6 Article 721 736 2072-6651 Hemodynamic Effects of Anthrax Toxins in the Rabbit Model and the Cardiac Pathology Induced by Lethal Toxin 2011-06-23 doi: 10.3390/toxins3060721 William S. Lawrence Jeffrey R. Marshall Diana L. Zavala Lori E. Weaver Wallace B. Baze Scott T. Moen Elbert B. Whorton Randy L. Gourley Johnny W. Peterson http://www.mdpi.com/2072-6651/3/6/697 Ribosome-inactivating proteins (RIPs) are a family of plant toxins that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death. RIPs are mostly divided in two types: Type 1 RIPs that are single-chain enzymatic proteins, and type 2 RIPs that consist of an active A chain (similar to a type 1 RIP) linked to a B chain with lectin properties. RIP-containing conjugates have been used in many experimental strategies against cancer cells, often showing great efficacy in clinical trials. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively utilized to construct anti-cancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. This review summarizes saporin-S6-containing conjugates and their application in cancer therapy, considering in-vitro and in-vivo studies both in animal models and in clinical trials. The review is structured on the basis of the targeting of hematological versus solid tumors and on the antigen recognized on the cell surface. http://www.mdpi.com/2072-6651/3/6/697 Wed, 22 Jun 2011 00:00:00 CEST Toxins 2011-06-22 3 6 Review 697 720 2072-6651 Immunotoxins and Other Conjugates Containing Saporin-S6 for Cancer Therapy 2011-06-22 doi: 10.3390/toxins3060697 Letizia Polito Massimo Bortolotti Manuela Pedrazzi Andrea Bolognesi http://www.mdpi.com/2072-6651/3/6/678 At this time, no “magic bullet” for solving the aflatoxin contamination problem in maize and cottonseed has been identified, so several strategies must be utilized simultaneously to ensure a healthy crop, free of aflatoxins. The most widely explored strategy for the control of aflatoxin contamination is the development of preharvest host resistance. This is because A. flavus infects and produces aflatoxins in susceptible crops prior to harvest. In maize production, the host resistance strategy has gained prominence because of advances in the identification of natural resistance traits. However, native resistance in maize to aflatoxin contamination is polygenic and complex and, therefore, markers need to be identified to facilitate the transfer of resistance traits into agronomically viable genetic backgrounds while limiting the transfer of undesirable traits. Unlike maize, there are no known cotton varieties that demonstrate enhanced resistance to A. flavus infection and aflatoxin contamination. For this reason, transgenic approaches are being undertaken in cotton that utilize genes encoding antifungal/anti-aflatoxin factors from maize and other sources to counter fungal infection and toxin production. This review will present information on preharvest control strategies that utilize both breeding and native resistance identification approaches in maize as well as transgenic approaches in cotton. http://www.mdpi.com/2072-6651/3/6/678 Tue, 21 Jun 2011 00:00:00 CEST Toxins 2011-06-21 3 6 Review 678 696 2072-6651 Developing Resistance to Aflatoxin in Maize and Cottonseed 2011-06-21 doi: 10.3390/toxins3060678 Jeffrey W. Cary Kanniah Rajasekaran Robert L. Brown Meng Luo Zhi-Yuan Chen Deepak Bhatnagar http://www.mdpi.com/2072-6651/3/6/672 Verocytotoxins VT1 and VT2, produced by Verocytotoxigenic Escherichia coli (VTEC), are encoded on temperate bacteriophages. Several studies reported the loss of the vtx genes after multiple subcultivation steps or long preservation. The objective of this study was to determine if the loss of the verocytotoxin genes can already occur during the first subcultivation step. Consequently, the stability of the vtx genes were tested in 40 isolates originating from 40 vtx-positive fecal samples after the first subcultivation step following the isolation procedure. The loss occurred in 12 out of 40 strains tested and was rather rare among the O157 strains compared to the non-O157 strains. This is the first study demonstrating that the loss of the verocytotoxin genes can already occur after the first subcultivation step. This may lead to an underestimation of VTEC positive samples. http://www.mdpi.com/2072-6651/3/6/672 Mon, 20 Jun 2011 00:00:00 CEST Toxins 2011-06-20 3 6 Short Note 672 677 2072-6651 Loss of vtx Genes after the First Subcultivation Step of Verocytotoxigenic Escherichia coli O157 and Non-O157 during Isolation from Naturally Contaminated Fecal Samples 2011-06-20 doi: 10.3390/toxins3060672 Maria-Adelheid Joris Karen Verstraete Koen De Reu Lieven De Zutter http://www.mdpi.com/2072-6651/3/6/660 Bacillus anthracis, similar to other bacterial pathogens, has evolved effective immune evasion strategies to prolong its survival in the host, thus ensuring the unchecked spread of the infection. This function is subserved by lethal (LT) and edema (ET) toxins, two exotoxins produced by vegetative anthrax bacilli following germination of the spores. The structure of these toxins and the mechanism of cell intoxication are topics covered by other reviews in this issue. Here we shall discuss how B. anthracis uses LT and ET to suppress the immune defenses of the host, focusing on T lymphocytes, the key players in adaptive immunity. We shall also summarize recent findings showing that, depending on its concentration, ET has the ability not only to suppress T cell activation but also to promote the polarization of CD4+ T cells to the Th2 and Th17 subsets, highlighting the potential use of this toxin as an immunomodulator. http://www.mdpi.com/2072-6651/3/6/660 Mon, 20 Jun 2011 00:00:00 CEST Toxins 2011-06-20 3 6 Review 660 671 2072-6651 T Cell Targeting by Anthrax Toxins: Two Faces of the Same Coin 2011-06-20 doi: 10.3390/toxins3060660 Silvia Rossi Paccani Cosima T. Baldari http://www.mdpi.com/2072-6651/3/6/647 Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B1 (AFB1), and aflatoxin G1 (AFG1) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB1 production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB1 production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB1 production by A. flavus EGP9 and AFG1 production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG1 production by A. parasiticus SSWT 2999. Changes in AFB1 production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology. http://www.mdpi.com/2072-6651/3/6/647 Fri, 17 Jun 2011 00:00:00 CEST Toxins 2011-06-17 3 6 Article 647 659 2072-6651 Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene 2011-06-17 doi: 10.3390/toxins3060647 Ahmed M. Abdel-Hadi Daniel P. Caley David R. F. Carter Naresh Magan http://www.mdpi.com/2072-6651/3/6/640 Shiga toxin-producing E. coli carrying the stx1 and/or stx2 genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low percentage of samples that have tested positive for Stx-producing microbes, suggesting that while deer may contain these microbes, their overall abundance in deer is low. In this study, quantitative PCR (qPCR) was utilized to test for the presence of stx genes in white-tailed deer fecal matter in western Pennsylvania. In this culture independent screening, nearly half of the samples tested positive for the stx2 gene, with a bias towards samples that were concentrated with stx2. This study, while limited in scope, suggests that deer may be a greater reservoir for stx than was previously thought. http://www.mdpi.com/2072-6651/3/6/640 Thu, 16 Jun 2011 00:00:00 CEST Toxins 2011-06-16 3 6 Brief Report 640 646 2072-6651 Detection of stx1 and stx2 Genes in Pennsylvanian White-Tailed Deer 2011-06-16 doi: 10.3390/toxins3060640 Whitney M. Kistler Surafel Mulugeta Steven A. Mauro http://www.mdpi.com/2072-6651/3/6/626 After ingestion via contaminated food or water, enterohaemorrhagic E. coli colonises the intestinal mucosa and produces Shiga toxins (Stx). No Stx-specific secretion system has been described so far, and it is assumed that Stx are released into the gut lumen after bacterial lysis. Human intestinal epithelium does not express the Stx receptor Gb3 or other Stx binding sites, and it remains unknown how Stx cross the intestinal epithelial barrier and gain access to the systemic circulation. This review summarises current knowledge about the influence of the intestinal environment on Stx production and release, Stx interaction with intestinal epithelial cells and intracellular uptake, and toxin translocation into underlying tissues. Furthermore, it highlights gaps in understanding that need to be addressed by future research. http://www.mdpi.com/2072-6651/3/6/626 Tue, 14 Jun 2011 00:00:00 CEST Toxins 2011-06-14 3 6 Review 626 639 2072-6651 Shiga Toxin Interaction with Human Intestinal Epithelium 2011-06-14 doi: 10.3390/toxins3060626 Stephanie Schüller http://www.mdpi.com/2072-6651/3/6/608 In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently “fine tunes” the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment. http://www.mdpi.com/2072-6651/3/6/608 Tue, 14 Jun 2011 00:00:00 CEST Toxins 2011-06-14 3 6 Review 608 625 2072-6651 Shiga Toxin: Expression, Distribution, and Its Role in the Environment 2011-06-14 doi: 10.3390/toxins3060608 Steven A. Mauro Gerald B. Koudelka http://www.mdpi.com/2072-6651/3/6/591 The pathological actions of anthrax toxin require the activities of its edema factor (EF) and lethal factor (LF) enzyme components, which gain intracellular access via its receptor-binding component, protective antigen (PA). LF is a metalloproteinase with specificity for selected mitogen-activated protein kinase kinases (MKKs), but its activity is not directly lethal to many types of primary and transformed cells in vitro. Nevertheless, in vivo treatment of several animal species with the combination of LF and PA (termed lethal toxin or LT) leads to morbidity and mortality, suggesting that LT-dependent toxicity is mediated by cellular interactions between host cells. Decades of research have revealed that a central hallmark of this toxicity is the disruption of key cellular barriers required to maintain homeostasis. This review will focus on the current understanding of the effects of LT on barrier function, highlighting recent progress in establishing the molecular mechanisms underlying these effects. http://www.mdpi.com/2072-6651/3/6/591 Tue, 14 Jun 2011 00:00:00 CEST Toxins 2011-06-14 3 6 Review 591 607 2072-6651 The Effects of Anthrax Lethal Toxin on Host Barrier Function 2011-06-14 doi: 10.3390/toxins3060591 Tao Xie Roger D. Auth David M. Frucht http://www.mdpi.com/2072-6651/3/6/566 Aflatoxin B1 is a common contaminant of poultry feeds in tropical and subtropical climates. Research during the last five decades has well established the negative effects of the mycotoxin on health of poultry. However, the last ten years of relevant data have accentuated the potential of low levels of aflatoxin B1 to deteriorate broiler performance. In this regard, any attempt to establish a dose-effect relationship between aflatoxin B1 level and broiler performance is also complicated due to differences in types of broilers and length of exposure to the mycotoxin in different studies. Contrary to the prevalent notion regarding literature saturation with respect to aflatoxicosis of chicken, many areas of aflatoxicosis still need to be explored. Literature regarding effects of the mycotoxin on the gastrointestinal tract in this regard is particular scanty and non-conclusive. In addition to these issues, the metabolism of aflatoxin B1 and recently proposed hypotheses regarding biphasic effects of the mycotoxin in broilers are briefly discussed. http://www.mdpi.com/2072-6651/3/6/566 Tue, 14 Jun 2011 00:00:00 CEST Toxins 2011-06-14 3 6 Review 566 590 2072-6651 Aflatoxin B1 in Affecting Broiler’s Performance, Immunity, and Gastrointestinal Tract: A Review of History and Contemporary Issues 2011-06-14 doi: 10.3390/toxins3060566 Agha W. Yunus Ebrahim Razzazi-Fazeli Josef Bohm http://www.mdpi.com/2072-6651/3/6/551 Animal feeding studies have demonstrated that clay additives, such as bentonites, can bind aflatoxins in ingested feed and reduce or eliminate the toxicity. Bentonite deposits are found throughout the world and mostly consist of expandable smectite minerals, such as montmorillonite. The surfaces of smectite minerals can be treated with organic compounds to create surface-modified clays that more readily bind some contaminants than the untreated clay. Montmorillonites treated with organic cations, such as hexadecyltrimethylammonium (HDTMA) and phenyltrimethylammonium (PTMA), more effectively remove organic contaminants, such as benzene and toluene, from water than untreated clay. Similarly, montmorillonite treated with PTMA (Kd = 24,100) retained more aflatoxin B1 (AfB1) from aqueous corn flour than untreated montmorillonite (Kd = 944). Feed additives that reduced aflatoxin toxicity in animal feeding studies adsorbed more AfB1 from aqueous corn flour than feed additives that were less effective. The organic cations HDTMA and PTMA are considered toxic and would not be suitable for clay additives used in feed or food, but other non-toxic or nutrient compounds can be used to prepare surface-modified clays. Montmorillonite (SWy) treated with choline (Kd = 13,800) and carnitine (Kd = 3960) adsorbed much more AfB1 from aqueous corn flour than the untreated clay (Kd = 944). A choline-treated clay prepared from a reduced-charge, high-charge montmorillonite (Kd = 20,100) adsorbed more AfB1 than the choline-treated high-charge montmorillonite (Kd = 1340) or the untreated montmorillonite (Kd = 293). Surface-modified clay additives prepared using low-charge smectites and nutrient or non-toxic organic compounds might be used to more effectively bind aflatoxins in contaminated feed or food and prevent toxicity. http://www.mdpi.com/2072-6651/3/6/551 Fri, 10 Jun 2011 00:00:00 CEST Toxins 2011-06-10 3 6 Article 551 565 2072-6651 Aflatoxin Toxicity Reduction in Feed by Enhanced Binding to Surface-Modified Clay Additives 2011-06-10 doi: 10.3390/toxins3060551 William F. Jaynes Richard E. Zartman http://www.mdpi.com/2072-6651/3/6/538 This research examined the expression patterns of 94 stress-related genes in seven maize inbred lines with differential expressions of resistance to aflatoxin contamination. The objective was to develop a set of genes/probes associated with resistance to A. flavus and/or aflatoxin contamination. Ninety four genes were selected from previous gene expression studies with abiotic stress to test the differential expression in maize lines, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6, using real-time RT-PCR. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups, which are highly expressed in the resistant Group 2 but down-regulated in susceptible Group 1. When further subjected to drought stress, Tex6 expressed more genes up-regulated and B73 has fewer genes up-regulated. The transcript patterns and interactions measured in these experiments indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as various host interactions with environmental factors, particularly, drought and high temperature. http://www.mdpi.com/2072-6651/3/6/538 Thu, 09 Jun 2011 00:00:00 CEST Toxins 2011-06-09 3 6 Article 538 550 2072-6651 Expression Analysis of Stress-Related Genes in Kernels of Different Maize (Zea mays L.) Inbred Lines with Different Resistance to Aflatoxin Contamination 2011-06-09 doi: 10.3390/toxins3060538 Tingbo Jiang Boru Zhou Meng Luo Hamed K. Abbas Robert Kemerait Robert Dewey Lee Brian T. Scully Baozhu Guo http://www.mdpi.com/2072-6651/3/6/520 We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD+ metabolites (nicotinamide, N1-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N1-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD+ from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD+ analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD+ analogue that inhibits IMP dehydrogenase in other cells. http://www.mdpi.com/2072-6651/3/6/520 Tue, 07 Jun 2011 00:00:00 CEST Toxins 2011-06-07 3 6 Article 520 537 2072-6651 4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate (4PyTP), a Novel NAD+ Metabolite Accumulating in Erythrocytes of Uremic Children: A Biomarker for a Toxic NAD+ Analogue in Other Tissues? 2011-06-07 doi: 10.3390/toxins3060520 Elena Synesiou Lynnette D. Fairbanks H. Anne Simmonds Ewa M. Slominska Ryszard T. Smolenski Elizabeth A. Carrey http://www.mdpi.com/2072-6651/3/6/504 Overt response to a single 6.25 mg dose of ochratoxin A (OTA) by oral gavage to 15 months male rats was progressive loss of weight during the following four days. Lost weight was restored within one month and animals had a normal life-span without OTA-related terminal disease. Decline in plasma OTA concentration only commenced four days after dosing, while urinary excretion of OTA and ochratoxin alpha was ongoing. During a temporary period of acute polyuria, a linear relationship between urine output and creatinine concentration persisted. Elimination of other common urinary solutes relative to creatinine was generally maintained during the polyuria phase, except that phosphate excretion increased temporarily. 1H NMR metabolomic analysis of urine revealed a progressive cyclic shift in the group principal components data cluster from before dosing, throughout the acute insult phase, and returning almost completely to normality when tested six months later. Renal insult by OTA was detected by 1H NMR within a day of dosing, as the most sensitive early indicator. Notable biomarkers were trimethylamine N-oxide and an aromatic urinary profile dominated by phenylacetylglycine. Tolerance of such a large acute insult by OTA, assessed by rat natural lifetime outcomes, adds a new dimension to toxicology of this xenobiotic. http://www.mdpi.com/2072-6651/3/6/504 Thu, 26 May 2011 00:00:00 CEST Toxins 2011-05-26 3 6 Article 504 519 2072-6651 1H NMR Spectroscopy-Based Metabolomic Assessment of Uremic Toxicity, with Toxicological Outcomes, in Male Rats Following an Acute, Mid-Life Insult from Ochratoxin A 2011-05-26 doi: 10.3390/toxins3060504 Peter G. Mantle Andrew W. Nicholls John P. Shockcor http://www.mdpi.com/2072-6651/3/5/489 The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K+ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca2+ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery. http://www.mdpi.com/2072-6651/3/5/489 Fri, 13 May 2011 00:00:00 CEST Toxins 2011-05-13 3 5 Article 489 503 2072-6651 Alpha-Latrotoxin Rescues SNAP-25 from BoNT/A-Mediated Proteolysis in Embryonic Stem Cell-Derived Neurons 2011-05-13 doi: 10.3390/toxins3050489 Mariano Mesngon Patrick McNutt http://www.mdpi.com/2072-6651/3/5/469 Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme. http://www.mdpi.com/2072-6651/3/5/469 Fri, 13 May 2011 00:00:00 CEST Toxins 2011-05-13 3 5 Review 469 488 2072-6651 Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach 2011-05-13 doi: 10.3390/toxins3050469 Jeeraphong Thanongsaksrikul Wanpen Chaicumpa http://www.mdpi.com/2072-6651/3/5/453 Ricin is a highly toxic type II ribosome-inactivating protein that has potential as a biochemical weapon and as the toxic component of immunotoxins. The unfolded protein response (UPR) is a survival response that helps cells to recover from endoplasmic reticulum (ER) stress. Failure to recover from ER stress leads to apoptosis. In yeast, ricin-A-chain (RTA), the enzymatic component of ricin, inhibits UPR. Our goals were to determine if RTA inhibits UPR in two epithelial cell lines and if this affects RTA cytotoxicity. RTA alone did not induce UPR. However, RTA inhibited both phosphorylation of inositol-requiring enzyme 1 (IRE1) and splicing of X-box binding protein1 mRNA by the UPR-inducing agent tunicamycin (Tm). The ability of dithiothreitol (DTT) to activate eukaryotic translation initiation factor 2 alpha (eIF2α), a component of the PERK pathway, was also inhibited by RTA. Treatment with RTA in combination with Tm or DTT inhibited protein synthesis more than either agent did alone in one cell line, while caspase cleavage was enhanced by the treatment combination in both cell lines. These data indicate that RTA is more cytotoxic when UPR is inhibited. This ability to inhibit UPR may enhance the potential of RTA as a therapeutic immunotoxin in solid tumors. http://www.mdpi.com/2072-6651/3/5/453 Tue, 10 May 2011 00:00:00 CEST Toxins 2011-05-10 3 5 Article 453 468 2072-6651 Inhibition of the Unfolded Protein Response by Ricin A-Chain Enhances Its Cytotoxicity in Mammalian Cells 2011-05-10 doi: 10.3390/toxins3050453 Chao-Ting Wang Amanda E. Jetzt Ju-Shun Cheng Wendie S. Cohick http://www.mdpi.com/2072-6651/3/5/442 Autoclaved oats were inoculated with a strain of Fusarium sporotrichioides or Fusarium poae. Moisture content of oats after inoculation was at 38%, incubation took place in standing culture at 28 °C. The A-type trichothecenes, 4,15-diacetoxyscirpenol (4,15-DAS), 15-monoacetoxyscirpenol (15-MAS), and scirpentriol (SCIRP) were analyzed by GC/MS. For each strain, three culture flasks were harvested at 2–3 day intervals starting immediately after inoculation. Total incubation time was 42 days (F. poae) and 56 days (F. sporotrichioides). Following peak accumulation, 4,15-DAS decreased below the detection limit for both strains, 15-MAS decreased below this limit for the isolate of F. sporotrichioides, for the isolate of F. poae it decreased to a level markedly below the peak value. SCIRP, after having peaked, decreased to some extent for the strain F. sporotrichioides, with a significant (P = 0.0029) negative linear regression of toxin content against culture age during this period. The content of 15-MAS, and in part also of 4,15-DAS, decreased along with an increase of SCIRP. This sequential accumulation pattern suggests the successive induction of esterases deacetylating 4,15-DAS and 15-MAS, as well as of enzymes involved in the metabolization of the parent alcohol, SCIRP. The results may explain, at least in part, the somewhat higher incidence in naturally contaminated compounds reported in the literature for SCIRP compared to 4,15-DAS and 15-MAS. http://www.mdpi.com/2072-6651/3/5/442 Mon, 09 May 2011 00:00:00 CEST Toxins 2011-05-09 3 5 Article 442 452 2072-6651 Accumulation Kinetics of Three Scirpentriol-Based Toxins in Oats Inoculated in Vitro with Isolates of Fusarium sporotrichioides and Fusarium poae 2011-05-09 doi: 10.3390/toxins3050442 Margit Schollenberger Hans-Martin Müller Melanie Liebscher Claudia Schlecker Melanie Berger Wilfried Hermann http://www.mdpi.com/2072-6651/3/5/420 The type 2 ribosome-inactivating proteins (RIPs) isolated from some species belonging to the Sambucus genus, have the characteristic that although being even more active than ricin inhibiting protein synthesis in cell-free extracts, they lack the high toxicity of ricin and related type 2 RIPs to intact cells and animals. This is due to the fact that after internalization, they follow a different intracellular pathway that does not allow them to reach the cytosolic ribosomes. The lack of toxicity of type 2 RIPs from Sambucus make them good candidates as toxic moieties in the construction of immunotoxins and conjugates directed against specific targets. Up to now they have been conjugated with either transferrin or anti-CD105 to target either transferrin receptor- or endoglin-overexpressing cells, respectively. http://www.mdpi.com/2072-6651/3/5/420 Fri, 29 Apr 2011 00:00:00 CEST Toxins 2011-04-29 3 5 Review 420 441 2072-6651 Use of Ribosome-Inactivating Proteins from Sambucus for the Construction of Immunotoxins and Conjugates for Cancer Therapy 2011-04-29 doi: 10.3390/toxins3050420 José M. Ferreras Lucía Citores Rosario Iglesias Pilar Jiménez Tomás Girbés http://www.mdpi.com/2072-6651/3/4/409 Precursor B cell acute lymphoblastic leukemia (pre-B ALL) affects five to six thousand adults and almost three thousand children every year. Approximately 25% of the children and 60% of the adults die from their disease, highlighting the need for new therapies that complement rather than overlap chemotherapy and bone marrow transplantation. Immunotherapy is a class of therapies where toxicities and mechanisms of action do not overlap with those of chemotherapy. Because CD19 is a B cell- restricted membrane antigen that is expressed on the majority of pre-B tumor cells, a CD19-based immunotherapy is being developed for ALL. In this study, the anti-tumor activities of immunotoxins (ITs) constructed by conjugating a murine monoclonal antibody (MAb), HD37, or its chimeric (c) construct to recombinant ricin toxin A chain (rRTA) were compared both in vitro using human pre-B ALL and Burkitt’s lymphoma cell lines and in vivo using a disseminated human pre-B ALL tumor cell xenograft model. The murine and chimeric HD37 IT constructs were equally cytotoxic to pre-B ALL and Burkitt’s lymphoma cells in vitro and their use in vivo resulted in equivalent increases in survival of SCID mice with human pre-B ALL tumors when compared with control mice. http://www.mdpi.com/2072-6651/3/4/409 Wed, 06 Apr 2011 00:00:00 CEST Toxins 2011-04-06 3 4 Article 409 419 2072-6651 A Comparison of the Anti-Tumor Effects of a Chimeric versus Murine Anti-CD19 Immunotoxins on Human B Cell Lymphoma and Pre-B Acute Lymphoblastic Leukemia Cell Lines 2011-04-06 doi: 10.3390/toxins3040409 Lydia K. Tsai Laurentiu M. Pop Xiaoyun Liu Ellen S. Vitetta http://www.mdpi.com/2072-6651/3/4/398 Ricin is a highly toxic protein present in the seeds of Ricinus communis (castor), grown principally as a source of high quality industrial lubricant and as an ornamental. Because ricin has been used for intentional poisoning in the past and could be used to contaminate food, there is a need for analytical methodology to detect ricin in food matrices. A monoclonal antibody-based method was developed for detecting and quantifying ricin in ground beef, a complex, fatty matrix. The limit of detection was 0.5 ng/g for the electrochemiluminescence (ECL) method and 1.5 ng/g for enzyme-linked immunosorbent assay (ELISA). The detection of nanogram per gram quantities of ricin spiked into retail samples of ground beef provides approximately 10,000-fold greater sensitivity than required to detect a toxic dose of ricin (>1 mg) in a 100 g sample. http://www.mdpi.com/2072-6651/3/4/398 Mon, 04 Apr 2011 00:00:00 CEST Toxins 2011-04-04 3 4 Article 398 408 2072-6651 Detection of Ricin Contamination in Ground Beef by Electrochemiluminescence Immunosorbent Assay 2011-04-04 doi: 10.3390/toxins3040398 David L. Brandon http://www.mdpi.com/2072-6651/3/4/369 Liposomes have long been effective delivery vehicles for transport of toxins to peripheral cancers. The combination of convection-enhanced delivery (CED) with liposomal toxins was originally proposed to circumvent the limited delivery of intravascular liposomes to the central nervous system (CNS) due to the blood-brain-barrier (BBB). CED offers markedly improved distribution of infused therapeutics within the CNS compared to direct injection or via drug eluting polymers, both of which depend on diffusion for parenchymal distribution. This review examines the basis for improved delivery of liposomal toxins via CED within the CNS, and discusses preclinical and clinical experience with these therapeutic techniques. How CED and liposomal technologies may influence future neurooncologic treatments are also considered. http://www.mdpi.com/2072-6651/3/4/369 Thu, 31 Mar 2011 00:00:00 CEST Toxins 2011-03-31 3 4 Review 369 397 2072-6651 The Use of Convection-Enhanced Delivery with Liposomal Toxins in Neurooncology 2011-03-31 doi: 10.3390/toxins3040369 Massimo S. Fiandaca Mitchel S. Berger Krystof S. Bankiewicz http://www.mdpi.com/2072-6651/3/4/356 Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions. http://www.mdpi.com/2072-6651/3/4/356 Tue, 29 Mar 2011 00:00:00 CEST Toxins 2011-03-29 3 4 Review 356 368 2072-6651 Cycle Inhibiting Factors (Cifs): Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells 2011-03-29 doi: 10.3390/toxins3040356 Frédéric Taieb Jean-Philippe Nougayrède Eric Oswald http://www.mdpi.com/2072-6651/3/4/345 The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications. http://www.mdpi.com/2072-6651/3/4/345 Thu, 24 Mar 2011 00:00:00 CET Toxins 2011-03-24 3 4 Article 345 355 2072-6651 Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity 2011-03-24 doi: 10.3390/toxins3040345 Enrico Ferrari Elizabeth S. Maywood Laura Restani Matteo Caleo Marco Pirazzini Ornella Rossetto Michael H. Hastings Dhevahi Niranjan Giampietro Schiavo Bazbek Davletov http://www.mdpi.com/2072-6651/3/3/309 Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins. http://www.mdpi.com/2072-6651/3/3/309 Tue, 22 Mar 2011 00:00:00 CET Toxins 2011-03-22 3 3 Review 309 344 2072-6651 Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes 2011-03-22 doi: 10.3390/toxins3030309 Olga Meiri Chaim Dilza Trevisan-Silva Daniele Chaves-Moreira Ana Carolina M. Wille Valéria Pereira Ferrer Fernando Hitomi Matsubara Oldemir Carlos Mangili Rafael Bertoni da Silveira Luiza Helena Gremski Waldemiro Gremski Andrea Senff-Ribeiro Silvio Sanches Veiga http://www.mdpi.com/2072-6651/3/3/294 Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition,since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism. http://www.mdpi.com/2072-6651/3/3/294 Mon, 21 Mar 2011 00:00:00 CET Toxins 2011-03-21 3 3 Review 294 308 2072-6651 Mechanism of Diphtheria Toxin Catalytic Domain Delivery to the Eukaryotic Cell Cytosol and the Cellular Factors that Directly Participate in the Process 2011-03-21 doi: 10.3390/toxins3030294 John R. Murphy http://www.mdpi.com/2072-6651/3/3/260 Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted. http://www.mdpi.com/2072-6651/3/3/260 Mon, 21 Mar 2011 00:00:00 CET Toxins 2011-03-21 3 3 Review 260 293 2072-6651 From Toxins Targeting Ligand Gated Ion Channels to Therapeutic Molecules 2011-03-21 doi: 10.3390/toxins3030260 Adak Nasiripourdori Valérie Taly Thomas Grutter Antoine Taly http://www.mdpi.com/2072-6651/3/3/242 Aggregatibacter actinomycetemcomitans has been described as a member of the indigenous oral microbiota of humans, and is involved in the pathology of periodontitis and various non-oral infections. This bacterium selectively kills human leukocytes through expression of leukotoxin, a large pore-forming protein that belongs to the Repeat in Toxin (RTX) family. The specificity of the toxin is related to its prerequisite for a specific target cell receptor, LFA-1, which is solely expressed on leukocytes. The leukotoxin causes death of different leukocyte populations in a variety of ways. It activates a rapid release of lysosomal enzymes and MMPs from neutrophils and causes apoptosis in lymphocytes. In the monocytes/macrophages, the toxin activates caspase-1, a cysteine proteinase, which causes a proinflammatory response by the activation and secretion of IL-1β and IL-18. A specific clone (JP2) of A. actinomycetemcomitans with enhanced leukotoxin expression significantly correlates to disease onset in infected individuals. Taken together, the mechanisms by which this toxin kills leukocytes are closely related to the pathogenic mechanisms of inflammatory disorders, such as periodontitis. Therapeutic strategies targeting the cellular and molecular inflammatory host response in periodontal diseases might be a future treatment alternative. http://www.mdpi.com/2072-6651/3/3/242 Mon, 21 Mar 2011 00:00:00 CET Toxins 2011-03-21 3 3 Review 242 259 2072-6651 Aggregatibacter actinomycetemcomitans Leukotoxin: A Powerful Tool with Capacity to Cause Imbalance in the Host Inflammatory Response 2011-03-21 doi: 10.3390/toxins3030242 Anders Johansson http://www.mdpi.com/2072-6651/3/3/218 The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity. http://www.mdpi.com/2072-6651/3/3/218 Wed, 16 Mar 2011 00:00:00 CET Toxins 2011-03-16 3 3 Article 218 241 2072-6651 Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes 2011-03-16 doi: 10.3390/toxins3030218 Tana L. Repella Mengfei Ho Tracy P. M. Chong Yuka Bannai Brenda A. Wilson