Toxins, Volume 5, Issue 2 (February 2013) – 14 articles , Pages 203-471

Various gating modifier toxins partition into membranes and interfere with the gating mechanisms of biological ion channels. For example, GsMTx4 potentiates gramicidin and several bacterial mechanosensitive channels whose gating kinetics are sensitive to mechanical properties of the membrane, whereas binding of HpTx2 shifts the voltage-activity curve of the voltage-gated potassium channel Kv4.2 to the right. The detailed process by which the toxin partitions into membranes has been difficult to probe using molecular dynamics due to the limited time scale accessible. Here we develop a protocol that allows the spontaneous assembly of a polypeptide toxin into membranes in atomistic molecular dynamics simulations of tens of nanoseconds. The protocol is applied to GsMTx4 and HpTx2. Both toxins, released in water at the start of the simulation, spontaneously bind into the lipid bilayer within 50 ns, with their hydrophobic patch penetrated into the bilayer beyond the phosphate groups of the lipids. It is found that the bilayer is about 2 Å thinner upon the binding of a GsMTx4 monomer. Such a thinning effect of GsMTx4 on membranes may explain its potentiation effect on gramicidin and mechanosensitive channels. Full article

Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi. Ergot alkaloid pathways vary among different fungal lineages, but the pathway intermediate chanoclavine-I is evolutionarily conserved among ergot alkaloid producers. At least four genes, dmaW, easF, easE, and easC, are necessary for pathway steps prior to chanoclavine-I; however, the sufficiency of these genes for chanoclavine-I synthesis has not been established. A fragment of genomic DNA containing dmaW, easF, easE, and easC was amplified from the human-pathogenic, ergot alkaloid-producing fungus Aspergillus fumigatus and transformed into Aspergillus nidulans, a model fungus that does not contain any of the ergot alkaloid synthesis genes. HPLC and LC-MS analyses demonstrated that transformed A. nidulans strains produced chanoclavine-I and an earlier pathway intermediate. Aspergillus nidulans transformants containing dmaW, easF, and either easE or easC did not produce chanoclavine-I but did produce an early pathway intermediate and, in the case of the easC transformant, an additional ergot alkaloid-like compound. We conclude that dmaW, easF, easE, and easC are sufficient for the synthesis of chanoclavine-I in A. nidulans and expressing ergot alkaloid pathway genes in A. nidulans provides a novel approach to understanding the early steps in ergot alkaloid synthesis. Full article

Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77), causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05–0.1 pM), inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation. Full article

Mycotoxins are secondary metabolites of fungi that can cause serious health problems in animals, and may result in severe economic losses. Deleterious effects of these feed contaminants in animals are well documented, ranging from growth impairment, decreased resistance to pathogens, hepato- and nephrotoxicity to death. By contrast, data with regard to their impact on intestinal functions are more limited. However, intestinal cells are the first cells to be exposed to mycotoxins, and often at higher concentrations than other tissues. In addition, mycotoxins specifically target high protein turnover- and activated-cells, which are predominant in gut epithelium. Therefore, intestinal investigations have gained significant interest over the last decade, and some publications have demonstrated that mycotoxins are able to compromise several key functions of the gastrointestinal tract, including decreased surface area available for nutrient absorption, modulation of nutrient transporters, or loss of barrier function. In addition some mycotoxins facilitate persistence of intestinal pathogens and potentiate intestinal inflammation. By contrast, the effect of these fungal metabolites on the intestinal microbiota is largely unknown. This review focuses on mycotoxins which are of concern in terms of occurrence and toxicity, namely: aflatoxins, ochratoxin A and Fusarium toxins. Results from nearly 100 published experiments (in vitro, ex vivo and in vivo) were analyzed with a special attention to the doses used. Full article

In 1998, during a toxicological surveillance of various marine fouling organisms in Hiroshima Bay, Japan, specimens of the ribbon worm, Cephalothrix simula (Nemertea: Palaeonemertea) were found. These ribbon worms contained toxins with extremely strong paralytic activity. The maximum toxicity in terms of tetrodotoxin (TTX) was 25,590 mouse units (MU) per gram for the whole worm throughout the monitoring period. The main toxic component was isolated and recrystallized from an acidified methanolic solution. The crystalline with a specific toxicity of 3520 MU/mg was obtained and identified as TTX by high performance liquid chromatography (HPLC)-fluorescent detection (FLD) (HPLC-FLD), electrospray ionization-mass spectrometry (ESI-MS), infrared (IR), nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC-MS). The highest toxicity of C. simula exceeded the human lethal dose per a single worm. A toxicological surveillance of C. simula from 1998 to 2005 indicated approximately 80% of the individuals were ranked as “strongly toxic” (≥1000 MU/g). Forty-eight percent of the specimens possessed toxicity scores of more than 2000 MU/g. Seasonal variations were observed in the lethal potency of C. simula. Specimens collected on January 13, 2000 to December 26, 2000 showed mean toxicities of 665–5300 MU/g (n = 10). These data prompted a toxicological surveillance of ribbon worms from other localities with different habitats in Japan, including Akkeshi Bay (Hokkaido) under stones on rocky intertidal beaches, as well as Otsuchi (Iwate) among calcareous tubes of serpulid polychaetes on rocky shores. Within twelve species of ribbon worms examined, only C. simula possessed extremely high toxicity. Therefore, C. simula appears to show generally high toxicity irrespective of their locality and habitat. Full article

Natural killer (NK) cells exert important immunoregulatory functions by releasing several inflammatory molecules, such as IFN-γ and members of chemokines, which include CCL3/MIP-1α and CCL4/MIP-1β. These cells also express heptahelical receptors, which are coupled to heterotrimeric G proteins that guide them into inflamed and injured tissues. NK cells have been shown to recognize and destroy transformed cells and virally-infected cells, but their roles in neurodegenerative diseases have not been examined in detail. In this review, I will summarize the effects of NK cells in two neurodegenerative diseases, namely multiple sclerosis and globoid cell leukodystrophy. It is hoped that the knowledge obtained from these diseases may facilitate building rational protocols for treating these and other neurodegenerative or autoimmune diseases using NK cells and drugs that activate them as therapeutic tools. Full article

Chemokines are important regulators of many different biological processes, including (i) inflammation with activation and local recruitment of immunocompetent cells; (ii) angiogenesis as a part of inflammation or carcinogenesis; and (iii) as a bridge between the coagulation system and inflammation/immune activation. The systemic levels of various chemokines may therefore reflect local disease processes, and such variations may thereby be used in the routine clinical handling of patients. The experience from patients with myeloproliferative diseases, and especially patients with acute myeloid leukemia (AML), suggests that systemic plasma/serum cytokine profiles can be useful, both as a diagnostic tool and for prognostication of patients. However, cytokines/chemokines are released by a wide range of cells and are involved in a wide range of biological processes; the altered levels may therefore mainly reflect the strength and nature of the biological processes, and the optimal clinical use of chemokine/cytokine analyses may therefore require combination with organ-specific biomarkers. Chemokine levels are also altered by clinical procedures, therapeutic interventions and the general status of the patients. A careful standardization of sample collection is therefore important, and the interpretation of the observations will require that the overall clinical context is considered. Despite these limitations, we conclude that analysis of systemic chemokine/cytokine profiles can reflect important clinical characteristics and, therefore, is an important scientific tool that can be used as a part of future clinical studies to identify clinically relevant biomarkers. Full article

A bradykinin-potentiating peptide (BPP) from Amazon Bothrops atrox venom with m/z 1384.7386 was identified and characterized by collision induced dissociation (CID) using an ESI-MS/MS spectra obtained in positive ion mode on a hybrid Qq-oaTOF mass spectrometer, Xevo G2 QTof MS (Waters, Manchester, UK). De novo peptide sequence analysis of the CID fragmentation spectra showed the amino acid sequence ZKWPRPGPEIPP, with a pyroglutamic acid and theoretical monoisotopic m/z 1384.7378, which is similar to experimental data, showing a mass accuracy of 0.6 ppm. The peptide is homologous to other BPP from Bothrops moojeni and was named as BPP-BAX12. Full article

The genes encoding Anthrax Toxin Receptors (ANTXRs) were originally identified based on expression in endothelial cells suggesting a role in angiogenesis. The focus of this review is to discuss what has been learned about the physiological roles of these receptors through evaluation of the Antxr knockout mouse phenotypes. Mice mutant in Antxr genes have defects in extracellular matrix homeostasis. We discuss how knowledge of physiological ANTXR function relates to what is already known about anthrax intoxication. Full article

Ca_v2.2 is a calcium channel subtype localized at nerve terminals, including nociceptive fibers, where it initiates neurotransmitter release. Ca_v2.2 is an important contributor to synaptic transmission in ascending pain pathways, and is up-regulated in the spinal cord in chronic pain states along with the auxiliary α2δ1 subunit. It is therefore not surprising that toxins that inhibit Ca_v2.2 are analgesic. Venomous animals, such as cone snails, spiders, snakes, assassin bugs, centipedes and scorpions are rich sources of remarkably potent and selective Ca_v2.2 inhibitors. However, side effects in humans currently limit their clinical use. Here we review Ca_v2.2 inhibitors from venoms and their potential as drug leads. Full article

Okadaic acid (OA) and its derivatives, which are produced by dinoflagellates of the genera Prorocentrum and Dinophysis, are responsible for diarrhetic shellfish poisoning in humans. In laboratory animals, these toxins cause epithelial damage and fluid accumulation in the gastrointestinal tract, and at high doses, they cause death. These substances have also been shown to be tumour promoters, and when injected into the brains of rodents, OA induces neuronal damage reminiscent of that seen in Alzheimer’s disease. OA and certain of its derivatives are potent inhibitors of protein phosphatases, which play many roles in cellular metabolism. In 1990, it was suggested that inhibition of these enzymes was responsible for the diarrhetic effect of these toxins. It is now repeatedly stated in the literature that protein phosphatase inhibition is not only responsible for the intestinal effects of OA and derivatives, but also for their acute toxic effects, their tumour promoting activity and their neuronal toxicity. In the present review, the evidence for the involvement of protein phosphatase inhibition in the induction of the toxic effects of OA and its derivatives is examined, with the conclusion that the mechanism of toxicity of these substances requires re-evaluation. Full article

Local chemodenervation with botulinum toxin (BoNT) injections to relax abnormally contracting muscles has been shown to be an effective and well-tolerated treatment in a variety of movement disorders and other neurological and non-neurological disorders. Despite almost 30 years of therapeutic use, there are only few studies of patients treated with BoNT injections over long period of time. These published data clearly support the conclusion that BoNT not only provides safe and effective symptomatic relief of dystonia but also long-term benefit and possibly even favorably modifying the natural history of this disease. The adverse events associated with chronic, periodic exposure to BoNT injections are generally minor and self-limiting. With the chronic use of BoNT and an expanding list of therapeutic indications, there is a need to carefully examine the existing data on the long-term efficacy and safety of BoNT. In this review we will highlight some of the aspects of long-term effects of BoNT, including efficacy, safety, and immunogenicity. Full article

Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP) consensus sequence in recombinant ricin toxin A-chain (RTA) that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N) and V76 (to I, or M) had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP(−) variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C) along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the T_m was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond. Full article

Secretory phospholipasesA₂ (sPLA₂s) form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA₂s from Vipera lebetina (VLPLA₂), Vipera berus berus (VBBPLA₂), and Naja naja oxiana (NNOPLA₂) venoms on (i) human platelets, (ii) four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis) and (iii) five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10) in vitro. sPLA₂s inhibited collagen-induced platelet aggregation: VBBPLA₂ IC₅₀ = 0.054, VLPLA₂ IC₅₀ = 0.072, NNOPLA₂ IC₅₀ = 0.814 μM. p-Bromophenacylbromide-inhibited sPLA₂ had no inhibitory action on platelets. 36.17 μM VBBPLA₂ completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA₂s (~0.7 μM and ~7 μM) towards cancer cells depended on both venom and cell type. VBBPLA₂ (7.2 μM) inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA₂s exhibited no inhibitory effect towards LNCaP cells and some effect (8%–20%) towards other cells. Thus, already sub-μM concentrations of sPLA₂s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA₂s and test cells, VBBPLA₂ was the most growth inhibitory towards Bacillus subtilis and K-562 cells. Full article