Toxins

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Open AccessArticle

Short Toxin-like Proteins Attack the Defense Line of Innate Immunity

by Yitshak Tirosh, Dan Ofer, Tsiona Eliyahu and Michal Linial

Toxins 2013, 5(7), 1314-1331; https://doi.org/10.3390/toxins5071314 - 23 Jul 2013

Cited by 13 | Viewed by 9475

ClanTox (classifier of animal toxins) was developed for identifying toxin-like candidates from complete proteomes. Searching mammalian proteomes for short toxin-like proteins (coined TOLIPs) revealed a number of overlooked secreted short proteins with an abundance of cysteines throughout their sequences. We applied bioinformatics and [...] Read more.

ClanTox (classifier of animal toxins) was developed for identifying toxin-like candidates from complete proteomes. Searching mammalian proteomes for short toxin-like proteins (coined TOLIPs) revealed a number of overlooked secreted short proteins with an abundance of cysteines throughout their sequences. We applied bioinformatics and data-mining methods to infer the function of several top predicted candidates. We focused on cysteine-rich peptides that adopt the fold of the three-finger proteins (TFPs). We identified a cluster of duplicated genes that share a structural similarity with elapid neurotoxins, such as α-bungarotoxin. In the murine proteome, there are about 60 such proteins that belong to the Ly6/uPAR family. These proteins are secreted or anchored to the cell membrane. Ly6/uPAR proteins are associated with a rich repertoire of functions, including binding to receptors and adhesion. Ly6/uPAR proteins modulate cell signaling in the context of brain functions and cells of the innate immune system. We postulate that TOLIPs, as modulators of cell signaling, may be associated with pathologies and cellular imbalance. We show that proteins of the Ly6/uPAR family are associated with cancer diagnosis and malfunction of the immune system. Full article

(This article belongs to the Special Issue Novel Properties of Well-Characterized Toxins)

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Figure 1
Classifier of animal toxins (ClanTox) results from mouse short proteome. Using 7337 short sequences as input to ClanTox resulted in 114 sequences (1.5%) that were scored as very high/high confidence levels. The dominant group from the high scored positive predictions are the β-defensins (beta defensin/neutrophil defensin domain) that account for 27% of the top predictions. Proteins that can be considered as TOLIPs include kazal protease inhibitors, metallothioneins, WAP proteins and several uncharacterized Epidermal growth factor (EGF) like proteins. An additional 304 sequences were scored at a moderate confidence level (P1, See Materials and Methods). A similar composition of domains was detected among these 304 moderate scored sequences. In addition, domains of “proline rich proteins” and “tripartite motif-containing proteins” were also found among the positively predicted list. The fraction of InterPro annotated proteins is indicated. Representatives of three-finger (TFP) fold proteins are distributed in all levels of positive predictions (P1–P3, see Materials and Methods). Full article ">Figure 2
Gene organization in mouse chromosome, 9qA4. A chromosomal region that covers 1.1 million nucleotides (position 35.6–36.7 M) is shown. This region covers testis related genes and previously uncharacterized short proteins. Applying structural prediction methods indicates that this genomic segment is rich with TOLIPs that belong to the TFP fold. The chromosomal arrangement is in accord with a recent duplication event. The information for each putative gene is summarized as follows: starting nucleotide: 35607093; transcript length: 4792 nucleotide; gene name: Pate4; strand orientation: -; detailed name: prostate and testis expressed 4. Full article ">Figure 3
The Ly-6/uPAR/α-neurotoxin domain. (A) Multiple sequence alignment of 28 proteins that contain a single LU domain from mouse. All Ly-6 related proteins are localized within 0.9 million nucleotides from chromosome 15 (position 74.7 M–75.6 M). The 10 conserved cysteines that form five-disulfide bridges are marked (below the sequences); (B) A representative fold for the Ly-6/uPAR/α-neurotoxin is the 3D structure of human CD59. This fold is a prototype for listed proteins of the LU domain; (C) The secondary structure organization of 78 amino acids of the human CD59 is shown. A similar organization of β-sheets and short α-helices is found in other proteins of the LU domain. Full article ">Figure 4
Domains’ composition of the uPAR/Ly6/CD59/snake toxin-receptor clan. The relative size of each domain family is shown in the central ring. The five domain families that compose the uPAR/Ly6/CD59/snake toxin-receptor superfamily are: (i) Toxin_1, which are proteins from elapid toxins, (ii) activin receptor, (iii) LU domain, (iv) PLA2_Inh and (v) BAMB1. The clan covers over 2900 proteins from 270 organisms. The pie charts display the compositions of the family domains for each of the five families in the clan. Proteins that are single domain are marked by a dashed pattern. All proteins of the Toxin_1 family are single domains. Note that a large fraction of the proteins in the UPAR_LY6, PF00021) are composed of two occurrences of the domain (UPAR_LY6 × 2) or a combination of the PLA2_Inh and UPAR_LY6. Full article ">Figure 5
Structural view on toxin-fold (SCOP g.7.1). SCOP classification is based on SCOP 1.75B. There are three families that compose the superfamily g.7.1 including: (i) g.7.1.1, snake venom toxins (28 protein domains) (ii) g.7.1.2, dendroaspin (one protein) and (iii) g.7.1.3, Extracellular domain of cell surface receptors (five protein domains). Representative examples are shown with their Protein Data Bank (PDB) identifier and the organism. The overall representatives share less than 40% sequence identity. Note that the structural similarity is high, with three loops and nearly all structure dominated by the β-strands. The length of most of these structural domains ranges from 65–85 amino acids. Full article ">Figure 6
Human Ly-6 proteins and their disease associations. The most significant 30 human uPAR/Ly6 (LU) genes that were implicated in disease and pathologies are shown (blue ovals). There are 48 human LU family members. Data on disease-gene association was extracted from the UniProtKB cross reference of NextBio. Only high score (score > 0.8) disease-gene associations supported by at least two genes from the LU domain family are reported. The relative expression level of LU domain proteins in the context of diseases is colored to indicate change in the gene expression. The induction and suppression are colored red and green, respectively. Several disease associations are based on genomics screening and GWAS (genome-wide association studies, black edges). We noted diseases such as “Wound of the skin” and “Diabetes type I” as “hub” connecting dominating genes. These diseases are the manifestation of an imbalance of the immune system. Full article ">

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Open AccessArticle

Development and Evaluation of Monoclonal Antibodies for the Glucoside of T-2 Toxin (T2-Glc)

by Chris M. Maragos, Cletus Kurtzman, Mark Busman, Neil Price and Susan McCormick

Toxins 2013, 5(7), 1299-1313; https://doi.org/10.3390/toxins5071299 - 19 Jul 2013

Cited by 19 | Viewed by 8020

Abstract

The interactions between fungi and plants can yield metabolites that are toxic in animal systems. Certain fungi are known to produce sesquiterpenoid trichothecenes, such as T-2 toxin, that are biotransformed by several mechanisms including glucosylation. The glucosylated forms have been found in grain [...] Read more.

The interactions between fungi and plants can yield metabolites that are toxic in animal systems. Certain fungi are known to produce sesquiterpenoid trichothecenes, such as T-2 toxin, that are biotransformed by several mechanisms including glucosylation. The glucosylated forms have been found in grain and are of interest as potential reservoirs of T-2 toxin that are not detected by many analytical methods. Hence the glucosides of trichothecenes are often termed “masked” mycotoxins. The glucoside of T-2 toxin (T2-Glc) was linked to keyhole limpet hemocyanin and used to produce antibodies in mice. Ten monoclonal antibody (Mab)-producing hybridoma cell lines were developed. The Mabs were used in immunoassays to detect T2-Glc and T-2 toxin, with midpoints of inhibition curves (IC₅₀s) in the low ng/mL range. Most of the Mabs demonstrated good cross-reactivity to T-2 toxin, with lower recognition of HT-2 toxin. One of the clones (2-13) was further characterized with in-depth cross-reactivity and solvent tolerance studies. Results suggest Mab 2-13 will be useful for the simultaneous detection of T-2 toxin and T2-Glc. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

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Open AccessArticle

Differences in the Regulation of Ochratoxin A by the HOG Pathway in Penicillium and Aspergillus in Response to High Osmolar Environments

by Dominic Stoll, Markus Schmidt-Heydt and Rolf Geisen

Toxins 2013, 5(7), 1282-1298; https://doi.org/10.3390/toxins5071282 - 19 Jul 2013

Cited by 43 | Viewed by 6907

Abstract

Penicillium verrucosum, P. nordicum and Aspergillus carbonarius are three important ochratoxin A producing species. P. verrucosum is in addition able to produce citrinin. It has been shown earlier that P. nordicum is adapted to NaCl rich environments like salt rich dry cured [...] Read more.

Penicillium verrucosum, P. nordicum and Aspergillus carbonarius are three important ochratoxin A producing species. P. verrucosum is in addition able to produce citrinin. It has been shown earlier that P. nordicum is adapted to NaCl rich environments like salt rich dry cured foods or even salines. In this organism, the biosynthesis of ochratoxin A plays an adaptive role in this habitat. P. verrucosum generally can be found on cereals, but occasionally also on salt rich dry cured foods. In contrast A. carbonarius usually cannot be found in NaCl rich environments, but it occurs in another environment with high concentration of solutes, e.g., in sugar rich substrates like grapes and grape juices. Usually osmotic challenging conditions activate the HOG MAP kinase signal cascade, which in turn activates various osmo-regulated genes. In the current analysis, it could be demonstrated that in case of P. nordicum and P. verrucosum the NaCl induced production of ochratoxin A is correlated to the phosphorylation status of the HOG MAP kinase. Just the opposite was true for A. carbonarius. In this case, also higher amounts of NaCl in the medium lead to an increased phosphorylation status of HOG, but no increase in ochratoxin biosynthesis was observed. In contrast to the Penicillia, higher NaCl concentrations lead to a rapid cessation of growth by A. carbonarius. High glucose concentrations have much less impact on growth and the phosphorylation of HOG. Full article

(This article belongs to the Special Issue Recent Advances in Ochratoxins Research)

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Growth rate of P. verrucosum, P. nordicum and A. carbonarius after growth on YES medium substituted with increasing amounts of NaCl for five days (Aspergillus) and nine days (Penicillium) at 25 °C in case of P. verrucosum and P. nordicum and at 30 °C in case of A. carbonarius. An amount of 10 µL of a spore suspension of 106 spores/mL were centrally spotted on an agar plate and incubated. The colony diameters were measured after six and nine days, respectively. Full article ">Figure 2
Relation of the production of ochratoxin A and citrinin (in case of P. verrucosum) determined by HPLC with the phosphorylation status of the HOG protein, determined by western blotting for P. nordicum (A); P. verrucosum (B); and A. carbonarius (C). The fungal strains were incubated on YES medium with increasing amounts of NaCl at 25 °C (Penicillia) or 30 °C (Aspergillus) for nine days. After that time, samples were withdrawn, subjected to HPLC or to western blotting. Full article ">Figure 3
Growth rate of A. carbonarius and P. nordicum on malt glucose medium (MEA medium). The growth rate was determined as described in legend to <a href="#toxins-05-01282-f001" class="html-fig">Figure 1</a>. Full article ">Figure 4
Influence of the glucose concentration on the biosynthesis of ochratoxin A and on the phosphorylation status of HOG by P. nordicum (A); and A. carbonarius (B). The fungal strains were grown for nine days on MEA medium with increasing amounts of glucose. After that time, samples were withdrawn and used in HPLC to determine the ochratoxin A produced and for western blot analysis to visualize the phosphorylation status of HOG. Full article ">Figure 5
Inactivation of the hog gene by ATMT. The cassette used for homologous integration (A) into the hog gene, was cloned in pPK2 [<a href="#B18-toxins-05-01282" class="html-bibr">18</a>]. LB, RB left and right border of the Ti DNA respectively, ∆hog5', ∆hog3' DNA regions from the upstream (5') and downstream (3') of the hog gene, hphB hygromycin resisstance gene from Streptomyces hygroscopicus. The vector was transformed into P. verrucosum BFE808 by A. tumefaciens mediated transformation. One of the resulting transformants (T4) was analyzed by TLC to visualize its ochratoxin A and citrinin production capacity (B) and to compare it with the wild type (WT). Citrinin shows the typical smear, which is due to the high number of hydroxyl groups which interfere with the silica gel under the conditions used. Transformants with a changed capacity to produce ochratoxin A were further analyzed by Southern blotting (C). For Southern blotting the chromosomal DNA of the wild type and the transformant T4 were digested with EcoR1, separated on an agarose gel (the vertical arrow indicates the direction of electrophoresis), transferred to a nylon membrane and hybridized to the hog gene probe. In the transformant a shift of the hybridization signal towards higher fragment lengths can be seen (C) indicating that the vector had integrated into the chromosomal hog gene and thereby had inactivated the gene. Full article ">

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Open AccessArticle

The Use of Feed Additives to Reduce the Effects of Aflatoxin and Deoxynivalenol on Pig Growth, Organ Health and Immune Status during Chronic Exposure

by Alexandra C. Weaver, M. Todd See, Jeff A. Hansen, Yong B. Kim, Anna L. P. De Souza, Teena F. Middleton and Sung Woo Kim

Toxins 2013, 5(7), 1261-1281; https://doi.org/10.3390/toxins5071261 - 17 Jul 2013

Cited by 90 | Viewed by 10204

Abstract

Three feed additives were tested to improve the growth and health of pigs chronically challenged with aflatoxin (AF) and deoxynivalenol (DON). Gilts (n = 225, 8.8 ± 0.4 kg) were allotted to five treatments: CON (uncontaminated control); MT (contaminated with 150 µg/kg [...] Read more.

Three feed additives were tested to improve the growth and health of pigs chronically challenged with aflatoxin (AF) and deoxynivalenol (DON). Gilts (n = 225, 8.8 ± 0.4 kg) were allotted to five treatments: CON (uncontaminated control); MT (contaminated with 150 µg/kg AF and 1100 µg/kg DON); A (MT + a clay additive); B (MT + a clay and dried yeast additive); and C (MT + a clay and yeast culture additive). Average daily gain (ADG) and feed intake (ADFI) were recorded for 42 days, blood collected for immune analysis and tissue samples to measure damage. Feeding mycotoxins tended to decrease ADG and altered the immune system through a tendency to increase monocytes and immunoglobulins. Mycotoxins caused tissue damage in the form of liver bile ductule hyperplasia and karyomegaly. The additives in diets A and B reduced mycotoxin effects on the immune system and the liver and showed some ability to improve growth. The diet C additive played a role in reducing liver damage. Collectively, we conclude that AF and DON can be harmful to the growth and health of pigs consuming mycotoxins chronically. The selected feed additives improved pig health and may play a role in pig growth. Full article

(This article belongs to the Special Issue Mycotoxins in Food and Feed)

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Mini-Review: Novel Therapeutic Strategies to Blunt Actions of Pneumolysin in the Lungs

by Rudolf Lucas, Istvan Czikora, Supriya Sridhar, Evgeny Zemskov, Boris Gorshkov, Umapathy Siddaramappa, Aluya Oseghale, Jonathan Lawson, Alexander Verin, Ferenc G. Rick, Norman L. Block, Helena Pillich, Maritza Romero, Martin Leustik, Andrew V. Schally and Trinad Chakraborty

Toxins 2013, 5(7), 1244-1260; https://doi.org/10.3390/toxins5071244 - 15 Jul 2013

Cited by 27 | Viewed by 8898

Abstract

Severe pneumonia is the main single cause of death worldwide in children under five years of age. The main etiological agent of pneumonia is the G⁺ bacterium Streptococcus pneumoniae, which accounts for up to 45% of all cases. Intriguingly, patients can [...] Read more.

Severe pneumonia is the main single cause of death worldwide in children under five years of age. The main etiological agent of pneumonia is the G⁺ bacterium Streptococcus pneumoniae, which accounts for up to 45% of all cases. Intriguingly, patients can still die days after commencing antibiotic treatment due to the development of permeability edema, although the pathogen was successfully cleared from their lungs. This condition is characterized by a dramatically impaired alveolar epithelial-capillary barrier function and a dysfunction of the sodium transporters required for edema reabsorption, including the apically expressed epithelial sodium channel (ENaC) and the basolaterally expressed sodium potassium pump (Na⁺-K⁺-ATPase). The main agent inducing this edema formation is the virulence factor pneumolysin, a cholesterol-binding pore-forming toxin, released in the alveolar compartment of the lungs when pneumococci are being lysed by antibiotic treatment or upon autolysis. Sub-lytic concentrations of pneumolysin can cause endothelial barrier dysfunction and can impair ENaC-mediated sodium uptake in type II alveolar epithelial cells. These events significantly contribute to the formation of permeability edema, for which currently no standard therapy is available. This review focuses on discussing some recent developments in the search for the novel therapeutic agents able to improve lung function despite the presence of pore-forming toxins. Such treatments could reduce the potentially lethal complications occurring after antibiotic treatment of patients with severe pneumonia. Full article

(This article belongs to the Special Issue Pore-Forming Toxins)

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Open AccessArticle

Fluoxetine Induced Suicidal Erythrocyte Death

by Kashif Jilani, Sigrid Enkel, Rosi Bissinger, Ahmad Almilaji, Majed Abed and Florian Lang

Toxins 2013, 5(7), 1230-1243; https://doi.org/10.3390/toxins5071230 - 15 Jul 2013

Cited by 57 | Viewed by 7593

Abstract

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic [...] Read more.

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca²⁺ activity ([Ca²⁺]_i). In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca²⁺]_i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca²⁺]_i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca²⁺. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca²⁺ activity. Full article

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Effect of fluoxetine on erythrocyte forward scatter. (A) Original histogram of forward scatter in erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 50 µM fluoxetine; (B) Arithmetic means ± SEM (n = 12) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) fluoxetine (1–50 µM). * (p < 0.05) and *** (p < 0.001) indicate significant difference from the absence of fluoxetine (ANOVA). Full article ">Figure 2
Effect of fluoxetine on phosphatidylserine exposure and hemolysis. (A) Original histogram of annexin V binding of erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 50 µM fluoxetine; (B) Arithmetic means ± SEM (n = 12) of erythrocyte annexin V binding following incubation for 48 h to Ringer solution without (white bar) or with (black bars) presence of fluoxetine (1–50 µM). For comparison, arithmetic means ± SEM (n = 4) of the percentage of hemolysis is shown as grey bars. ** (p < 0.01) and *** (p < 0.001) indicate significant differences from the absence of fluoxetine (ANOVA). Full article ">Figure 3
Effect of fluoxetine on erythrocyte cytosolic Ca2+ concentration. (A) Original histogram of Fluo-3 fluorescence intensity (arbitrary units) in erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 50 µM fluoxetine; (B) Arithmetic means ± SEM (n = 12) of the Fluo-3 mean fluorescence intensity (MFI) (arbitrary units) in erythrocytes exposed for 48 h to Ringer solution without (white bar) or with (black bars) fluoxetine (1–50 µM). *** (P < 0.001) indicate significant difference from the absence of fluoxetine (ANOVA). Full article ">Figure 4
Effect of Ca2+ withdrawal on fluoxetine induced annexin V binding. Arithmetic means ± SEM (n = 4) of the percentage of annexin V binding erythrocytes after a 48 h treatment with Ringer solution without (white bar) or with (black bars) 50 µM fluoxetine in the presence (left bars, + Calcium) and absence (right bars, − Calcium) of calcium. *** (p < 0.001) indicate significant difference from the absence of fluoxetine (ANOVA), # (p < 0.05) indicates significant difference from the respective values in the presence of Ca2+. Full article ">

580 KiB

Open AccessArticle

Degradation of Aflatoxin B₁ during the Fermentation of Alcoholic Beverages

by Tomonori Inoue, Yasushi Nagatomi, Atsuo Uyama and Naoki Mochizuki

Toxins 2013, 5(7), 1219-1229; https://doi.org/10.3390/toxins5071219 - 28 Jun 2013

Cited by 34 | Viewed by 8798

Abstract

Aflatoxin B₁ (AFB₁) is a contaminant of grain and fruit and has one of the highest levels of carcinogenicity of any natural toxin. AFB₁ and the fungi that produce it can also contaminate the raw materials used for beer [...] Read more.

Aflatoxin B₁ (AFB₁) is a contaminant of grain and fruit and has one of the highest levels of carcinogenicity of any natural toxin. AFB₁ and the fungi that produce it can also contaminate the raw materials used for beer and wine manufacture, such as corn and grapes. Therefore, brewers must ensure strict monitoring to reduce the risk of contamination. In this study, the fate of AFB₁ during the fermentation process was investigated using laboratory-scale bottom and top beer fermentation and wine fermentation. During fermentation, cool wort beer samples and wine must samples were artificially spiked with AFB₁ and the levels of AFB₁ remaining after fermentation were analyzed. AFB₁ levels were unchanged during both types of fermentation used for beer but were reduced to 30% of their initial concentration in wine. Differential analysis of the spiked and unspiked wine samples showed that the degradation compound was AFB_2a, a hydrated derivative of AFB₁. Thus, the results showed that the risk of AFB₁ carryover was still present for both types of beer fermentation but was reduced in the case of wine fermentation because of hydration. Full article

(This article belongs to the Special Issue Mycotoxins in Food and Feed)

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Toxins, Volume 5, Issue 7 (July 2013) – 7 articles , Pages 1219-1331

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