Toxins

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Open AccessArticle

by Elisabeth Lang, Paola Modicano, Markus Arnold, Rosi Bissinger, Caterina Faggio, Majed Abed and Florian Lang

Toxins 2013, 5(10), 1918-1931; https://doi.org/10.3390/toxins5101918 - 23 Oct 2013

Cited by 44 | Viewed by 7513

Abstract

Background: Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane [...] Read more.

Background: Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺-concentration ([Ca²⁺]_i) and activation of p38 kinase. The present study explored, whether thioridazine elicits eryptosis. Methods: [Ca²⁺]_i has been estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, and hemolysis from hemoglobin release. Results: A 48 hours exposure to thioridazine was followed by a significant increase of [Ca²⁺]_i (30 µM), decrease of forward scatter (30 µM), and increase of annexin-V-binding (?12 µM). Nominal absence of extracellular Ca²⁺ and p38 kinase inhibitor SB203580 (2 µM) significantly blunted but did not abolish annexin-V-binding following thioridazine exposure. Conclusions: Thioridazine stimulates eryptosis, an effect in part due to entry of extracellular Ca²⁺ and activation of p38 kinase. Full article

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255 KiB

Open AccessReview

Cyanobacteria and Cyanotoxins: From Impacts on Aquatic Ecosystems and Human Health to Anticarcinogenic Effects

by Giliane Zanchett and Eduardo C. Oliveira-Filho

Toxins 2013, 5(10), 1896-1917; https://doi.org/10.3390/toxins5101896 - 23 Oct 2013

Cited by 239 | Viewed by 16451

Abstract

Cyanobacteria or blue-green algae are among the pioneer organisms of planet Earth. They developed an efficient photosynthetic capacity and played a significant role in the evolution of the early atmosphere. Essential for the development and evolution of species, they proliferate easily in aquatic [...] Read more.

Cyanobacteria or blue-green algae are among the pioneer organisms of planet Earth. They developed an efficient photosynthetic capacity and played a significant role in the evolution of the early atmosphere. Essential for the development and evolution of species, they proliferate easily in aquatic environments, primarily due to human activities. Eutrophic environments are conducive to the appearance of cyanobacterial blooms that not only affect water quality, but also produce highly toxic metabolites. Poisoning and serious chronic effects in humans, such as cancer, have been described. On the other hand, many cyanobacterial genera have been studied for their toxins with anticancer potential in human cell lines, generating promising results for future research toward controlling human adenocarcinomas. This review presents the knowledge that has evolved on the topic of toxins produced by cyanobacteria, ranging from their negative impacts to their benefits. Full article

(This article belongs to the Special Issue Toxins and Carcinogenesis)

461 KiB

Open AccessArticle

Aflatoxin, Fumonisin and Shiga Toxin-Producing Escherichia coli Infections in Calves and the Effectiveness of Celmanax^®/Dairyman’s Choice™ Applications to Eliminate Morbidity and Mortality Losses

by Danica Baines, Mark Sumarah, Gretchen Kuldau, Jean Juba, Alberto Mazza and Luke Masson

Toxins 2013, 5(10), 1872-1895; https://doi.org/10.3390/toxins5101872 - 23 Oct 2013

Cited by 19 | Viewed by 8000

Abstract

Mycotoxin mixtures are associated with Shiga toxin-producing Escherichia coli (STEC) infections in mature cattle. STEC are considered commensal bacteria in mature cattle suggesting that mycotoxins provide a mechanism that converts this bacterium to an opportunistic pathogen. In this study, we assessed the mycotoxin [...] Read more.

Mycotoxin mixtures are associated with Shiga toxin-producing Escherichia coli (STEC) infections in mature cattle. STEC are considered commensal bacteria in mature cattle suggesting that mycotoxins provide a mechanism that converts this bacterium to an opportunistic pathogen. In this study, we assessed the mycotoxin content of hemorrhaged mucosa in dairy calves during natural disease outbreaks, compared the virulence genes of the STECs, evaluated the effect of the mucosal mycotoxins on STEC toxin expression and evaluated a Celmanax^®/Dairyman’s Choice™ application to alleviate disease. As for human infections, the OI-122 encoded nleB gene was common to STEC genotypes eliciting serious disease. Low levels of aflatoxin (1–3 ppb) and fumonisin (50–350 ppb) were detected in the hemorrhaged mucosa. Growth of the STECs with the mycotoxins altered the secreted protein concentration with a corresponding increase in cytotoxicity. Changes in intracellular calcium indicated that the mycotoxins increased enterotoxin and pore-forming toxin activity. A prebiotic/probiotic application eliminated the morbidity and mortality losses associated with the STEC infections. Our study demonstrates: the same STEC disease complex exists for immature and mature cattle; the significance of the OI-122 pathogenicity island to virulence; the significance of mycotoxins to STEC toxin activity; and, finally, provides further evidence that prebiotic/probiotic applications alleviate STEC shedding and mycotoxin/STEC interactions that lead to disease. Full article

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

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Effect of Celmanax®/Dairyman’s Choice™ applications on STEC shedding in calves from three production sites (A,B,C) at day 0 and day 7–14 (n = 3; *** p = 0.001). Full article ">Figure 2
Impact of Celmanax® and Dairyman’s Choice™ on the cytotoxicity of mycotoxin extracts from calf feed rations (n = 3; 0, extract alone; C, extract + 0.1% Celmanax®; DC, extract + 0.1% Dairyman’s Choice™ calf starter; *** p = 0.001). Full article ">Figure 3
Effect of STEC-secreted protein composition on intracellular Ca2+ concentrations (340/380 fluorescence) in bovine liver cells. Ca2+ signaling in response to STEC-secreted proteins produced in M9 medium with normal 1 mM extracellular Ca2+ in the medium. The cells were loaded with fura-2/AM and stored in balanced salt solutions with or without Ca2+ to achieve a baseline before addition of the secreted proteins. Each value represents the Ca2+ mobilization for the protein composition secreted by STEC grown in M9 medium evoked in about 10 cells. Data are presented for each STEC involved in the infections at production site A (A1 = O145 STEC, A2 = ExPEC), B (B1–B3 = O177 STEC) and C (C1 = O177 STEC, C2 = O174 STEC). Recordings were performed at 37 °C and the experiment was repeated twice. Full article ">Figure 4
Effect of STEC-secreted protein composition on intracellular Ca2+ concentrations (340/380 fluorescence) in bovine liver cells. Ca2+ signaling in response to STEC-secreted proteins produced in the absence or presence of aflatoxin (0.02 ppb) or fumonisin (700 ppb) with normal 1 mM extracellular Ca2+ in the medium. The cells were loaded with fura-2/AM and stored in balanced salt solutions with or without Ca2+ to achieve a baseline before addition of the secreted proteins. Each value represents the difference in calcium mobilization of the mycotoxin-treated and untreated STEC-secreted protein composition evoked intracellular Ca2+ concentrations in about 10 cells. Data are presented for each STEC involved in the infections at production site A (A1 = O145 STEC, A2 = ExPEC), B (B1–B3 = O177 STEC) and C (C1 = O177 STEC, C2 = O174 STEC). Recordings were performed at 37 °C and the experiment was repeated twice. Full article ">

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Open AccessArticle

Assessment of the Functional Regions of the Superantigen Staphylococcal Enterotoxin B

by Lily Zhang and Thomas J. Rogers

Toxins 2013, 5(10), 1859-1871; https://doi.org/10.3390/toxins5101859 - 22 Oct 2013

Cited by 6 | Viewed by 5977

Abstract

The functional activity of superantigens is based on capacity of these microbial proteins to bind to both the ?-chain of the T cell receptor (TcR) and the major histocompatibility complex (MHC) class II dimer. We have previously shown that a subset of the [...] Read more.

The functional activity of superantigens is based on capacity of these microbial proteins to bind to both the ?-chain of the T cell receptor (TcR) and the major histocompatibility complex (MHC) class II dimer. We have previously shown that a subset of the bacterial superantigens also binds to a membrane protein, designated p85, which is expressed by renal epithelial cells. This binding activity is a property of SEB, SEC1, 2 and 3, but not SEA, SED, SEE or TSST. The crystal structure of the tri-molecular complex of the superantigen staphylococcal enterotoxin B (SEB) with both the TcR and class II has previously been reported. However, the relative contributions of regions of the superantigen to the overall functional activity of this superantigen remain undefined. In an effort to better define the molecular basis for the interaction of SEB with the TcR ?-chain, we report studies here which show the comparative contributions of amino- and carboxy-terminal regions in the superantigen activity of SEB. Recombinant fusion proteins composed of bacterial maltose-binding protein linked to either full-length or truncated toxins in which the 81 N-terminal, or 19 or 34 C-terminal amino acids were deleted, were generated for these studies. This approach provides a determination of the relative strength of the functional activity of the various regions of the superantigen protein. Full article

(This article belongs to the Special Issue Enterotoxins: Microbial Proteins and Host Cell Dysregulation)

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358 KiB

Open AccessArticle

Mouse in Vivo Neutralization of Escherichia coli Shiga Toxin 2 with Monoclonal Antibodies

by Luisa W. Cheng, Thomas D. Henderson II, Stephanie Patfield, Larry H. Stanker and Xiaohua He

Toxins 2013, 5(10), 1845-1858; https://doi.org/10.3390/toxins5101845 - 22 Oct 2013

Cited by 23 | Viewed by 7180

Abstract

Shiga toxin-producing Escherichia coli (STEC) food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS). Besides supportive care, there currently are [...] Read more.

Shiga toxin-producing Escherichia coli (STEC) food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS). Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs). We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t1/2 ? to be 3 min and the clearance phase or t1/2 ? to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections. Full article

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Standard curve of Stx2 spiked in mouse serum. Known standards ranging from 10 to 1,000 pg/mL of Stx2 in control sera (pooled healthy mouse sera) were used to determine the concentration of Stx2 in unknown blood samples. The linear regression of the standard curve has a correlation coefficient (R2) of 1. The LOD of 10 pg/mL was determined by the addition of 3 times standard deviation to the mean background signal and is denoted here with a dashed line at 5984 relative luminescent counts. Full article ">Figure 2
Biologic half-lives of Stx2 in mouse serum. Stx2 was introduced into mice by iv. Sera was taken at 2, 5, 10, 20, 30 min and 1, 1.5, 2, 3, 6 and 8 h after intoxication and the Stx2 concentration was determined based on standard curves plotted in non-linear regression of the second polynomial (Prism 6). The fast distribution phase t1/2 α and slow clearance phase t1/2 β were determined using the same program. The mean values for each time point were plotted along with the standard error of the mean (SEM) with n ≥ 5. Full article ">Figure 3
Monoclonal antibody protection of mice from Stx2. Mice (n ≥ 10) were treated with different doses of single mAb or with a combination of anti-Stx2 mAbs (A. Stx2-1; B. Stx2-2; C. Stx2-5; D. Stx2-6; E. Stx2-4 and F. 3 mAbs, 1:1:1 of Stx2-1, Stx2-2, and Stx2-5) about 30 min prior to administration with a lethal dose (3 ip mouse LD50) of Stx2. The percentage of survival of mice was plotted over time. Control mice were treated with sterile PBS instead of mAb. Full article ">Figure 4
Survival of mice treated with mAbs before and after Stx2 intoxication. A. Mice were treated with a lethal dose of Stx2 followed by treatment with a mAb combination against Stx2 at 2, 5, 10, 20, 30 min and 1 h. B. Mice were treated with the same combination of mAbs against Stx2 at 4, 5, 6, 7, 8 weeks before injection with Stx2. Full article ">Figure 5
Clearance of Stx2 by monoclonal antibodies. Mice were treated iv with 100 ng of Stx2. They were then treated with (inverted triangles) or without (circles) a combination of mAbs Stx2-1, Stx2-2, and Stx2-5 at 2 min after toxin injection. Sera were obtained at 2, 5, 10, 20, 30 min and 1, and 2 h. MAbs accelerated Stx2 clearance, eliminating toxin from the bloodstream within minutes. The mean values for each time point were plotted along with the standard error of the mean (SEM) with n = 3 for no mAb controls and n = 6 for mAb treated mice. Full article ">

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Open AccessArticle

Comparison of Clean-Up Methods for Ochratoxin A on Wine, Beer, Roasted Coffee and Chili Commercialized in Italy

by Ambra Prelle, Davide Spadaro, Aleksandra Denca, Angelo Garibaldi and Maria Lodovica Gullino

Toxins 2013, 5(10), 1827-1844; https://doi.org/10.3390/toxins5101827 - 22 Oct 2013

Cited by 38 | Viewed by 8152

Abstract

The most common technique used to detect ochratoxin A (OTA) in food matrices is based on extraction, clean-up, and chromatography detection. Different clean-up cartridges, such as immunoaffinity columns (IAC), molecular imprinting polymers (MIP), Mycosep™ 229, Mycospin™, and Oasis^® HLB (Hydrophilic Lipophilic balance) [...] Read more.

The most common technique used to detect ochratoxin A (OTA) in food matrices is based on extraction, clean-up, and chromatography detection. Different clean-up cartridges, such as immunoaffinity columns (IAC), molecular imprinting polymers (MIP), Mycosep™ 229, Mycospin™, and Oasis^® HLB (Hydrophilic Lipophilic balance) as solid phase extraction were tested to optimize the purification for red wine, beer, roasted coffee and chili. Recovery, reproducibility, reproducibility, limit of detection (LOD) and limit of quantification (LOQ) were calculated for each clean-up method. IAC demonstrated to be suitable for OTA analysis in wine and beer with recovery rate >90%, as well as Mycosep™ for wine and chili. On the contrary, MIP columns were the most appropriate to clean up coffee. A total of 120 samples (30 wines, 30 beers, 30 roasted coffee, 30 chili) marketed in Italy were analyzed, by applying the developed clean-up methods. Twenty-seven out of 120 samples analyzed (22.7%: two wines, five beers, eight coffees, and 12 chili) resulted positive to OTA. A higher incidence of OTA was found in chili (40.0%) more than wine (6.6%), beers (16.6%) and coffee (26.6%). Moreover, OTA concentration in chili was the highest detected, reaching 47.8 µg/kg. Furthermore, three samples (2.5%), two wines and one chili, exceeded the European threshold. Full article

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

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1357 KiB

Open AccessArticle

Treatment with the Hyaluronic Acid Synthesis Inhibitor 4-Methylumbelliferone Suppresses SEB-Induced Lung Inflammation

by Robert J. McKallip, Harriet F. Hagele and Olga N. Uchakina

Toxins 2013, 5(10), 1814-1826; https://doi.org/10.3390/toxins5101814 - 17 Oct 2013

Cited by 27 | Viewed by 7104

Abstract

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for SEB-induced inflammation. In the current study we investigated the potential use [...] Read more.

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for SEB-induced inflammation. In the current study we investigated the potential use of the hyaluronic acid synthase inhibitor 4-methylumbelliferone (4-MU) on staphylococcal enterotoxin B (SEB) induced acute lung inflammation. Culturing SEB-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production as well as an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from SEB-induced lung injury. Specifically, 4-MU treatment led to a reduction in SEB-induced HA levels, reduction in lung permeability, and reduced pro-inflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target hyaluronic acid production may be an effective treatment for the inflammatory response following exposure to SEB. Full article

(This article belongs to the Special Issue Enterotoxins: Microbial Proteins and Host Cell Dysregulation)

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4-MU inhibits SEB-induced leukocyte proliferation and cytokine production in vitro. Spleen cells from C57BL/6 mice were treated with 4-MU (0.1, 0.5, and 1.0 mM) or vehicle control (DMSO) and then stimulated with SEB (2 μg/mL). The effect of 4-MU on the proliferative response and cytokine production was determined 48 h later by MTT assay (A) and cytometric bead array (B), respectively. Asterisks indicate statistically significant difference when compared with vehicle controls, p ≤ 0.05. Full article ">Figure 2
4-MU treatment leads to increased apoptosis in SEB-exposed leukocytes in vitro. Spleen cells from C57BL/6 mice were treated with 4-MU (0.1, and 0.5 mM) or vehicle control (DMSO) and then stimulated with SEB (2 μg/mL). The effect of 4-MU on SEB-induced apoptosis was determined 48 h later by Annexin V/PI (A) and TUNEL (B) assays, respectively. The level of apoptosis in individual immune cell subsets was determined by staining the spleen cells with phenotype-specific fluorescently-labeled mAbs followed by TUNEL staining (C). Full article ">Figure 3
4-MU treatment suppresses SEB-induced hyaluronic acid synthase expression and accumulation of soluble HA in the lungs. The effect of 4-MU on soluble HA levels in the lungs of SEB-exposed mice was determined by treating the mice with 4-MU (450 mg/mouse i.p.) or vehicle control (5% gum arabic) one day prior and on the day of SEB exposure (20 µg/50 μL PBS). The levels of lung hyaluronic acid and mRNA levels of HAS were determined 24 h later. HAS mRNA levels in whole lung extracts were determined by real-time RT-PCR (A). Hyaluronic acid levels in bronchoalveolar lavage fluid (BALF) were determined by ELISA (B). Asterisks indicate statistically significant difference when compared with the levels from vehicle-exposed mice, p ≤ 0.05. Number sign indicates statistically significant difference when compared to PBS exposed mice, p ≤ 0.05. Full article ">Figure 4
The effect of 4-MU on SEB-induced vascular permeability in vivo was determined by exposing mice to SEB, as described in the Material and Methods section, and treating the mice with 4-MU (450 mg/mouse i.p.) or Vehicle (5% gum arabic i.p.) one day prior, and on the day of, SEB exposure. Vascular permeability was determined as described Materials and Methods. Asterisks indicate statistically significant difference when compared with the Vehicle-treated controls, p ≤ 0.05. Full article ">Figure 5
4-MU treatment suppresses SEB-induced inflammatory cytokine production in the lungs. The effect of 4-MU on SEB-induced inflammatory cytokine production in vivo was determined by treating the mice with 4-MU (450 mg/mouse i.p.) or vehicle (300 µL 5% gum arabic/mouse i.p.) one day prior to, and on the day of, SEB exposure. Following 4-MU treatment mice were exposed to SEB or PBS, as described in the Material and Methods section. The levels of BALF cytokine protein levels and total lung cytokine mRNA and were determined 24 h later. Cytokine mRNA levels in whole lung extracts were determined by real-time RT-PCR (A). The protein levels of cytokines in BALF were determined using a cytokine bead array (B). Asterisks indicate statistically significant difference when compared to the cytokine levels from SEB-exposed vehicle-treated mice, p < 0.05. Full article ">

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Open AccessArticle

Appraisal of Antiophidic Potential of Marine Sponges against Bothrops jararaca and Lachesis muta Venom

by Camila Nunes Faioli, Thaisa Francielle Souza Domingos, Eduardo Coriolano De Oliveira, Eládio Flores Sanchez, Suzi Ribeiro, Guilherme Muricy and Andre Lopes Fuly

Toxins 2013, 5(10), 1799-1813; https://doi.org/10.3390/toxins5101799 - 17 Oct 2013

Cited by 9 | Viewed by 6846

Abstract

Snakebites are a health problem in many countries due to the high incidence of such accidents. Antivenom treatment has regularly been used for more than a century, however, this does not neutralize tissue damage and may even increase the severity and morbidity of [...] Read more.

Snakebites are a health problem in many countries due to the high incidence of such accidents. Antivenom treatment has regularly been used for more than a century, however, this does not neutralize tissue damage and may even increase the severity and morbidity of accidents. Thus, it has been relevant to search for new strategies to improve antiserum therapy, and a variety of molecules from natural sources with antiophidian properties have been reported. In this paper, we analyzed the ability of ten extracts from marine sponges (Amphimedon viridis, Aplysina fulva, Chondrosia collectrix, Desmapsamma anchorata, Dysidea etheria, Hymeniacidon heliophila, Mycale angulosa, Petromica citrina, Polymastia janeirensis, and Tedania ignis) to inhibit the effects caused by Bothrops jararaca and Lachesis muta venom. All sponge extracts inhibited proteolysis and hemolysis induced by both snake venoms, except H. heliophila, which failed to inhibit any biological activity. P. citrina inhibited lethality, hemorrhage, plasma clotting, and hemolysis induced by B. jararaca or L. muta. Moreover, other sponges inhibited hemorrhage induced only by B. jararaca. We conclude that Brazilian sponges may be a useful aid in the treatment of snakebites caused by L. muta and B. jararaca and therefore have potential for the discovery of molecules with antiophidian properties. Full article

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Effect of sponges’ extracts on proteolysis induced by B. jararaca or L. muta venom. Marine sponges (132 μg/mL) were incubated for 30 min at room temperature with 34 μg/mL B. jararaca (Panel A) or with 32 μg/mL L. muta (Panel B), and then proteolysis test performed. M. angulosa (column 1), C. collectrix (column 2), T. ignis (column 3), A. fulva (column 4), D. etheria (column 5), D. anchorata (column 6), A. viridis (column 7), P. citrina (column 8), P. janeirensis (column 9), H. heliophila (column 10). Data are means ± SE of two individual experiments (n = 3). Full article ">Figure 2
Effect of the sponge extracts on hemorrhage induced by B. jararaca or L. muta venom. The sponge extracts (220 µg/g) were incubated for 30 min. at room temperature with 20 µg/g B. jararaca (Panel A) or with 10 µg/g L. muta venom (Panel B), then the mixtures were injected into mice and the hemorrhage test results evaluated, as described in the methods. Columns are: Venom with DMSO (Column C), M. angulosa (column 1), C. collectrix (column 2), T. ignis (column 3), A. fulva (column 4), D. etheria (column 5), D. anchorata (column 6), A. viridis (column 7), P. citrina (column 8), P. janeirensis (column 9), H. heliophila (column 10). Data are expressed as means SEM of two individual experiments (n = 3). Panel C: B. jararaca venom (20 µg/g) was injected i.d., and 15 min. later, the sponge extracts M. angulosa (Ma), D. anchorata (Da), P. citrine (Pc) and T. ignis (Ti), 110 µg/g (black columns) or 220 µg/g (white columns) were injected i.d. or i.v. (220 µg/g, hatched columns). * Significance level (p < 0.05) when compared to columns C. Full article ">Figure 3
Effect of the sponge extracts on hemolysis induced by B. jararaca or L. muta venom. For Panel A, the sponge extracts (100 µg/mL) were incubated with 50 µg/mL B. jararaca and for Panel B, sponges (50 µg/mL) were incubated with 25 µg/mL L. muta, then hemolytic tests were performed. Columns are: Venoms incubated with M. angulosa (column 1), or with C. collectrix (column 2), or with T. ignis (column 3), or with A. fulva (column 4), or with D. etheria (column 5), or with D. anchorata (column 6), or with A. viridis (column 7), or with P. citrina (column 8), or with P. janeirensis (column 9), or with H. heliophila (column 10). Data are expressed as means SEM of three individual experiments (n = 3). Full article ">Figure 4
Effect of the sponge extracts on coagulation induced by B. jararaca or L. muta. B. jararaca (Panel A) or L. muta (Panel B) venoms were incubated with 0.5% DMSO (column 0), M. angulosa (column 1), C. collectrix (column 2), T. ignis (column 3), A. fulva (column 4), D. etheria (column 5), D. anchorata (column 6), A. viridis (column 7), P. citrina (column 8), P. janeirensis (column 9), H. heliophila (column 10), for 30 min at room temperature. After the mixture was added to the plasma, the clotting time was recorded, as described in the Methods. Data are expressed as means SEM of three individual experiments (n = 3). * Significance level (p < 0.05) when compared to column 0. Full article ">Figure 5
Effect of the sponge extracts on the edematogenic activity induced by L. muta venom. L. muta (0.4 µg/g) venom was incubated for 30 min at room temperature with 4.8 µg/g sponge extracts: M. angulosa (column 1), T. ignis (column 2), A. fulva (column3), D. anchorata (column 4), A. viridis (column 5), P. citrina (column 6), P. janeirensis (column 7), or H. heliophila (column 8). Data are expressed as means SEM of three individual experiments (n = 3). Full article ">

1177 KiB

Open AccessArticle

Molecular Characterization of Lys49 and Asp49 Phospholipases A₂ from Snake Venom and Their Antiviral Activities against Dengue virus

by Alzira B. Cecilio, Sergio Caldas, Raiana A. De Oliveira, Arthur S. B. Santos, Michael Richardson, Gustavo B. Naumann, Francisco S. Schneider, Valeria G. Alvarenga, Maria I. Estevão-Costa, Andre L. Fuly, Johannes A. Eble and Eladio F. Sanchez

Toxins 2013, 5(10), 1780-1798; https://doi.org/10.3390/toxins5101780 - 15 Oct 2013

Cited by 42 | Viewed by 8257

Abstract

We report the detailed molecular characterization of two PLA₂s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA₂s, named BlK-PLA₂ and BlD-PLA₂, are composed [...] Read more.

We report the detailed molecular characterization of two PLA₂s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA₂s, named BlK-PLA₂ and BlD-PLA₂, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA₂s, and show a high degree of sequence similarity to homologous snake venom PLA₂s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA₂s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA₂s were assessed by quantitative Real-Time PCR. Bl-PLA₂s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA₂s as tools of research. Full article

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Mass spectrometry analysis of native BlD-PLA2 and BlK-PLA2 from B. leucurus venom (top and bottom panels, respectively) performed in Matrix assisted desorption/ionization-time-of flight (MALDI-TOF-MS). Full article ">Figure 2
Multiple amino acid sequence alignment of BlPLA2s with svPLA2s homologous. The one letter code for amino acid nomenclature is used. Proteins compared and their UniProt or GeneBank (GB) accession numbers: K49 (piratoxin II, P82287) from Bothrops pirajai: K49 (bthtx I, Q90249) from B. jararacussu: K49 (BlK-PLA2, P86975) from B. leucurus: K49 (myotoxin II, P24605) from B. asper: K49 (myotoxin II, Q91834) from B. moojeni: K49 (O57385) from Deinagkistrodon (formerly Agkistrodon) acutus: R49 (Q28681) from Protobothrops elegans: S49 (P48650) from Echis carinatus; D49 (BlD-PLA2, P86974) from B. leucurus: D49 (vipoxin complex, P04084) from Vipera ammodytes meridionalis: D49 (C9E7C4) from Crotalus durissus cascavella: D49 (Q7ZTA6) from C.v.viridis: D49 (Q2H228) from B. erythromelas: D49 (GB/KC544002) from B. neuwiedi: D49 (O42191) from Gloydius (formerly Agkistrodon) halys pallas. (*) is used for identical residues (:) for conserved ones and (.) for semi-conserved substitutions among all sequences in the alignment. Gaps were introduced to maximize the sequence homology, as indicated in part (A) and (B), respectively. Full article ">Figure 3
2-DE SDS-PAGE pattern of venom proteins from B. leucurus. (A) 60 µg of total proteins were isoelectrically focused (pI range 3–10) followed by separation by SDS-PAGE (15% gel) and Coomassie blue staining. Protein spots of approx. 14 kDa correspond to the position of Bl-PLA2 isoforms are indicated by arrows, (B) Immunoblotting of venom proteins separated by 2D-SDS-PAGE with anti-BlK-PLA2 IgG. Arrows indicates the reactivity of the antibody with the approx. 14 kDa PLA2s. Full article ">Figure 4
Reactivity of purified rabbit anti BlK-PLA2 IgG against BlD- and BlK-PLA2 isoforms. (A) reduced SDS-PAGE (15% gel) of: 1, molecular mass markers; 2, crude venom (20 µg); 3, BlK-PLA2 (5 µg); 4, BlD-PLA2 (5 µg); (B) immunoblotting of anti BlK-PLA2 IgG against crude venom (1), BlK-PLA2 (2) and BlD-PLA2 (3). (C) Reactivity ofanti BlK-PLA2 IgG against several snake venoms examined by ELISA. 96-well microtitration plates were precoated with 0.5 µg/mL of Bl-PLA2s (pool), Bothrops species, L. muta, C. d. terrificus and Micrurus lemniscatus. Anti BlK-PLA2 IgG was added at different dilutions. Binding was visualized by incubation with peroxidase-coupled anti-rabbit IgG (diluted 1:12,000) and subsequent peroxidase-catalyzed conversion of O-phenylenediamine (OPD). The absorbance of pre-immune serum (control) was subtracted. Data shown represent the average of two independent experiments, with error bars indicating the maximum and minimum deviation from the average. Full article ">Figure 5
Phylogenetic tree for the multiple-sequence alignment of svPLA2s listed in <a href="#toxins-05-01780-f002" class="html-fig">Figure 2</a> (Viperidae), and some proteins from Elapidae and Hydrophiidae snakes. The length of the horizontal scale represents 30% divergence. Phylogenetic distances branch points are indicated. Full article ">Figure 6
DENV and RnaseP standard curves generated from transcribed RNAs. (A) Curves were generated from seven serial dilutions of transcribed DENV and RnaseP RNAs from 107 copy number/µL. The black lines refer to DENV RNA and the gray lines to RnaseP RNA (exogenous control). (B,C) Standard curves were generated from the linear region of each amplification curve. Efficiency of amplification for each primer set was determined using the equation: Efficiency (E) = 10(−1/sl°pe), being EDENV = 91.34%, RDENV = 0.999, ERnaseP = 95%, RRnaseP = 0.999. Full article ">Figure 7
(A) Cellular viability after the incubation of LLC-MK2 cells with different concentrations of Bl-PLA2s (Pool) and their isoforms BlK- and BlD-PLA2 for 48 h. The higher concentrations without significant toxicity (p < 0.05) were considered as the maximum non-toxic concentration (MNTC). Data represent the mean of three independent experiments performed in triplicate. Means with different symbols denote significant differences (p < 0.05). (B) Antiviral activity assessed by quantitative Real-Time PCR. Data represent the mean of two independent experiments performed in triplicate. Full article ">

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Open AccessArticle

Isolation and Molecular Characterization of Two Lectins from Dwarf Elder (Sambucus ebulus L.) Blossoms Related to the Sam n1 Allergen

by Pilar Jimenez, Patricia Cabrero, José E. Basterrechea, Jesús Tejero, Damian Cordoba-Diaz and Tomas Girbes

Toxins 2013, 5(10), 1767-1779; https://doi.org/10.3390/toxins5101767 - 14 Oct 2013

Cited by 16 | Viewed by 7210

Abstract

Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two D-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named [...] Read more.

Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two D-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named ebulin blo (A-B toxin) and SELblo (B-B lectin)—blo from blossoms—were subjected to molecular characterization and analysis by MALDI-TOF mass spectrometry and tryptic peptide fingerprinting. Both lectins share a high degree of amino acid sequence homology with Sambucus lectins related to the Sam n1 allergen. Ebulin blo, but not SELblo, was highly toxic by nasal instillation to mice. Overall, our results suggested that both lectins would belong to an allergen family exemplified by Sam n1 and could trigger allergy responses. Furthermore, they raise a concern about ebulin blo toxicity. Full article

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(a) Picture showing blossoms, early green fruits and early green stalks of S. ebulus. (b) SDS-PAGE of raw extracts of blossoms, early green fruits and early green stalks; 20 µL of extract concentrated by ultrafiltration were loaded into each well. Full article ">Figure 2
(a) Affinity chromatography of raw protein extract on acid-treated Sepharose 6B. (b) Chromatography of affinity-bound protein on Superdex 75. (c) SDS-PAGE of proteins purified from S. ebulus blossoms. The amounts of protein per well were ebulin f (11.5 µg), SELfd (11.1 µg), ebulin blo (9.5 µg) and SELblo (10.8 µg). The markers were, from top to bottom: SNAI (Mr 136 kDa), BSA (Mr 68 kDa), Ng b (Mr 58 kDa), ovalbumin (Mr 45 kDa), SNAIV (Mr 30 kDa) and trypsin inhibitor (Mr 20 kDa). 2-ME, 2-mercaptoethanol. Full article ">Figure 3
Mass spectrometry profiles of tryptic peptides from ebulin blo (a) and SELblo (b). Full article ">Figure 4
Alignment of amino acid sequences of some tryptic peptides of blossom lectins with ebulin l and SNAld. Boxed are sequences found in tryptic peptides from the allergen Sam n1 of S. nigra pollen. In red are coincident sequences. Highlighted in yellow and grey are tryptic peptides obtained from ebulin blo and SELblo, respectively. Full article ">Figure 5
Effects of the instillation of ebulin blo in Swiss mice on the evolution of survival (a) and relative body weight loss (b). Dashed line and circles: instillation of 2.5 mg/kg body weight of ebulin blo; continuous line and squares: instillation of 5 mg/kg body weight of ebulin blo; dotted line: intraperitoneal administration of 5 mg/kg body weight of ebulin f. Full article ">

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Open AccessReview

Toxicity of Ochratoxin A and Its Modulation by Antioxidants: A Review

by Valeria Sorrenti, Claudia Di Giacomo, Rosaria Acquaviva, Ignazio Barbagallo, Matteo Bognanno and Fabio Galvano

Toxins 2013, 5(10), 1742-1766; https://doi.org/10.3390/toxins5101742 - 11 Oct 2013

Cited by 166 | Viewed by 12096

Abstract

Ochratoxin A (OTA) is a mycotoxin involved in the development of different types of cancers in rats, mice and humans. A growing number of in vitro and in vivo studies has been collected and has described evidence compatible with a role for oxidative [...] Read more.

Ochratoxin A (OTA) is a mycotoxin involved in the development of different types of cancers in rats, mice and humans. A growing number of in vitro and in vivo studies has been collected and has described evidence compatible with a role for oxidative stress in OTA toxicity and carcinogenicity. Because the contribution of the oxidative stress response in the development of cancers is well established, a role in OTA carcinogenicity is plausible. Several studies have been performed to try to counteract the adverse effects of oxygen radicals generated under OTA-exposure. A number of molecules with various antioxidant properties were tested, using in vivo or in vitro models. Protection against OTA-induced DNA damage, lipid peroxidation, as well as cytotoxicity were observed, further confirming the link between OTA toxicity and oxidative damage. These studies demonstrated that antioxidants are able to counteract the deleterious effects of chronic consumption or exposure to OTA and confirmed the potential effectiveness of dietary strategies to counteract OTA toxicity. Full article

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

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Open AccessArticle

Small Chemical Chromatin Effectors Alter Secondary Metabolite Production in Aspergillus clavatus

by Christoph Zutz, Agnieszka Gacek, Michael Sulyok, Martin Wagner, Joseph Strauss and Kathrin Rychli

Toxins 2013, 5(10), 1723-1741; https://doi.org/10.3390/toxins5101723 - 7 Oct 2013

Cited by 49 | Viewed by 8912

Abstract

The filamentous fungus Aspergillus clavatus is known to produce a variety of secondary metabolites (SM) such as patulin, pseurotin A, and cytochalasin E. In fungi, the production of most SM is strongly influenced by environmental factors and nutrients. Furthermore, it has been shown [...] Read more.

The filamentous fungus Aspergillus clavatus is known to produce a variety of secondary metabolites (SM) such as patulin, pseurotin A, and cytochalasin E. In fungi, the production of most SM is strongly influenced by environmental factors and nutrients. Furthermore, it has been shown that the regulation of SM gene clusters is largely based on modulation of a chromatin structure. Communication between fungi and bacteria also triggers chromatin-based induction of silent SM gene clusters. Consequently, chemical chromatin effectors known to inhibit histone deacetylases (HDACs) and DNA-methyltransferases (DNMTs) influence the SM profile of several fungi. In this study, we tested the effect of five different chemicals, which are known to affect chromatin structure, on SM production in A. clavatus using two growth media with a different organic nitrogen source. We found that production of patulin was completely inhibited and cytochalasin E levels strongly reduced, whereas growing A. clavatus in media containing soya-derived peptone led to substantially higher pseurotin A levels. The HDAC inhibitors valproic acid, trichostatin A and butyrate, as well as the DNMT inhibitor 5-azacytidine (AZA) and N-acetyl-D-glucosamine, which was used as a proxy for bacterial fungal co-cultivation, had profound influence on SM accumulation and transcription of the corresponding biosynthetic genes. However, the repressing effect of the soya-based nitrogen source on patulin production could not be bypassed by any of the small chemical chromatin effectors. Interestingly, AZA influenced some SM cluster genes and SM production although no Aspergillus species has yet been shown to carry detectable DNA methylation. Full article

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SM production in A. clavatus. Production of brevianamid F, cytochalasin E, patulin, pseurotin A and territrem B of A. clavatus grown for 72 h in FM1 (black bars) and FM2 (grey bars). Values are mean values ± standard deviation of three independent biological replicates, * indicates statistical significant difference between SM production in FM1 and FM2 (p < 0.05), n.d.: not detectable. Full article ">Figure 2
Effect of SCCEs on SM production. Cytochalasin E (panel A), patulin (panel B) and pseurotin A (panel C) production in A. clavatus grown for 72 h in FM1 in the absence (control) and presence of VPA, TSA, butyrate, AZA, and GlcNAc alone or in combination with VPA, TSA, butyrate and AZA. Values are mean values ± standard deviation of three independent biological replicates, * indicates statistical significant difference compared to the control (p < 0.05). Full article ">Figure 3
Effect of SCCEs on cytochalasin E production and ccsA expression. Cytochalasin E production (panel A) and ccsA expression (panel B) in A. clavatus grown for 48 h (black bars) and 72 h (grey bars) in FM2 in the absence (control) and presence of VPA, TSA, butyrate, AZA, and GlcNAc alone or in combination with VPA, TSA, butyrate and AZA. Values are mean values ± standard deviation of three independent biological replicates, * indicates statistical significant difference compared to the control (p < 0.05). Full article ">Figure 4
Effect of SCCEs on patK expression. PatK expression in A. clavatus grown for 48 h in FM2 in the absence (control) and presence of VPA, TSA, butyrate, AZA, and GlcNAc alone or in combination with VPA, TSA, butyrate and AZA. Patulin production was not detectable after 48 and 72 h, and no patK expression was detectable after 72 h. Values are mean values ± standard deviation of three independent biological replicates, * indicates statistical significant difference compared to the control (p < 0.05). Full article ">Figure 5
Effect of SCCEs on pseurotin A production and psoA expression. Pseurotin A production (panel A) and psoA expression (panel B) in A. clavatus grown for 48 h (black bars) and 72 h (grey bars) in FM2 in the absence (control) and presence of VPA, TSA, butyrate, AZA, and GlcNAc alone or in combination with VPA, TSA, butyrate and AZA. Values are mean values ± standard deviation of three independent biological replicates, * indicates statistical significant difference compared to the control (p < 0.05). Full article ">

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Open AccessReview

Saporin-S6: A Useful Tool in Cancer Therapy

by Letizia Polito, Massimo Bortolotti, Daniele Mercatelli, Maria Giulia Battelli and Andrea Bolognesi

Toxins 2013, 5(10), 1698-1722; https://doi.org/10.3390/toxins5101698 - 7 Oct 2013

Cited by 95 | Viewed by 11885

Abstract

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the [...] Read more.

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials. Full article

(This article belongs to the Special Issue Toxins and Carcinogenesis)

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463 KiB

Open AccessArticle

Faces of a Changing Climate: Semi-Quantitative Multi-Mycotoxin Analysis of Grain Grown in Exceptional Climatic Conditions in Norway

by Silvio Uhlig, Gunnar Sundstøl Eriksen, Ingerd Skow Hofgaard, Rudolf Krska, Eduardo Beltrán and Michael Sulyok

Toxins 2013, 5(10), 1682-1697; https://doi.org/10.3390/toxins5101682 - 27 Sep 2013

Cited by 120 | Viewed by 9826

Abstract

Recent climatological research predicts a significantly wetter climate in Southern Norway as a result of global warming. Thus, the country has already experienced unusually wet summer seasons in the last three years (2010–2012). The aim of this pilot study was to apply an [...] Read more.

Recent climatological research predicts a significantly wetter climate in Southern Norway as a result of global warming. Thus, the country has already experienced unusually wet summer seasons in the last three years (2010–2012). The aim of this pilot study was to apply an existing multi-analyte LC-MS/MS method for the semi-quantitative determination of 320 fungal and bacterial metabolites in Norwegian cereal grain samples from the 2011 growing season. Such knowledge could provide important information for future survey and research programmes in Norway. The method includes all regulated and well-known mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone. In addition, a wide range of less studied compounds are included in the method, e.g., Alternaria toxins, ergot alkaloids and other metabolites produced by fungal species within Fusarium, Penicillium and Aspergillus. Altogether, 46 metabolites, all of fungal origin, were detected in the 76 barley, oats and wheat samples. The analyses confirmed the high prevalence and relatively high concentrations of type-A and -B trichothecenes (e.g., deoxynivalenol up to 7230 µg/kg, HT-2 toxin up to 333 µg/kg). Zearalenone was also among the major mycotoxins detected (maximum concentration 1670 µg/kg). Notably, several other Fusarium metabolites such as culmorin, 2-amino-14,16-dimethyloctadecan-3-ol and avenacein Y were co-occurring. Furthermore, the most prevalent Alternaria toxin was alternariol with a maximum concentration of 449 µg/kg. A number of Penicillium and Aspergillus metabolites were also detected in the samples, e.g., sterigmatocystin in concentrations up to 20 µg/kg. Full article

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

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Toxins, Volume 5, Issue 10 (October 2013) – 14 articles , Pages 1682-1931

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