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Thomas Reinard - One of the best experts on this subject based on the ideXlab platform.
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Production of a single-chain variable fragment antibody against Fumonisin B1.
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin−biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentia...
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Production of a single-chain variable fragment antibody against Fumonisin B1
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin-biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.
Raghubir P. Sharma - One of the best experts on this subject based on the ideXlab platform.
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Ceramide synthase inhibition by Fumonisin B1 treatment activates sphingolipid-metabolizing systems in mouse liver.
Toxicological sciences : an official journal of the Society of Toxicology, 2006Co-Authors: Hirofumi Suzuki, Neelesh Sharma, Raghubir P. SharmaAbstract:Sphingolipids are important components of cell structure and cell signaling. Both external and internal stimuli can alter levels of cellular sphingolipids by regulating enzyme activities associated with sphingolipid metabolism. Fumonisin B1, mycotoxin produced by Fusarium verticillioides, is a reportedly specific inhibitor of ceramide synthase. In order to test our hypothesis whether ceramide synthase inhibition by Fumonisin B1 alters other sphingolipidmetabolizing enzymes, we investigated the changes in free sphingoid bases and sphingomyelin (SM) and activities of key enzymes for their metabolism, sphingomyelinase (SMase), serine palmitoyltransferase (SPT), and sphingosine kinase (SPHK) in mouse liver. The hepatic free sphingoid bases increased significantly following five daily treatments with Fumonisin B1 in mice. The activity of acidic SMase was enhanced by Fumonisin B1, accompanied with a decrease in liver SM content. The expression and activities of SPT and SPHK1 in liver increased significantly following Fumonisin B1 treatment. Another hepatotoxicant acetaminophen caused liver regeneration similar to Fumonisin B1 but did not produce similar effects on liver sphingolipid-metabolizing enymes, suggesting that activation of sphingolipid metabolism was not a consequence of hepatocye regeneration. Data suggest that ceramide synthase inhibition by Fumonisin B1 treatment stimulates sphingolipid-metabolizing systems to maintain a balance of cellular sphingolipids.
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Amelioration of Fumonisin B1 hepatotoxicity in mice by depletion of T cells with anti-Thy-1.2.
Toxicology, 2006Co-Authors: Neelesh Sharma, Raghubir P. SharmaAbstract:Abstract Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B1 perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies in male mice indicated that Fumonisin B1-induced hepatotoxicity is modulated by the localized activation of cytokines in liver macrophages and other cell types. In the current study, male athymic nude mice and their wild type counterparts (WT), the latter with or without depletion of T cells, were treated subcutaneously with Fumonisin B1 at 2.25 mg/kg/day for 5 days and sampled 24 h after the last injection. Depletion of T cells in WT was achieved by a single intravenous injection of 50 μg monoclonal antibody against Thy-1.2 surface antigen of mature peripheral T lymphocytes 24 h before the first Fumonisin B1 treatment. The depletion of T cells nearly abolished Fumonisin B1-mediated liver toxicity as indicated by the near normal concentrations of circulating liver enzymes and by enumeration of apoptotic hepatocytes. There was no difference in the Fumonisin B1-induced elevation in circulating liver enzymes between WT and nude mice. Fumonisin B1-induced mRNA expression of tumor necrosis factor α and interleukin-1α was observed in nude and WT mice but not in T cell-depleted mice. Hepatotoxic response to Fumonisin B1 was unaltered in mice lacking natural killer cells. This study suggested that T cells and corresponding proinflammatory cytokines have a vital role in mediating Fumonisin B1-induced hepatic toxicity.
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Sphingosine kinase activity confers resistance to apoptosis by Fumonisin B1 in human embryonic kidney (HEK-293) cells.
Chemico-biological interactions, 2004Co-Authors: Neelesh Sharma, Raghubir P. SharmaAbstract:Abstract Fumonisin B1 induces cytotoxicity in sensitive cells by inhibiting ceramide synthase due to its structural similarity to the long-chain backbones of sphingolipids. The resulting accumulation of sphingoid bases has been established as a mechanism for Fumonisin B1 cytotoxicity. We found that despite the accumulation of sphinganine, human embryonic kidney (HEK-293) cells are resistant to Fumonisin B1 toxicity; 25 μM Fumonisin B1 exposure for 48 h did not increase apoptosis in these cells, while it did so in sensitive porcine kidney epithelial (LLC-PK1) cells. In this study, dl -threo-dihydrosphingosine, the sphingosine kinase inhibitor (SKI), considerably increased the sensitivity of HEK-293 cells to Fumonisin B1. Treatment of these cells with 25 μM Fumonisin B1 and 2.5 μM SKI increased apoptosis. Sphingoid bases, sphinganine or sphingosine, added to cell cultures induced apoptosis by themselves and their effects were potentiated by SKI or Fumonisin B1. Addition of physiological amounts of sphingosine-1-phosphate prevented the toxic effects induced by SKI inhibition and Fumonisin B1. Results indicated that HEK-293 cells are resistant to Fumonisin B1 due to rapid formation of sphingosine-1-phosphate that imparts survival properties. Taken together, these findings suggest that sphingoid base metabolism by sphingosine kinase may be a critical event in rendering the HEK-293 cells relatively resistant to Fumonisin B1-induced apoptosis.
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Augmented Fumonisin B1 toxicity in co-cultures: evidence for crosstalk between macrophages and non-parenchymatous liver epithelial cells involving proinflammatory cytokines
Toxicology, 2004Co-Authors: Neelesh Sharma, Raghubir P. SharmaAbstract:Fumonisin B1, a common mycotoxin produced by Fusarium verticillioides found in corn, causes several fatal animal diseases. Liver and kidney are target organs of Fumonisin B1 in laboratory animals, but primary rodent hepatocytes and liver slices were resistant to Fumonisin B1-induced cytotoxic effects. We have shown that Fumonisin B1 induces expression of tumor necrosis factor (TNF)alpha, interferon (IFN)gamma, and interleukine (IL) 12, in mouse liver. In various models of acute liver injury, a positive amplification loop involving TNFalpha, IFNgamma, and IL-12 has been implied that involves Kupffer cells (macrophages), hepatic lymphocytes and non-parenchymatous liver epithelial cells (NPECs). In the current study, cellular interactions in Fumonisin B1-induced toxicity were investigated, using co-cultures of murine macrophages (J774A.1) and NPECs (NMuLi). Treatment of the co-cultures with Fumonisin B1-produced cytotoxicity, whereas either J774A.1 or NMuLi cultures alone showed no response to the mycotoxin. Accumulation of sphinganine occurred to the similar extent in individual cultures as well as co-cultures. Expression of TNFalpha and IL-12 was increased in co-cultures but not in individual cultures. Transfer of conditioned medium from Fumonisin B1-treated J774A.1 cells to NMuLi cultures produced an increase in IFNgamma expression in NMuLi cells. Results indicated that macrophages and liver epithelial cells interact in response to Fumonisin B1 and potentiate the cytokines expression, which may have implications in making hepatocytes responsive to cytotoxicity of Fumonisin B1.
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Sphingoid bases and their phosphates: transient activation and delayed repression of protein kinase C isoforms and their possible involvement in Fumonisin B1 cytotoxicity.
Toxicology, 2003Co-Authors: Neera V. Gopee, Raghubir P. SharmaAbstract:Fumonisin B1, a potent inhibitor of ceramide synthase, leads to accumulation of sphinganine, and later sphingosine, in vivo and in vitro. Fumonisin B1 modulates the activity of protein kinase C (PKC), however, which metabolite of disrupted sphingolipid metabolism is involved, has not been ascertained. In the present study, we evaluated the modulation of PKC by sphingolipid bases and their metabolites using exogenous sphingolipid analogues in porcine renal epithelial (LLC-PK1) cells. In preliminary studies we found that Fumonisin B1 (1 μM) selectively and transiently activated PKCα, whereas Fumonisin B1 concentrations of 1–50 μM at 48 h repressed PKC-α, -δ, -e and -ζ isoforms in a concentration-dependent manner. Addition of exogenous sphinganine-1-phosphate (1 μM for 5 min) alone stimulated cytosolic to membrane translocation of PKCα. Co-exposure of Fumonisin B1 with N,N-dimethylsphingosine, an inhibitor of sphingosine/sphinganine kinase, prevented the effects of Fumonisin B1 on PKCα. Sphinganine, sphingosine, sphingosine-1-phosphate and ceramide (all at 1 μM) added exogenously, did not alter PKCα cytosolic to membrane translocation at 5 min. Fumonisin B1 (10 μM), sphinganine, sphingosine and ceramide (1 μM each) significantly repressed PKC-α and -δ isoforms at 48 h, whereas all the exogenously added sphingolipids significantly repressed PKC-e and ζ similar to Fumonisin B1. Co-exposure of myriocin with Fumonisin B1 prevented the delayed inhibitory effects of Fumonisin B1 on PKC isoforms in LLC-PK1 cells. This study demonstrated that selective and transient activation of PKCα may be due to the Fumonisin B1-induced accumulation of the bioactive sphinganine-1-phosphate, whereas the long-term repression of PKC isoforms may be predominantly due to the accumulation of sphinganine or its metabolite, and to a lesser extent sphingosine or its metabolite in LLC-PK1 cells. These findings suggest that the direct or indirect modulation of PKC by these sphingolipids is involved at least in part in the action of Fumonisin B1.
Björn Lauer - One of the best experts on this subject based on the ideXlab platform.
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Production of a single-chain variable fragment antibody against Fumonisin B1.
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin−biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentia...
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Production of a single-chain variable fragment antibody against Fumonisin B1
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin-biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.
Ilka Ottleben - One of the best experts on this subject based on the ideXlab platform.
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Production of a single-chain variable fragment antibody against Fumonisin B1.
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin−biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentia...
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Production of a single-chain variable fragment antibody against Fumonisin B1
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin-biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.
Hans-jörg Jacobsen - One of the best experts on this subject based on the ideXlab platform.
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Production of a single-chain variable fragment antibody against Fumonisin B1.
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin−biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentia...
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Production of a single-chain variable fragment antibody against Fumonisin B1
Journal of Agricultural and Food Chemistry, 2005Co-Authors: Björn Lauer, Ilka Ottleben, Hans-jörg Jacobsen, Thomas ReinardAbstract:The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin Fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free Fumonisin B1 and Fumonisin-biotin conjugate. Also Fumonisin B2 was bound by the scFv. Modeling of both scFv and Fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.
