Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane.
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Protein kinase D (PKD) is a cytosolic serine-threonine kinase that binds to the trans-Golgi network (TGN) and regulates the fission of transport carriers specifically destined to the cell surface. PKD was found to bind diacylglycerol (DAG), and this binding was necessary for its recruitment to the TGN. Reducing cellular levels of DAG inhibited PKD recruitment and blocked protein transport from the TGN to the cell surface. Thus, a DAG-dependent, PKD-mediated signaling regulates the formation of transport carriers from the TGN in mammalian cells. (+info)
Immunobiological effects of fumonisin B1 in experimental subchronic mycotoxicoses in rats.
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Fumonisin B1 (FB1), the principal secondary metabolite produced by the fungus Fusarium verticillioides (Gibberella fujikuroi mating population A), is a potent toxin that can be found in fungus-contaminated corn and corn-based food products. We have investigated the immunobiological effects of subchronic dietary exposure to FB1 in male Wistar rats. Animals were fed with diets containing 0 (control) or 100 ppm of FB1 for 12 weeks. The total FB1 intake on day 90 was 810 mg/kg of body weight. Food consumption, body weight, and body weight gain on day 90 were reduced in animals exposed to FB1. Histopathologic changes consisted of histiocytic perivascular infiltrate and an increased number of Kupffer cells in the liver, necrosis and apoptosis of tubular epithelial cells in the kidney, and increased mitotic figures and lymphocytic infiltrate in the small intestine. Serum enzyme alkaline phosphatase was significantly elevated in rats fed FB1, while triglyceride levels decreased compared to controls. Treatment with FB1 in vivo or in vitro did not have a significant effect on mitogen-induced proliferation of spleen mononuclear cells. However, increased levels of interleukin-4 (IL-4) and decreased levels of IL-10 were released by these cells in culture compared to controls. FB1 in vivo or in vitro decreased the hydrogen peroxide (H(2)O(2)) released by peritoneal macrophages, while no changes in levels of superoxide anion produced by total peritoneal cells were detected. The results from the present work demonstrate that subchronic FB1 intake could affect the small intestine and alter the interleukin profile and some main functions of macrophages in antitumor activity. (+info)
Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin.
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To test the hypothesis that regulation of palmitate metabolism, through carnitine palmitoyl transferase-1 (CPT-1) or through alterations of glycolysis, was involved in the pathway of palmitate-mediated cell death, cardiomyocytes were cultured from 7-day-old chick embryos. Palmitate-induced cell death, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was enhanced by carnitine, a cofactor needed for palmitate transport into mitochondria via CPT-1. Carnitine co-incubation with palmitate significantly (P < 0.01) increased the amount of apoptotic cells, assessed by propidium iodine staining and fluorescent-activated cell sorting analysis compared with treatment with either palmitate or carnitine alone. The CPT-1 inhibitor oxfenicine significantly (P < 0.05) blocked the cell death induced by the combination of palmitate and carnitine. The short-chain saturated fatty acid capric acid (100 microM), which is not likely transported by CPT-1, did not significantly affect cell viability, whereas the C18 saturated fatty acid stearic (100 microM) significantly (P < 0.01) reduced cell viability and to a similar extent as palmitate. In contrast, there was no significant alteration of palmitate-induced cell death by cotreatment with 100 nM insulin + 2 g/l glucose or 1 mM lactate, which promote ATP generation by glycolysis rather than fatty acid oxidation. Fumonisin did not alter palmitate-induced cell death or apoptosis, suggesting that the effect of palmitate was not operative through increased ceramide synthesis. These results suggest that oxidation of palmitate through CPT-1 is involved in the production of apoptosis in cardiomyocytes. (+info)
De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.
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Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C(6) ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 microm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C(6) ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis. (+info)
Toxicology and carcinogenesis studies of fumonisin B1 (cas no. 116355-83-0) in F344/N rats and B6C3F1 mice (feed studies).
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[formula: see text] Fumonisin B1 is a mycotoxin produced by the fungus Fusarium moniliforme, one of the major species found in corn. There are no known commercial or medical uses of fumonisin B1. Fumonisin B1 was nominated by the FDA Center for Food Safety and Applied Nutrition for study because of its occurrence in corn and corn-based products in the United States and its toxicity in field exposure of horses and pigs. Male and female F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV-) mice were exposed to fumonisin B1 (92% pure) in feed for 28 days or (greater than 96% pure) for 2 years. 28-DAY STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 99, 163, 234, or 484 ppm fumonisin B1 for 28 days. There were no exposure-related deaths in rats. The mean body weights of the 484 ppm groups were significantly less (-16%) than those of the controls. Dietary concentrations of 99, 163, 234, and 484 ppm fumonisin B1 resulted in average daily doses of 12, 20, 28, and 56 mg fumonisin B1/kg body weight for males and females. Additional groups of male and female rats were exposed to the same concentrations of fumonisin B1 for 28 days for clinical pathology studies. The concentrations of creatinine, cholesterol, triglycerides, and total bile acids, as well as activities of the enzymes alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and gamma-glutamyltransferase, were generally significantly greater in the 484 ppm groups than in the control groups at all time points, indicating hyperlipidemia and a hepatic effect. Fumonisin B1 is an inhibitor of ceramide synthase, resulting in an interruption of de novo sphingolipid synthesis. This enzyme inhibition results in increased levels of sphinganine (or increased sphinganine:sphingosine ratio) in tissues and urine. Urinary sphinganine was increased in groups of males exposed to 163 ppm or greater, while urinary sphinganine was increased in all exposed groups of females. The kidney weights, relative to body weight, of all exposed groups of rats were less than those of the control groups, decreasing by approximately 11% in the females and 20% in the males. Apoptosis and degeneration of the kidney were observed in all exposed males and in most females exposed to 163 ppm or greater. The incidences of minimal to mild apoptosis, degeneration, and mitotic alteration of the liver were significantly increased in 234 and 484 ppm males and in females exposed to 163 ppm or greater. The incidences of bile duct hyperplasia were significantly increased in males and females in the 484 ppm groups. In the core study, male rats in all exposed groups and females exposed to 163 ppm or greater had significantly increased percentages of hepatocytes in one or more proliferative (non-G0) states. 28-DAY STUDY IN MICE: Groups of 12 male and 12 female mice were fed diets containing 0, 99, 163, 234, or 484 ppm fumonisin B1 for 28 days. There were no exposure-related deaths in mice. The mean body weights of the 484 ppm groups of males were significantly less than those of the controls. Feed consumption by males exposed to 484 ppm was less than that by the controls; dietary concentrations of 99, 163, 234, and 484 ppm fumonisin B1 resulted in average daily doses of approximately 19, 31, 44, and 93 mg/kg for males and 24, 41, 62, and 105 mg/kg for females. Additional groups of male and female mice were exposed to the same concentrations of fumonisin B1 for 28 days for clinical pathology studies. Cholesterol and total bile acid concentrations and alanine aminotransferase and alkaline phosphatase activities were increased at 484 ppm, indicating hyperlipidemia and a hepatic effect. Urinary sphinganine concentrations and sphinganine/sphingosine ratios were increased in 484 ppm male mice. In 484 ppm males and all exposed groups of females, the incidences of hepatocellular necrosis, diffuse periportal hypertrophy, and diffuse centrilobular hyperplasia, as well as hyperplasia of the bile canaliculi and Kupffer cells, were generally significantly greater than those in the controls. Core study males exposed to 99, 163, or 234 ppm had significantly increased incidences of hepatocellular cytoplasmic alteration. Hepatocytes of 484 ppm male mice and all exposed groups of female mice were induced into proliferative (non-G0) states. 2-YEAR STUDY IN RATS: Groups of 48 male and 48 female rats (40 for 5 ppm groups) were fed diets containing 0, 5, 15, 50, or 150 ppm fumonisin B1 (males) or 0, 5, 15, 50, or 100 ppm fumonisin B1 (females) (equivalent to average daily doses of approximately 0.25, 0.76, 2.5, or 7.5 mg/kg to males and 0.31, 0.91, 3.0, or 6.1 mg/kg to females) for 105 weeks. Additional groups of four male and four female rats were exposed to the same concentrations as the core study animals and were evaluated at 6, 10, 14 or 26 weeks. Survival, Body Weights, and Feed Consumption Survival, mean body weights, and feed consumption of exposed male and female rats were generally similar to the controls throughout the study. Clinical Pathology Findings Sphinganine/sphingosine ratios were increased in the urine of 15, 50 and 150 ppm males and 50 and 100 ppm females exposed to fumonisin B1 for up to 26 weeks. The sphinganine/sphingosine ratios were also increased in kidney tissue of 50 and 150 ppm males (85- and 119-fold) and 50 and 100 ppm females (7.8- and 22-fold) at 2 years. Cell Proliferation Analyses Renal tubule epithelial cell proliferation was increased in 50 and 150 ppm male rats exposed to fumonisin B1 for up to 26 weeks. Renal tubule epithelial cell proliferation was marginally increased in 100 ppm females. Organ Weights and Pathology Findings Kidney weights of 50 and 150 ppm males were less than those of the controls at 6, 10, 14, and 26 weeks and at 2 years. Kidney weights of 100 ppm females were less than those of the controls at 26 weeks, and kidney weights of 15, 50, and 100 ppm females were less than those of the controls at 2 years. At 2 years, there was a significant increase in the incidences of renal tubule adenoma from none in the groups receiving 15 ppm or less to five of 48 in 150 ppm males. Renal tubule carcinomas were not present in male rats receiving 15 ppm or less and occurred in seven of 48 and 10 of 48 male rats in the 50 and 150 ppm groups, respectively. Incidences of apoptosis of the renal tubule epithelium were generally significantly increased in males exposed to 15 ppm or greater for up to 26 weeks. The incidences of focal renal tubule epithelial hyperplasia were significantly increased in 50 and 150 ppm males at 2 years. 2-YEAR STUDY IN MICE: Groups of 48 male and 48 female mice were fed diets containing 0, 5, 15, 80, or 150 ppm (males) or 0, 5, 15, 50, or 80 ppm (females) fumonisin B1 (equivalent to average daily doses of approximately 0.6, 1.7, 9.7, or 17.1 mg/kg to males or 0.7, 2.1, 7.1, or 12.4 mg/kg to females) for 105 weeks. Additional groups of four male and four female mice were exposed to the same concentrations as the core study animals and were evaluated at 3, 7, 9, or 24 weeks. Survival, Body Weights, and Feed Consumption Survival of males and females in the 15 ppm groups and of 5 ppm females was significantly greater and survival of 80 ppm males and females was significantly less than that of the control groups. Mean body weights and feed consumption of exposed mice were generally similar to the controls. Organ Weights and Pathology Findings Liver weights, relative to body weight, were increased 1.3- and 2.9-fold in 50 and 80 ppm females at 2 years. At 2 years, the incidences of hepatocellular adenoma in 50 and 80 ppm females were significantly greater than those in the controls and occurred with a positive trend. Similarly, the incidences of hepatocellular carcinoma increased from none in the groups receiving 0, 5, or 15 ppm fumonisin B1 to 10 of 47 females at 50 ppm and nine of 45 females at 80 ppm. The incidences of hepatocellular hypertrophy were significantly increased in 15, 80, and 150 ppm males and in 50 and 80 ppm females at 2 years. The incidences of hepatocellular apoptosis were significantly increased in 50 and 80 ppm females at 2 years. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenic activity of fumonisin B1 in male F344/N rats based on the increased incidences of renal tubule neoplasms. There was no evidence of carcinogenic activity of fumonisin B1 in female F344/N rats exposed to 5, 15, 50, or 100 ppm. There was no evidence of carcinogenic activity of fumonisin B1 in male B6C3F1 mice exposed to 5, 15, 80, or 150 ppm. There was clear evidence of carcinogenic activity of fumonisin B1 in female B6C3F1 mice based on the increased incidences of hepatocellular neoplasms. The sphinganine/sphingosine ratios were increased in the urine and the kidney tissue of rats receiving diets containing fumonisin B1. There was evidence of apoptosis and increased cell proliferation of the renal tubule epithelium in exposed rats, particularly in those groups of males that developed renal tubule neoplasms. Increased incidences of hyperplasia of the renal tubule epithelium also occurred in these groups of male rats. In mice exposed to the higher concentrations of fumonisin B1, males and females had increased incidences of hepatocellular hypertrophy and females had increased incidences of hepatocellular apoptosis. (+info)
The lysosomal transport of prosaposin requires the conditional interaction of its highly conserved d domain with sphingomyelin.
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Lysosomal prosaposin (65 kDa) is a nonenzymic protein that is transported to the lysosomes in a mannose 6-phosphate-independent manner. Selective deletion of the functional domains of prosaposin indicates that the D domain and the carboxyl-terminal region are necessary for its transport to the lysosomes. Inhibitors of sphingolipid biosynthesis, such as fumonisin B(1) (FB(1)) and tricyclodecan-9-yl xanthate potassium salt (D609), also interfere with the trafficking of prosaposin to lysosomes. In this study, we examine sphingomyelin as a direct candidate for the trafficking of prosaposin. Chinese hamster ovary and COS-7 cells overexpressing prosaposin or an albumin/prosaposin construct were incubated with these inhibitors, treated with sphingolipids, and then immunostained. Sphingomyelin restored the immunostaining in lysosomes in both FB(1)- and D609-treated cells and ceramide reestablished the immunostaining in FB(1)-treated cells only. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits glycosphingolipids, had no effect on the immunostaining pattern. To determine whether sphingomyelin has the same effect on the transport of endogenous prosaposin, testicular explants were treated with FB(1) and D609. Sphingomyelin restored prosaposin immunogold labeling in the lysosomes of FB(1)- and D609-treated Sertoli cells, whereas ceramide restored the label in FB(1) treatment only. Albumin linked to the D and COOH-terminal domains of prosaposin was used as a dominant negative competitor. The construct blocked the targeting of prosaposin and induced accumulation of membrane in the lysosomes, demonstrating that the construct uses the same transport pathway as endogenous prosaposin. In conclusion, our results showed that sphingomyelin, the D domain, and its adjacent COOH-terminal region play a crucial role in the transport of prosaposin to lysosomes. Although the precise nature of this lipid-protein interaction is not well established, it is proposed that sphingomyelin microdomains (lipid rafts) are part of a mechanism ensuring correct intercellular trafficking of prosaposin. (+info)
Overexpression of the tomato Asc-1 gene mediates high insensitivity to AAL toxins and fumonisin B1 in tomato hairy roots and confers resistance to Alternaria alternata f. sp. lycopersici in Nicotiana umbratica plants.
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The sphinganine-analog mycotoxins (SAMs) fumonisin B1 and AAL toxins are inhibitors of eukaryotic sphinganine N-acyltransferase in vitro. Treatment of eukaryotes with SAMs generally results in an accumulation of sphingoid base precursors and a depletion of complex sphingolipids. The asc,asc genotypes of tomato (Lycopersicon esculentum) and Nicotiana umbratica are sensitive to SAMs and host of the AAL toxin-producing fungus Alternaria alternata f. sp. lycopersici. Codominant insensitivity to SAMs in tomato is mediated by the Asc-1 gene, and sensitivity is associated with a frame-shift mutation present in asc-1. We investigated the function of Asc-1 in mediating insensitivity to SAMs and resistance to the fungus by overexpression of asc-1 and Asc-1. In this study, it is shown that overexpression of these genes did not lead to visual symptoms in tomato hairy roots and N. umbratica plants. Overexpression of asc-1 did not influence the (in)sensitivity to SAMs. Overexpression of Asc-1 in SAM-sensitive hairy roots and N. umbratica plants, however, mediated a high insensitivity to SAMs and resistance to plant infection by Alternaria alternata f. sp. lycopersici. (+info)
Fumonisin B(1) is genotoxic in human derived hepatoma (HepG2) cells.
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Fumonisin B(1) (FB(1)), a widespread Fusarium toxin which is frequently found in corn, causes liver tumors in laboratory rodents and is a suspected human carcinogen. The compound was tested in micronucleus (MN) and single cell gel electrophoresis (SCGE) assays in human derived hepatoma (HepG2) cells and caused a pronounced dose-dependent genotoxic effect at exposure concentrations > or = 25 microg/ml. In contrast, no induction of his(+) revertants was found in Salmonella microsome assays with strains TA98, TA100, TA102, TA1535 and TA1537 upon addition of HepG2-derived enzyme (S9) mix in liquid incubation assays with identical exposure concentrations. Taken together, our results indicate that FB(1) is clastogenic in human derived cells. This observation supports the assumption that this compound may act as a genotoxic carcinogen in humans. (+info)