Ligation of the WC1 receptor induces gamma delta T cell growth arrest through fumonisin B1-sensitive increases in cellular ceramide.
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Ceramide is a powerful regulator of cell fate, inducing either apoptosis or growth arrest. We have previously shown that an Ab to the gammadelta T cell-specific orphan receptor, WC1, is able to induce growth arrest in proliferating IL-2-dependent gammadelta T cells. We now show that this WC1-mediated growth arrest is associated with an increase in cellular ceramide, in the absence of any measurable changes in acidic/neutral sphingomyelinase activity. Moreover, cell-permeable analogues of ceramide also mimicked WC1-induced growth arrest along with an associated decrease in pocket protein expression and phosphorylation status. An important role for ceramide in WC1-induced growth arrest was confirmed by demonstrating that the specific ceramide synthase inhibitor fumonisin B1 blocked WC1-induced growth arrest and the associated molecular effects on the pocket proteins. Finally, we observed constitutive expression of both antiapoptotic factors bcl-2 and bcl-X, the former having increased expression upon WC1 stimulation. It is therefore proposed that ligation of WC1 leads to an accumulation in cellular ceramide through activation of ceramide synthase. This in turn results in a decreased overall expression of the pocket proteins pRb and p107, their hypophosphorylation, and an eventual growth arrest of the gammadelta T cell. To our knowledge, these results demonstrate for the first time that cell surface receptor-mediated ceramide synthase activation can affect cell fate through increases in cellular ceramide and provide further evidence that the orphan receptor WC1 regulates gammadelta T cell biology through a novel signaling pathway. (+info)
Protective antigens and mechanisms of anti-Candida immunity.
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Life threatening fungal diseases are now frequent in a substantial fraction of the immunocompromised host population. The toxicity and the relative scarcity of efficacious antifungal drugs highlight the need for developing alternative or integrative immunoprophylactic and therapeutic tools; among them the need to develop prophylactic or therapeutic vaccines against candidiasis, a widespread mucosal or deep-seated infection caused primarily by the fungus Candida albicans, are of clear priority. Vaccination is a highly beneficial medical practice, and probably the most cost-effective measure against disease onset and progression. It is based on the use of microbial antigens capable of conferring protection in a susceptible target host. To date, only a handful of Candida albicans antigens have been produced and very few of them have been thoroughly investigated for immunogenicity and protection in experimental models of candidiasis. Thus, approaches to the molecular, biochemical and functional characterization of novel C. albicans encoded molecules are most welcome to improve the perspective of developing in the near future an effective vaccine against C. albicans. Identification of anti-Candida vaccine candidates must take into account the diversity of Candida diseases, the various underlying mechanisms of protection as well as the major immune dysfunctions observed as predisposing factors for disease. Antigens to be considered possible vaccine candidates include members of the aspartyl proteinase (Sap2) family and the 65kDa mannoprotein (MP65) antigen. An additional molecule of C. albicans which has not yet been identified but deserves great consideration as a vaccine candidate is the yeast-killer toxin receptor (KTR). Initial experimental evidence strongly suggests that the above antigens are able to elicit protective immunity against mucosal and/or systemic candidiasis. A series of molecular, biochemical and immunological studies aimed at validating and strengthening this initial evidence are in progress, with the ultimate goal of producing recombinant or natural antigens that can be assessed for their ability to elicit a protective immunity in animal models and the mechanisms whereby protection is achieved, with emphasis on determination of immune correlates of protection. (+info)
Assessment of in vitro mutagenicity in Salmonella and in vivo genotoxicity in mice of the mycotoxin fumonisin B(1).
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Fumonisin B(1) (FB(1)), a mycotoxin produced by Fusarium moniliforme, is a contaminant of cereals with various and complex cellular effects. FB(1) induces liver cancer in rats and has been linked to esophageal cancer in South Africa and China. The mechanisms of FB(1)-induced carcinogenesis are uncertain and the information on FB(1) mutagenic properties is limited and controversial. FB(1) contamination levels in maize and wheat from Chile were found to be similar to those in other countries. FB(1) was devoid of activity in gene mutation assays with Salmonella typhimurium strains TA100, TA102 and TA98. However, i.p. injection of FB(1) induced an increased frequency of micronuclei in mouse bone marrow polychromatic erythrocytes at 25 and 100 mg/kg. We conclude that FB(1) induces in vivo genotoxicity in the absence of in vitro mutagenicity in Salmonella. (+info)
Isolation, purification, and characterization of a killer protein from Schwanniomyces occidentalis.
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The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40 degrees C. The killer protein was chromosomally encoded. Mannan, but not beta-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations. (+info)
Stachybotrys chartarum: current knowledge of its role in disease.
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Stachybotrys chartarum is one of several species of filamentous fungi capable of producing mycotoxins under certain environmental conditions. In some observational studies, the growth of this toxigenic mold in the indoor environment has been implicated as a cause of building-related illness. Following reports of a cluster of cases of pulmonary hemosiderosis and hemorrhage associated with exposure to Stachybotrys, public health measures have been recommended which have far-reaching implications. Although the hazards associated with exposure to some mycotoxins have been well studied, the health risks from environmental exposure to Stachybotrys remain poorly defined. The purpose of this review is to critically evaluate the current body of epidemiologic knowledge regarding Stachybotrys and to increase physician awareness regarding this emerging environmental health issue. (+info)
Preparation of 14C-labeled sterigmatocystin in liquid media.
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14C-labeled sterigmatocystin was prepared from surface cultures of Aspergillus versicolor A-18074 maintained in liquid media by multiple additions of (1-14C)acetate to the cultures. The highest yield of 7.75 mg/10 ml was found with a sucrose-asparagine-ammonium medium in which more than 3% of the radioactivity of the added (1-14C)acetate was recovered ithe purified (ring-14C) sterigmatocystin. The method offers an easy way to prepare 14C-labeled sterigmatocystin for studies of this mycotoxin. (+info)
Ochratoxin A-induced tumor formation: is there a role of reactive ochratoxin A metabolites?
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Ochratoxin A is a nephrotoxic and tumorigenic mycotoxin which contaminates a variety of food items, resulting in chronic human exposure. Biotransformation reactions have been implicated in the tumorigenicity of ochratoxin A. The biotransformation of ochratoxin A by cytochromes P450 and other mammalian enzymes was investigated to optimize conditions for bacterial mutagenicity testing. Metabolite formation was assessed by HPLC with UV and fluorescence detection and by LC/MS/MS. When ochratoxin A was incubated with liver microsomes from rats and mice, formation of 4R- and 4S-hydroxyochratoxin A was observed at very low rates. However, oxidation of ochratoxin A was not observed using kidney microsomes from rats and mice. Significantly higher rates of oxidation were seen in liver microsomes from rats pretreated with 3-methylcholanthrene and dexamethasone. Other reported or postulated that ochratoxin A-metabolites were not formed in detectable concentrations. Human cytochromes P450 (3A4, 1A2, and 2C9-1 Supersomes((R))) also showed very low activity with ochratoxin A (<60 fmole/min x pmol P450). Other enzyme systems used to study possible biotransformation of ochratoxin A were rat and human liver and kidney S-9 fortified with NADPH and glutathione, semipurified glutathione S-transferases, horseradish peroxidase, and soybean lipoxygenase; none of these resulted in detectable biotransformation of ochratoxin A. Using rat liver microsomes with high activity for ochratoxin A oxidation and the other enzyme systems to activate ochratoxin A for mutagenicity testing in the Ames test, mutagenicity was not observed in Salmonella typhimurium TA 100 and TA 2638. The obtained results suggest that oxidative biotransformation of ochratoxin A occurs at low rates, is catalyzed by cytochromes P450, and is unlikely to form reactive intermediates capable of binding to DNA. (+info)
Specific binding of vf14-3-3a isoform to the plasma membrane H+-ATPase in response to blue light and fusicoccin in guard cells of broad bean.
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The plasma membrane H(+)-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform(s) bound to the H(+)-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b, vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H(+)-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H(+)-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H(+)-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis revealed that the H(+)-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H(+)-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H(+)-ATPase in guard cells. (+info)