Intracellular Expression of a Single Domain Antibody Reduces Cytotoxicity of 15-Acetyldeoxynivalenol in Yeast

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V_HH) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V_HH immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V_HH within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 μg·ml⁻¹. We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 μg·ml⁻¹. The proof of concept established within this eukaryotic system suggests that in planta V_HH expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.     

Enhancement of antioxidant production in <Emphasis Type="Italic">Spirulina platensis</Emphasis> under oxidative stress

The present study examined the possibility of increasing the contents of some bioactive compounds of Spirulina platensis cultivated in medium containing various hydrogen peroxide concentrations (2, 4, 6 and 8 mM) as a model for environmental stress. A positive correlation was observed between the increase of H₂O₂ and increasing amounts of cellular lipophilic antioxidants (total carotenoids and α-tocopherol) and hydrophilic antioxidants [glutathione (GSH) and ascorbic acid (AsA)]. HPLC profile of carotenoids revealed that algae responded to the change of H₂O₂ exposure by the accumulation of higher amounts of β-carotene, astaxanthine, luteine, zeaxanthin and cryptoxanthin. S. platensis showed significant linear increase in activities of antioxidant enzymes, i.e., catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX) and superoxide dismutase (SOD), with increasing H₂O₂ concentrations. A pronounced increase of oxidative lesions’ indexes [thiobarbituric acid reactive substances (TBARS) and paramagnetic radical-EPR signal] was found in algal grown at 8 mM H₂O₂. These data revealed that S. platensis behaved with different strategies against H₂O₂ exposure which is dose dependent and their response strongly correlated with the scavenging enzymes (SOD, CAT, PX and APX) and antioxidant compounds (GSH, AsA, β-carotene, astaxanthine and α-tocopherol) in the antioxidant defense systems. Therefore, S. platensis could be considered as good candidates for successful cultivation in artificial open ponds under different environmental conditions, as high value health foods, functional foods and as source of wide spectrum of nutrients.     

Synthesis and Antiviral Activity of Some N-Pentopyranosyl-2-Pyridinethiones

Abstract

A novel synthesis of 1-(β-D-pentopyranosyl)pyridinethione nucleosides utilizing pyridine-2(1H)-thiones and α-bromoxylose or β-bromoarabinose triacetate as starting components is described. The free nucleosides were tested for their potential activity against HIV and different types of tumor virus.     

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)(1) against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight(2) alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCE's)(3) in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCE's, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.     

Molecular approaches to origin,ancestry and domestication history of crop plants: Barley and clover as examples

Knowledge of the origin and domestication history of crop plants is important for studies aiming at avoiding the erosion of genetic resources due to the loss of ecotypes and landraces and habitats and increased urbanization. Such knowledge also strengthens the capacity of modern farming system to develop and scale-up the domestication of high value potential crops that can be achieved by improving the knowledge that help to identify and select high value plant species within their locality, identify and apply the most appropriate propagation techniques for improving crops and integrate improved crop species into the farming systems. The study of domestication history and ancestry provide means for germplasm preservation through establishment of gene banks, largely as seed collections, and preservation of natural habitats. Information about crop evolution and specifically on patterns of genetic change generated by evolution prior, during, and after domestication, is important to develop sound genetic conservation programs of genetic resources of crop plants and also increases the efficiency of breeding programs. In recent years, molecular approaches have contributed to our understanding of the aspects of plant evolution and crops domestication. In this article, aspects of crops domestication are outlined and the role of molecular data in elucidating the ancestry and domestication of crop plants are outlined. Particular emphasis is given to the contribution of molecular approaches to the origin and domestication history of barley and the origin and ancestry of the Egyptian clover.     

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.