In vitro Interactions of Thallium with Components of the Glutathione-dependent Antioxidant Defence System

We investigated the hypothesis that thallium (Tl) interactions with the glutathione-dependent antioxidant defence system could contribute to the oxidative stress associated with Tl toxicity. Working in vitro with reduced glutathione (GSH), glutathione reductase (GR) or glutathione peroxidase (GPx) in solution, we studied the effects of Tl⁺ and Tl³⁺ (1-25 μM) on: (a) the amount of free GSH, investigating whether the metal binds to GSH and/or oxidizes it; (b) the activity of the enzyme GR, that catalyzes GSH regeneration; and (c) the enzyme GPx, that reduces hydroperoxide at expense of GSH oxidation. We found that, while Tl⁺ had no effect on GSH concentration, Tl³⁺ oxidized it. Both cations inhibited the reduction of GSSG by GR and the diaphorase activity of this enzyme. In addition, Tl³⁺per se oxidized NADPH, the cofactor of GR. The effects of Tl on GPx activity depended on the metal charge: Tl⁺ inhibited GPx when cumene hydroperoxide (CuOOH) was the substrate, while Tl³⁺-mediated GPx inhibition occurred with both substrates. The present results show that Tl interacts with all the components of GSH/GSSG antioxidant defence system. Alterations of this protective pathway could be partially responsible for the oxidative stress associated with Tl toxicity.     

9-(2-Deoxy-ß-D-xylofuranosyl)adenine and 1-(2-Deoxy-ß-D-xylofuranosyl)thymine: Phosphorylation and Stability

Abstract

Phosphorylation of 1-(2-deoxy-β-D-xylofuranosyl)thymine (1) or 9-(2-deoxy-β-D-xylofuranosyl)adenine (3) with phosphoryl chloride gives the cyclic 3′,5′-phosphates (2 and 4a) but not the 5′-monophosphates 8a or 8b. The latter are obtained by phosphorylation of the 3′-0-benzoylated 2′-deoxy-β-D-xylonucleosides (7a, b) and subsequent base-catalyzed removal of the benzoyl groups. Compound 3, as the parent dA, depurinates in acidic medium, a reaction which is facilitated in the case of the N⁶-benzoyl derivative 9b and reduced after the introduction of an amidine protecting group. N-Glycosylic bond hydrolysis of 2′-deoxy-β-D-xylofuranosyl nucleosides is enhanced by a factor of two compared to 2′-deoxy-β-D-ribofuranosyl nucleosides.     

Syntheses of 1-[2-(Phosphonomethoxy)Alkyl]Thymine Monophosphates and an Evaluation of their Inhibitory Activity Toward Human Thymidine Phosphorylase

A series of new monophosphates of 1-[2-(phosphonomethoxy)alkyl]thymines, such as PMPTp_, 3-MeO-PMPTp, HPMPTp, and FPMPTp, were synthesized and tested for their ability to inhibit human thymidine phosphorylase. Kinetic measurements of enzyme activity were performed using thymidine and inorganic phosphate as the substrates. The data show that some monophosphates provide a considerable increase of the multisubstrate inhibitory effect. The highest inhibitory potency was found with (R)-FPMPTp 4c (K _i ^dT = 4.09 ± 0.47 μM, K _i(P_i) = 2.13 ± 0.29 μM) and (R) 3-MeO-PMPTp 4d (K _i ^dT = 5.78 ± 0.71 μM, K _i(P_i) = 2.71 ± 0.37 μM).     

Microbial Environment Affects Innate Immunity in Two Closely Related Earthworm Species Eisenia andrei and Eisenia fetida

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.     

The Pneumatron: An automated pneumatic apparatus for estimating xylem vulnerability to embolism at high temporal resolution

Xylem vulnerability to embolism represents an important trait to determine species distribution patterns and drought resistance. However, estimating embolism resistance frequently requires time-consuming and ambiguous hydraulic lab measurements. Based on a recently developed pneumatic method, we present and test the “Pneumatron”, a device that generates high time-resolution and fully automated vulnerability curves. Embolism resistance is estimated by applying a partial vacuum to extract air from an excised xylem sample, while monitoring the pressure change over time. Although the amount of gas extracted is strongly correlated with the percentage loss of xylem conductivity, validation of the Pneumatron was performed by comparison with the optical method for Eucalyptus camaldulensis leaves. The Pneumatron improved the precision of the pneumatic method considerably, facilitating the detection of small differences in the (percentage of air discharged [PAD] < 0.47%). Hence, the Pneumatron can directly measure the 50% PAD without any fitting of vulnerability curves. PAD and embolism frequency based on the optical method were strongly correlated (r² = 0.93) for E. camaldulensis. By providing an open source platform, the Pneumatron represents an easy, low-cost, and powerful tool for field measurements, which can significantly improve our understanding of plant–water relations and the mechanisms behind embolism.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Nitric oxide as a key component in hormone-regulated processes

Nitric oxide (NO) is a small gaseous molecule, with a free radical nature that allows it to participate in a wide spectrum of biologically important reactions. NO is an endogenous product in plants, where different biosynthetic pathways have been proposed. First known in animals as a signaling molecule in cardiovascular and nervous systems, it has turned up to be an essential component for a wide variety of hormone-regulated processes in plants. Adaptation of plants to a changing environment involves a panoply of processes, which include the control of CO₂ fixation and water loss through stomatal closure, rearrangements of root architecture as well as growth restriction. The regulation of these processes requires the concerted action of several phytohormones, as well as the participation of the ubiquitous molecule NO. This review analyzes the role of NO in relation to the signaling pathways involved in stomatal movement, plant growth and senescence, in the frame of its interaction with abscisic acid, auxins, gibberellins, and ethylene.