C. Channa Reddy

Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA

ABSTRACT The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an ∼50-fold reduction in Se-GPx1 activity and an ∼20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.

A compact wireless gas sensor using a carbon nanotube/PMMA thin film chemiresistor

The design and development of a compact wireless gas sensor with a surface modified multiwalled carbon nanotube (f-CNT) chemiresistor as the sensing element is presented in this paper. f-CNT/polymer composite sensing film is patterned on a printed circuit board and is integrated to the wireless system. The change in resistance of the CNT/polymer composite film due to exposure of different gases is utilized as the principle of this gas sensor. The response for different organic vapors are evaluated and it is observed that the f-CNT/PMMA composite film shows fast response and change in resistance of the order of 102–103 due to its surface modification.

Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on in vitro lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it has no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.

The anti-inflammatory effects of selenium are mediated through 15-Deoxy-Δ12, 14-prostaglandin J2 in macrophages

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-κB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-κB cascade, IκB-kinase β (IKKβ) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKβ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKβ activity. Further analysis revealed that inhibition of IKKβ activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKβ, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-γ in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium.

Evidence for the occurrence of selenium-independent glutathione peroxidase activity in rat liver microsomes.

Abstract Rat liver microsomes exhibit selenium-independent glutathione peroxidase activity which is associated with glutathione S-transferase activity. The peroxidase activity is not due to contamination with either soluble selenium-dependent or selenium-independent glutathione peroxidase activities of the cytosol. N-Ethylmaleimide treatment which stimulates rat liver microsomal glutathione transferase activity concomitantly stimulates the glutathione peroxidase activity. In contrast, N-ethylmaleimide depresses both enzyme activities of the cytosol. A protein exhibiting both glutathione peroxidase and glutathione transferase activity was isolated from the microsomes and purified to homogeneity by DEAE cellulose ion-exchange and S-hexylglutathione Sepharose 6B affinity chromatography.

Inhibition of 5-lipoxygenase by vitamin E

Purified 5‐lipoxygenase from potato tubers was inhibited strongly by vitamin E and its analogs. The inhibition by d‐α‐tocopherol was found to be irreversible and non‐competitive with respect to arachidonic acid. An IC50 of 5μM was calculated for d‐α‐tocopherol. The inhibition appears to be unrelated to its antioxidant function. Binding studies with 14C‐labelled d‐α‐tocopherol revealed that there is a strong interaction between vitamin E and 5‐lipoxygenase. Tryptic digestion and peptide mapping of 5‐lipoxygenase‐vitamin E complex indicate that vitamin E binds strongly to a single peptide. These studies suggest that cellular vitamin E levels may have profound influence on the formation of leukotrienes.

Selenium attenuates pro-inflammatory gene expression in macrophages.

Selenium (Se) is an important element required for the optimal functioning of the immune system. Particularly in macrophages, which play a pivotal role in immune regulation, Se acts as a major antioxidant in the form of selenoproteins to mitigate the cytotoxic effects of reactive oxygen species. Here we describe the role of Se as an anti-inflammatory agent and its effect on the macrophage signal transduction pathways elicited by bacterial endotoxin, LPS. Our studies demonstrate that supplementation of Se to macrophages (Se-deficient) leads to a significant decrease in the LPS-induced expression of two important pro-inflammatory genes, cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-alpha) via the inhibition of MAP kinase pathways. Furthermore, Se-deficiency in mice exacerbated the LPS-mediated infiltration of macrophages into the lungs suggesting that Se status is a crucial host factor that regulates inflammation. In summary, our results indicate that Se plays an important role as an anti-inflammatory agent by tightly regulating the expression of pro-inflammatory genes in immune cells.

Nuclear factor-κB mediates over-expression of cyclooxygenase-2 during activation of RAW 264.7 macrophages in selenium deficiency

Selenium (Se) is an essential micronutrient for all mammalian species and is associated with a variety of physiological functions, notably immune system, in the form of selenoproteins. Inadequate Se nutrition has been linked to various diseases, including rheumatoid arthritis, cardiomyopathy, and cancer. Important to this discussion is that cyclooxygenase-2 (COX-2) is over-expressed in all the aforesaid pathologies; however, a casual relationship between Se status and COX-2 expression remains to be established. The present study is based on the hypothesis that oxidant stress, a consequence of Se deficiency, lowers the activation potential of the redox-sensitive transcription factor, NF-kappaB, and that the activated NF-kappaB is required for the altered expression of COX-2. To test this hypothesis, we have investigated the relationship between Se status and COX-2 expression in response to LPS stimulation in RAW 264.7, a macrophage-like cell line. In Se-deficient cells, the Se-dependent glutathione peroxidase activity (Se-GPx), a measure of Se status, was markedly reduced and the overall oxidative stress was significantly higher than Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation, we found 2-3-folds higher COX-2 protein expression as well as higher PGE2 levels in Se-deficient cells than Se-supplemented cells. In comparison, COX-1 protein expression was not affected by either LPS stimulation or Se status. Following LPS stimulation, the nuclear localization of NF-kappaB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2. This is the first report demonstrating an inverse relationship between Se status and the expression of COX-2.

Purification of arachidonate 5-lipoxygenase from potato tubers

Publisher Summary The chapter briefly describes the assay, the purification, and the properties of arachidonate 5-1ipoxygenase (EC 1.13.11.34) from potato tubers. Lipoxygenases (EC 1.13. 11. 12) are a group of closely related enzymes that appear to be widely distributed in plants and mammalian tissues. One of the most physiologically important lipoxygenases and the subject of greater interest in recent years is 5-lipoxygenase, which catalyzes the first committed step in leukotriene (LT) biosynthesis. Information on the mechanistic details of this enzyme-catalyzed reaction is scant because of limitations in obtaining sufficient quantity of the purified enzyme in stable form from mammalian sources. Therefore, potato tubers are frequently employed as the source of large amounts of 5-1ipoxygenase. The mechanism of dioxygenation of polyunsaturated fatty acids (PUFA) catalyzed by lipoxygenases involves the following three main steps: (1) Abstraction of a hydrogen atom from the double allylic methylene carbon atom (the first and rate-limiting step), (2) conjugation of double bonds followed by rearrangement of the radical electron, and (3) addition of molecular oxygen (this occurs at a diffusion-controlled rate under ambient O 2 concentrations). Based on this mechanism, it is possible to determine the lipoxygenase activity by measuring the following: (1) absorbance of conjugated diene at 235 nm, (2) O 2 uptake by means of a Clark oxygen electrode, and (3) product formation by means of high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC). Although each one of these methods can be used for monitoring enzyme activity during the course of purification of 5 -lipoxygenase from potato tubers, the polarographic method is found to be the most convenient and reproducible.

Mechanism of Interaction of Vitamin E and Glutathione in the Protection against Membrane Lipid Peroxidation

Although the function of vitamin E as the major, if not only, chain-breaking, lipidsoluble antioxidant in biological membranes has been established,’ its mode( s) of action has yet to be elucidated. One proposed mechanism of vitamin E function involves a possible glutathione-dependent system that inhibits in vitro lipid peroxidation. Previously, we have described a rat liver microsomal system that inhibited both enzymatic and nonenzymatic lipid peroxidation in the presence of both glutathione and microsoma1 vitamin E., The GSH-dependent inhibition of lipid peroxidation was not observed in microsomes obtained from vitamin E-deficient rats and was independent of the selenium status of the animal. This inhibition, however, was not seen in heatdenatured microsomes, despite the continued presence of vitamin E. We therefore suggested that a microsomal heat-labile protein was likely involved in the mediation of the vitamin E-dependent, glutathione inhibition of lipid peroxidation. Other groups have also recently reported the presence of a glutathione-dependent liver microsomal factor that inhibited lipid per~xidation.’-~ The experiments reported here were designed to investigate the nature of the GSH-dependent inhibition of lipid peroxidation in the presence or absence of vitamin E. Weanling male Long-Evans hooded rats were fed chemically defined diets either deficient or supplemented with 150 IU dl-a-tocopheryl acetate/kg diet for 7 weeks? In vitro lipid peroxidation of liver microsomes was monitored by the formation of thiobarbituric acid (TBA)-reactive products, using both NADPH-dependent enzymatic and ascorbate/ADP-dependent nonenzymatic procedures, and by the consumption of 0, from the reaction medium. As shown in FIGURE lA, 5 mM GSH inhibited lipid peroxidation in this system for approximately 45 minutes. Interestingly, the addition of both GSH and oxidized glutathione (GSSG) resulted in a synergistic interaction that inhibited lipid peroxidation to a greater extent than the addition of GSH alone. GSSG alone did not inhibit lipid peroxidation of rat liver microsomes, and additional experiments (data not shown) revealed that GSH was oxidized to a limited extent to GSSG, whereas the conversion of GSSG to GSH was not observed. A possible explanation for this effect