myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol.

myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron enzyme that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with K(m) and k(cat) values of 5.9 mM and 11 min(-1) respectively. The pI was estimated to be 4.5. Preincubation with 1 mM Fe(2+) and 2 mM cysteine was essential for the enzyme's activity. D-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated K(m) of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.

Enzyme-catalyzed and enzyme-triggered pathways in dioxygenation of 1-monolinoleoyl-rac-glycerol by potato tuber lipoxygenase.

It was shown for the first time that potato tuber lipoxygenase (ptLOX) catalyzed the aerobic oxidation of 1-monolinoleoyl-rac-glycerol (mLG) in a mixed micellar reaction solution with the non-ionic detergent monododecyl ether of decaoxyethylene glycol. No hydrolysis of mLG occurred during the reaction. The four major reaction products obtained at 23 degrees C were identified as 1-[9-hydroperoxy-10E,12Z-octadecadienoyl]-rac-glycerol (9-(E,Z)HPODE-GE, 41%), 1-[13-hydroperoxy-9Z,11E-octadecadienoyl]-rac-glycerol (13-(Z,E)-HPODE-GE, 17%), and their all-trans isomers ( approximately 21% each). The molar fraction of all-trans isomers depended on the temperature of the reaction solution; it was found that at 0 degrees C their molar fractions were approximately 15.5% each, while 9-(E,Z)HPODE-GE and 13-(Z,E)-HPODE-GE gave 42% and 27%, respectively, of the overall product. A free radical scavenger, 4-hydroxy-TEMPO, dramatically increased the molar fraction of 9-(E,Z)HPODE-GE, yielding 83% at 23 degrees C, at the expense of all other products. Chiral HPLC of 9-(E,Z)HPODE-GE formed in the presence of 4-hydroxy-TEMPO revealed that it was composed of approximately 94% S and approximately 6% (R) isomers. This assures largely a uniform orientation of mLG molecules in the ptLOX active center, with their methyl end most likely deepened into the protein globule. The second major product, 13-(Z,E)-HPODE-GE, which yielded approximately 9% of the total product formed in the presence of 4-hydroxy-TEMPO, was racemic, and so were the all-trans isomers. Therefore, the last three cannot be considered the true products of the enzyme reaction, which is known to be stereospecific. It appears that they were formed as a result of (i) leakage of the pentadienyl radicals from the ptLOX active center and their subsequent non-enzymatic dioxygenation, and/or (ii) leakage of the peroxyl radicals leading to a free radical chain reaction affording all positional, geometrical and stereoisomers of the products. This reaction resembles ptLOX oxidation of another non-ionizable substrate, linoleyl alcohol [I.A. Butovich, S.M. Luk'yanova, C.C. Reddy, Arch. Biochem. Biophys. 378 (2000) 65-77], and differed substantially from oxidation of ionizable linoleic acid. Consequently, formation of large amounts of the non-specific oxidation products might be considered a universal characteristic of ptLOX oxidation of non-ionizable compounds.

Lowering dietary saturated fat and total fat reduces the oxidative susceptibility of LDL in healthy men and women.

The present study examined the effects of reducing dietary total fat and saturated fat (SFA) on LDL oxidative susceptibility in 27 healthy men and women (age 24-65 y). Each subject consumed each of three diets for 8 wk: an average American diet (AAD, 34% energy from fat, 15% from SFA), a Step-1 diet (29% fat, 9% SFA) and a very low SFA diet (Low-Sat, 25% fat, 6% SFA). In vitro LDL oxidation was assessed by copper-mediated oxidation, as measured by the kinetics of conjugated diene formation and lipid peroxide formation. Compared with the AAD, plasma LDL-cholesterol (LDL-C) and HDL cholesterol levels were 8% lower (P: = 0.16 and P: = 0.11, respectively), in subjects when they consumed the Step-1 diet and 11% (P: < 0.03) and 14% (P: < 0.057) lower, respectively, when they consumed the Low-Sat diet. Conjugated diene production and oxidation rate were 7% (P: < 0. 05) and 9% (P: < 0.05) lower, respectively. The reduction of lipid peroxide formation was 9% (P: < 0.05) in subjects when they consumed the Low-Sat diet vs. the AAD. In addition, lipid peroxide and conjugated diene formation were positively correlated with plasma total and LDL-C and apolipoprotein B (apo B) levels (r = 0.5-0.6, P: < 0.001), suggesting that quantity of LDL is an important determinant of oxidative modification. Furthermore, at the same level of apo B or LDL-C, LDL from subjects when they consumed either Step-1 or Low-Sat diets was less susceptible (P: < 0.05) to oxidation than those when they consumed the AAD, suggesting that qualitative changes also affect LDL oxidative susceptibility. Therefore, the benefits of lowering dietary SFA may extend beyond decreasing LDL-C levels and include favorable qualitative changes in LDL that further decrease risk of coronary heart disease.

Oxidation of linoleyl alcohol by potato tuber lipoxygenase: kinetics and positional, stereo, and geometrical (cis, trans) specificity of the reaction.

The dioxygenation of linoleyl alcohol (LAL) by potato tuber lipoxygenase leads to formation of two positional isomeric products--9- and 13-hydroperoxyoctadecadien-1-ols (Butovich, I. A., Luk'yanova, S. M., and Reddy, C. C. (1998) Biochem. Biophys. Res. Commun. 249, 344-349). In the present study, we examined the stereospecificity and double-bond conformation of primary dioxygenation products of LAL catalyzed by potato lipoxygenase. In contrast to the product profiles of linoleic acid oxidation by potato lipoxygenase, oxidation of LAL led to all possible positional (9- and 13-), stereo, and geometrical (cis,trans and all-trans) isomers in equimolar mixtures at 25 degrees C. The reaction appears to proceed through an enzyme-catalyzed formation of a pentadiene carbon-centered radical followed by resonance stabilization of the radical and molecular oxygen insertion in an enzyme-dependent as well as an enzyme-independent pathway. A strict positional, stereo, and geometrical specificity of the dioxygenation products of LAL oxidation appears to be maintained when the reaction occurs at the active site of the enzyme. However, when the pentadiene carbon-centered radical of LAL is dissociated from the active site of the enzyme, it appears to be nonenzymatically transformed into a mixture of all possible positional and geometrical stereoisomers of primary dioxygenation products. The latter pathway was effectively blocked by the free radical scavenger 4-hydroxy-TEMPO, which substantially reduced the production of all-trans hydroperoxyoctadecadienols. In the presence of the scavenger, 9(S)-hydroperoxy-10E,12Z-octadecadien-1-ol was the predominant LAL oxidation product, representing approximately 80% of the total conjugated dienes, with 13(S)-hydroxy-9Z,11E-octadecadien-1-ol the expected product of reverse orientation of the substrate at the active site, accounting for approximately 10%. A similar pattern in oxidation of LAL was observed when the reactions were carried out at 0 degrees C.

Altered eicosanoid biosynthesis in selenium-deficient endothelial cells.

Selenium (Se) is an integral part of the Se-dependent glutathione peroxidase (Se-GSH-Px) catalytic domain. By modulating the cellular levels of fatty acid hydroperoxides, Se-GSH-Px can influence key enzymes of arachidonic acid cascade, in this case cyclooxygenase (COX) and lipoxygenase (LOX). To investigate this phenomenon, the effects of cellular Se status on the enzymatic oxidation of arachidonic acid were investigated in bovine mammary endothelial cells (BMEC), which were cultured in either Se-deficient (-Se) or Se-adequate (+Se) media. When stimulated with calcium ionophore A23187, BMEC produced eicosanoids of both COX and LOX pathways. Compared with the Se-adequate cells, the production of prostaglandin I(2) (PGI(2)), prostaglandin F(2) (PGF(2alpha)), and prostaglandin E(2) (PGE(2)) was significantly decreased in Se-deficient cells, whereas the production of thromboxane A(2) (TXA(2)) was markedly increased in the -Se BMEC cultures. Although the enzymatic oxidation of arachidonic acid by the LOX pathway was found to be relatively less than by the COX pathway, the BMEC cultured in -Se media produced significantly more 15-hydroperoxyeicosatetraenoic acid (15-HPETE) than the +Se cells produced. Based on these results, we postulate that cellular Se status plays an important regulatory role in the enzymatic oxidation of arachidonic acid by the COX and LOX pathways. The altered eicosanoid biosynthesis, especially the overproduction of 15-HPETE, in -Se BMEC may be one of the underlying biochemical phenomena responsible for vascular dysfunction during Se deficiency.

Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium.

Oxidation of linoleyl alcohol by potato tuber lipoxygenase: possible mechanism and the role of carboxylic group in substrate binding.

We have studied the aerobic oxidation of linoleyl alcohol (LAL) by potato tuber lipoxygenase in the presence of 0.02% (w/v) non-ionic detergent Lubrol PX (and its analog C12E10) and 0.1 mM sodium dodecyl sulfate to investigate the role of carboxylic group in substrate binding. While the enzyme displayed a comparable affinity toward LA and LAL, the rate of LAL oxidation was approximately one-fourth of that of linoleic acid. The pH-profile of the reaction suggests that the rate of LAL oxidation is controlled by two ionizable groups with pKa values of 5.3 and 7.5, with optimal pH being 6.4+/-0.1. Since LAL is not ionizable at this pH, we conclude that the rate of the reaction is controlled by two ionogenic groups of the enzyme. The primary dioxygenation product(s) of LAL had a maximal absorbance at 233+/-1 nm. The products have been isolated, catalytically hydrogenated with H2 over Pd on carbon, and analyzed by GC-MS. Two major equimolar products were found to be 9- and 13-hydroxystearyl alcohols, indicating that 9- and 13-hydroperoxylinoleyl alcohols are the primary dioxygenation products. Based on these results we propose that the carboxyl group of polyunsaturated fatty acid may not be involved in substrate binding of potato tuber lipoxygenase.

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.

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 approximately 50-fold reduction in Se-GPx1 activity and an approximately 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.

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber.

We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs. Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs. We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column. Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate. The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX. When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences.

Selenium and vitamin E deficiency impair transferrin receptor internalization but not IL-2, IL-2 receptor, or transferrin receptor expression.

Vitamin E and Se deficiency increase the risk of disease by impairing the immune response. To aid in the understanding of how vitamin E and Se deficiency reduce immune competence, this study examined several mechanisms necessary for lymphocyte proliferation. Weanling rats were fed a vitamin E-deficient, selenium-deficient, or control diet for 8 weeks. At this time splenic mononuclear cells were isolated and stimulated with concanavalin A for 48 h. Although the percentage of lymphocytes and monocytes capable of proliferating were consistent among the dietary groups, lymphocyte proliferation was decreased significantly in vitamin E- and selenium-deficient rats. This decrease in proliferation was not associated with alterations in interleukin-2, interleukin-2 receptor, or transferrin receptor expression. However, stimulated cells from vitamin E- and Se-deficient rats internalized few if any transferrin receptors. Reduced transferrin receptor internalization may limit lymphocyte expansion by depleting the intracellular iron stores needed for cellular function and proliferation.

Effects of vitamin E and selenium on antioxidant defense in rat heart.

Heart mitochondria, isolated from rats fed diets deficient or supplemented with vitamin E (E) and/or selenium (Se), were subjected to time-course assays of lipid peroxidation stimulated by ascorbate/ADP/Fe3+. Mitochondria depleted of alpha-tocopherol (alpha-TH) peroxided rapidly as assessed by formation of thiobarbituric acid reactive substances (TBARS). Formation of TBARS was strongly inhibited in mitochondria from rats fed diets supplemented with E. Selenium deficiency, reduced glutathione (GSH), glutathione disulfide (GSSG) or GSH + GSSG did not affect the course of lipid peroxidation in mitochondria from rats supplemented or deficient in E. Combined E and Se deficiency resulted in significantly lower total (oxidized+reduced) mitochondrial coenzyme Q-9 (CoQ-9) concentration compared with control rats supplemented with dietary E and Se. Time-course changes in mitochondrial alpha-TH and total CoQ-9 during oxidizing conditions were minor in +E rats. Total CoQ-9 was reduced substantially, however, during the course of lipid peroxidation in mitochondria depleted of alpha-TH. Selenium-dependent glutathione peroxidase (Se-GSHPx) activity was reduced by approximately 96% in heart cytosol, and to a somewhat lesser extent in mitochondria, by dietary Se deficiency. Non-Se GSHPx activity was not detected in heart cytosol but was detected in very small amounts in heart mitochondria. Glutathione S-transferase activity of heart cytosol was decreased in E and/or Se deficiency. The results of these experiments indicate that membrane alpha-TH was most effective in inhibiting lipid peroxidation in heart mitochondria.

Classical selenium-dependent glutathione peroxidase expression is decreased secondary to iron deficiency in rats.

While there are reports that classical selenium-dependent glutathione peroxidase (Se-GPX1) activity is decreased during iron deficiency, the relationship between tissue iron status and Se-GPX1 activity remains speculative. This study was undertaken to investigate the mechanism for the decrease in Se-GPX1 activity during iron deficiency. Male weanling Sprague-Dawley rats were given free access to either an iron-deficient or an iron-adequate diet for eight weeks, after which blood, livers, kidneys, hearts, brains and testes were surgically excised. During iron deficiency, Se-GPX1 mRNA levels in liver tissue were decreased by approximately 55%. Similarly, the concentration of immunoreactive Se-GPX1 protein and total selenium-dependent glutathione peroxidase (Se-GPX) activity were decreased by 55% and 60%, respectively. In kidney, heart and brain total Se-GPX activities were depressed as much as 33%. Selenium concentration in liver was reduced by 42%, whereas the decrease in Se concentrations in kidney, heart, and brain ranged from 17 to 25%. Concentrations of plasma Se also were reduced by 18%, but testes showed little change in either Se-GPX activity or Se concentration during iron deficiency. Results suggest that the synthesis of Se-GPX1 protein is decreased during iron deficiency possibly due to pretranslational regulation.

Dietary vitamin E and selenium effects on resistance to oxidative stress in rat liver mitochondria.

Mitochondria-rich fractions isolated from livers of rats fed diets differing in their vitamin E (E) and/or selenium (Se) contents were subjected to NADPH/ADP/Fe(3+)- dependent assays of lipid peroxidation. Addition of GSH resulted in an inhibition, or lag period, of lipid peroxidation in mitochondria from rats supplemented with E. This effect was independent of the Se status of the rats. Addition of GSH + GSSG did not potentiate the lag period over that observed with GSH alone. Significant changes in mitochondrial alpha-TH during lipid peroxidation, either in the presence or absence of GSH, were not observed. Total protein thiol (PrSH) content of native mitochondria was lower in rats fed a diet deficient in both E and Se, compared to the other dietary groups. Addition of GSH or GSH + GSSG maintained mitochondrial PrSH at higher levels during lipid peroxidation than in control assays without added GSH/GSSG. Addition of GSSG alone decreased PrSH in mitochondria prepared from all rats regardless of their E or Se status. Reduced ubiquinone-9 (U-9) and the % of total U-9 and U-10 in the reduced form were significantly decreased in liver tissue from rats fed the diet deficient in both E and Se.

Increased thromboxane A2 synthesis by rat lung neutrophils during selenium deficiency.

Modulation of cellular hydroperoxide levels is considered one of the important physiological mechanisms for regulating the synthesis of prostaglandins (PGs) and leukotrienes (LTs) in mammalian cells. Both vitamin E and selenium (Se) have the potential to affect the concentration of peroxides and, thus, the biosynthesis of eicosanoids. To gain insight into some of the molecular mechanisms underlying the regulation of the arachidonic acid cascade by vitamin E and Se, we have investigated the influence of altered vitamin E and Se nutrition on the ability of polymorphonuclear leukocytes (PMNs) derived from endotoxin-challenged lung to secrete arachidonic acid metabolites. Selenium deficiency had no significant effect (p > 0.05) on lavage fluid levels of thromboxane (TX) B2, LTB4 or LTC4. Vitamin E deficiency, however, led to a significant increase in LTB4 recovered from lavage fluid while having no effect on TXB2. In contrast, Se deficiency, although producing no discernible effects on the production of LTB4, resulted in a significant increase in the release of TXB2 by PMNs. An increase in TXB2 release was seen in both in vitro-stimulated and nonstimulated PMNs. Vitamin E deficiency appeared to induce an enhancement of LTB4 release by PMNs but the increase was not statistically significant. No detectable levels of LTC4 were found in PMN cultures stimulated with either zymosan or A23187. Thus, these studies indicate that deficiencies of either Se or vitamin E lead to alterations in the metabolism of arachidonic acid in the lung.

11-hydroperoxyeicosatetraenoic acid is the major dioxygenation product of lipoxygenase isolated from hairy root cultures of Solanum tuberosum.

The profile of primary dioxygenation products of arachidonic acid catalyzed by lipoxygenase isolated from hairy root cultures of Solanum tuberosum treated with a fungal elicitor was compared to that obtained for the enzyme from potato tubers. 11-Hydroperoxyeicosatetraenoic acid (11-HPETE) was the most abundant dioxygenation product formed followed by 8- and 5-HPETEs in the decreasing order of abundance. In contrast, 5-HPETE is the predominant oxidation product of lipoxygenase from potato tubers. Differences in the defense requirements of storage tuber as compared to roots may be the basis of the differences in regio-specificity demonstrated in this work.

Selenium deficiency alters the lipoxygenase pathway and mitogenic response in bovine lymphocytes.

We investigated the effect of altered selenium (Se) nutrition on arachidonic acid oxidation in immune cells. Experiments were conducted with peripheral blood lymphocytes obtained from dairy cattle fed diets either supplemented with or deficient in Se. The results indicate that the concanavalin A-stimulated lymphocyte proliferation was significantly lower in Se-deficient cows. When stimulated by calcium ionophore A23187, the lymphocytes derived from Se-deficient cows produced significantly less 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) than those obtained from Se-supplemented cows. When included in cell cultures from animals fed +Se diets, 5-HETE and LTB4 elicited a partial reversal of the inhibition of lymphocyte proliferation by either hydrocortisone or nordihydroguaiaretic acid. Based on this information, we postulate that dietary Se status, which in turn determines tissue Se concentration, plays an important role in the regulation of arachidonate metabolism by way of the 5-lipoxygenase pathway. This may be one of the biochemical mechanisms underlying the inhibition of lymphocyte proliferation and the decrease in resistance to infectious diseases observed in Se-deficient animals.

Active transcription of the selenium-dependent glutathione peroxidase gene in selenium-deficient rats.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, Ec.1.11.1.9) is the best characterized selenoenzyme in higher animals. However, neither the mechanism whereby selenium (Se) becomes incorporated into the enzyme nor the level at which the expression of Se-GSH-Px gene is regulated by Se is fully understood. In the current investigation, we have determined the relative rates of the transcription of the Se-GSH-Px gene in purified liver nuclei isolated from rats fed on Se-supplemented or Se-deficient diets. No significant difference in the transcription rates appeared in these two groups. These results are consistent with the previous observations that active message for Se-GSH-Px- that is, translatable mRNA for Se-GSH-Px- is present in Se-deficient tissues (Li et al., J. Biol. Chem., 265, 108-113, 1990). The data also suggest that the alteration of Se-GSH-Px activity and the corresponding protein and mRNA levels in rats subjected to dietary Se manipulation can be attributed only to post-transcriptional regulation.

Increased 12-HETE production in bovine lymphocytes during selenium deficiency.

When peripheral blood lymphocytes were incubated with arachidonic acid in the presence of Ca++ ionophore (A23187), the cells from the selenium-deficient dairy cows produced significantly greater quantities of 12-hydroxyeicosatetraenoic acid (12-HETE) than the cells from the selenium-supplemented animals. The major product of reaction was verified as 12-HETE by cochromatography with a 12-HETE standard on an HPLC and structural analysis by GC-MS. Additionally, concanavalin A-stimulated lymphocyte proliferation was significantly decreased in cells from the Se-deficient cows. Furthermore, 12-HETE generated by the A23187-stimulated lymphocytes inhibited lymphocyte proliferation when added to Se-supplemented cell cultures. These observations suggest that self-regulation of lymphocyte proliferation might be mediated by 12-HETE production, especially during an altered nutritional state such as Se deficiency.

Dietary selenium effects on milk eicosanoid concentration in dairy cows during coliform mastitis.

The effect of selenium deficiency on the product profile of arachidonic acid oxidation by enzymatic pathways in Holstein cows with experimentally-induced coliform mastitis was investigated. The animals were fed dairy rations containing 0.05 mg Se/kg dry matter, with the supplemented group receiving additional Se to increase the dietary concentration to approximately 0.35 mg Se/kg dry matter. Cows were inoculated intracisternally with 30 colony-forming-units of Escherichia coli at 14-16 weeks of lactation. Eicosanoids and bacteria numbers were recorded at various intervals of time for 60 h postinoculation. Milk from cows fed the Se-depleted diet had significantly higher (p less than 0.05) concentrations of TXB2 between 24 and 48 h and 6-keto-PGF1 alpha between 24 and 60 h postinoculation. Milk PGE2 concentration was significantly higher in the Se-deficient group at 24 h, whereas LTB4 was higher between 36 and 60 h postinoculation in the Se-deficient cows (p less than 0.05). Milk bacteria numbers were significantly higher between 16 and 24 h postinoculation in the Se-deficient group and three of the four cows in this group required euthanasia, whereas all four cows in the Se-supplemented group recovered without therapeutic intervention. These data indicate marked effects of dietary Se on milk eicosanoid concentrations in response to an E. coli infection. The changes in eicosanoid concentrations may be associated with the altered pathogenesis and outcome of mastitis in a Se-deficient state.