Are prostatic biopsies necessary in men aged > or =80 years?

OBJECTIVE: To examine whether prostatic biopsies are necessary in all men aged > or =80 years, as men found to have prostate cancer are frequently treated with a 'watch and wait' policy or with hormonal withdrawal alone, and biopsies are associated with a small but significant complication rate. PATIENTS AND METHODS: The findings on a digital rectal examination (DRE), the prostate-specific antigen (PSA) level, the biopsy and staging bone scan results for all men aged > or = 80 years who had prostatic biopsies over a 3-year period were reviewed, together with those in a group of men aged <80 years for comparison. All biopsy samples had been examined in one of three histopathology units, and 33 consultant urological surgeons contributed. RESULTS: In all, 210 biopsies from 205 men aged > or = 80 years were identified, of whom 163 (79%) had biopsy-confirmed prostate cancer. All 29 men with a PSA level of > or = 100 ng/mL, 98% of 47 with > or = 50 ng/mL, 97% of 76 with > or = 30 ng/mL and 92% of 101 with > or = 20 ng/mL had biopsy cores containing cancer; 63% of men with a PSA level of <20 ng/mL had cancer on biopsy. In men with cancer and a PSA level of > or = 30 ng/mL, 92% had Gleason grade > or = 7 and 93% were treated with hormonal withdrawal alone. In all men with cancer the DRE was abnormal in 91%, the mean number of positive cores was 59% and the bone scan was positive in 18%. The DRE was abnormal in 77% of men with benign biopsies. CONCLUSIONS: In men aged > or = 80 years with a PSA level of > or = 30 ng/mL, at least 97% had prostate cancer, >90% of whom had high-grade disease, and nearly all with cancer received active pharmacological treatment. In the vast majority of these men prostate biopsies did not alter their cancer management. The value of prostatic biopsy in this age group, with a PSA level of > or = 30 ng/mL, is questionable.

Open-air-nesting honey bees Apis dorsata and Apis laboriosa differ from the cavity-nesting Apis mellifera and Apis cerana in brood hygiene behaviour.

The cavity-nesting Apis mellifera and Apis cerana bees detect, uncap, and remove diseased brood. The hygiene behaviour of open-air-nesting bees Apis dorsata and Apis laboriosa was investigated in India and Nepal. Sealed A. dorsata pupae were pin-killed or deep-frozen. The workers removed 73 or 37% of damaged pin-killed pupae depending on the diameter of the pins, and only 7% of the frozen undamaged pupae. Migrating A. dorsata and A. laboriosa left unopened the sealed brood in deserted combs. Thus, A. dorsata and A. laboriosa do not open undamaged cells with dead brood. This behaviour is a more efficient mechanism in preventing the spread of diseases and parasitic mites than uncapping and removing dead pupae by A. mellifera and A. cerana. It may be beneficial for migrating A. dorsata and A. laboriosa to temporarily disuse part of the comb cells in exchange for arresting the mites there and thus reducing the increase of their population.

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.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

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.

Comparative mitogenic potencies of EGF and TGF alpha and their dependence on receptor-limitation versus ligand-limitation.

Transforming growth factor alpha (TGF alpha) has been reported to be a more potent agonist when compared to epidermal growth factor (EGF) in several systems while acting via their common receptor, the epidermal growth factor receptor (EGFR). It has been postulated that this increased potency is mediated by the increased recycling of EGFR upon activation by TGF alpha as against receptor activation by EGF. The authors test this hypothesis by simultaneously measuring mitogenesis and the dynamics of surface receptor number in response to these ligands in NR6 mouse fibroblasts expressing the EGFR. The data demonstrates that increased receptor recycling due to endosomal dissociation of TGF alpha can indeed realise an increased mitogenic potency relative to EGF under appropriate cellular and experimental conditions (i.e. situations in which the increase in the number of occupied receptors due to receptor sparing by TGF alpha represents additional mitogenic signalling capacity). However, this difference in receptor trafficking does not uniquely determine the relative potencies of these ligands since TGF alpha is a less potent mitogen compared to EGF when experimental conditions are dominated by the effects of ligand trafficking on growth factor availability. Thus, the relative potencies of these growth factors are determined in a given context by the relative importance of ligand and receptor trafficking effects which determine the availability of these signalling components. These results are consistent with a suggested model of hormone responsiveness which favours dissociative ligands (such as TGF alpha) in receptor-limited situations and non-dissociative ligands (such as EGF) in the case of ligand limitation.

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.

Isolation and characterization of an enzyme from sheep seminal vesicles that catalyzes the glutathione-dependent reduction of prostaglandin H2 to prostaglandin F2 alpha.

An enzyme which catalyzes the direct 2-electron reduction of prostaglandin H2 to prostaglandin F2 alpha has been purified from the microsomes of sheep seminal vesicles. This enzyme, called prostaglandin endoperoxide reductase, was found to be a monomer of 16,500 molecular mass. The activity of the enzyme was dependent on reduced glutathione, enhanced by heat-treatment, and inhibited by sulfhydryl reagents. The enzyme is not a glutathione S-transferase nor does it utilize prostaglandin D2 as a substrate, and thus is distinct from previously characterized prostaglandin F2 alpha biosynthetic enzymes. The protein also catalyzes the reduction of cumene hydroperoxide, but not hydrogen peroxide. Thus, this microsomal prostaglandin endoperoxide reductase may play an important role in the synthesis of prostaglandin F2 alpha in some tissues.

Glutathione-dependent factors and inhibition of rat liver microsomal lipid peroxidation.

The effects of reduced glutathione (GSH) and glutathione disulfide (GSSG) on lipid peroxidation were investigated in rat liver microsomes containing deficient or adequate amounts of alpha-tocopherol (alpha-TH). Rates of formation of thiobarbituric acid reactive substances (TBARS) as well as rates of consumption of alpha-TH and O2 were decreased by GSH and were more pronounced in the NADPH-dependent assay system than in the ascorbate-dependent system. The GSH-dependent inhibition of lipid peroxidation was potentiated by GSSG in the NADPH-dependent assay system, but it had no effect in the nonenzymatic system. Diphenyliodonium chloride, an inhibitor of NADPH cytochrome P-450 reductase, completely prevented lipid peroxidation in the NADPH-dependent assay system whereas it had no effect on the ascorbate-dependent system. This is further evidenced by the fact that purified rat liver microsomal NADPH cytochrome P-450 reductase (EC 1.6.2.4) was inhibited approximately 24% and 52% by 5 mM GSH and 5 mM GSH + 2.5 mM GSSG, respectively. Glutathione disulfide alone had no effect on reductase activity. Similarly, other disulfides such as cystine, cystamine and lipoic acid were without effect on reductase activity. These results clearly delineate different mechanisms underlying the combined effects of GSH and GSSG on microsomal lipid peroxidation in rat liver. One mechanism involves recycling of microsomal alpha-TH by GSH during oxidative stress via a labile protein, ostensibly associated with "free radical reductase" activity. A second glutathione-dependent mechanism appears to be mediated through the inhibition of NADPH cytochrome P-450 reductase. The enhanced inhibition by GSH + GSSG of microsomal lipid peroxidation in the NADPH-dependent assay system suggests suppression of the initiation phase at the level of NADPH cytochrome P-450 reductase which is independent of microsomal alpha-TH.

Evidence for G-protein-dependent and G-protein-independent activation of phospholipase D in lymphocytes.

Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independent of protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys. Res. Commun. 171, 955-962, 1990; 217, 908-915, 1995). In the present study, we examined the effects of prostagladins (PGs), E2, F2 alpha, D2, and H2 on PLD activity as measured by conversion of [1-14C] arachidonic acid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of PEt at an optimal concentration of 10 microM with relative stimulatory effect on the order of PGE2 > PGF2 alpha > PGH2 > PGD2. The PGE2-stimulated formation of PEt was dose-dependent in the range of 0.1 to 10 microM and was not inhibited by PKC inhibitors staurosporine and K252a. When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLD activation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly, the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF and TPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent of PKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways.

Engineering epidermal growth factor for enhanced mitogenic potency.

Successful use of growth factors in therapeutic and bioprocessing applications requires overcoming two attenuation mechanisms: growth factor depletion and receptor down-regulation. Current ameliorative strategies use physiologically inappropriate high growth-factor concentrations, along with periodic media refeeding in vitro and reinjection or controlled-release devices in vivo. We demonstrate a new approach derived from understanding how these attenuation mechanisms arise from ligand/receptor trafficking processes. Specifically, a recombinant epidermal growth factor (EGF) mutant with reduced receptor binding affinity is a more potent mitogenic stimulus for fibroblasts than natural EGF or transforming growth factor alpha because of its altered trafficking properties.