Selective and potent inhibition of different hepatic UDP-glucuronosyltransferase activities by omega,omega,omega-triphenylalcohols and UDP derivatives.

A homologous series of omega,omega,omega-triphenylalcohols and corresponding omega,omega,omega-triphenylalkyl-UDP derivatives was synthesized and tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes, with 1-naphthol, testosterone and bilirubin as substrates. Introduction of the UDP moiety in the triphenylalcohols increased their inhibition potency markedly toward the isoforms which glucuronidate 1-naphthol and testosterone, but strongly decreased that toward bilirubin. The inhibiting potency of the UDP-derivatives increased as a function of the length of the hydrocarbon chain. The best inhibitor 7,7,7-triphenylheptyl-UDP showed an I50 of 30 and 10 microM for 1-naphthol and testosterone glucuronidation, respectively; even a 1 mM concentration of the compound had little, if any, effect on bilirubin glucuronidation. The inhibition by 7,7,7-triphenylheptyl-UDP was mixed-type toward 1-naphthol, and non competitive toward testosterone (apparent K(i) 30 microM and 1.7 microM, respectively); on the other hand, the inhibition was competitive toward the common substrate UDP-glucuronic acid (apparent K(i) 1.9-1.2 microM). In addition, 7,7,7-triphenylheptyl-UDP (0.25-0.50 mM) almost inhibited glucuronidation of 1-naphthol and testosterone catalyzed by the recombinant rat liver UGT-2B1 and human liver UGT-1A1, whose cDNA has been expressed in V79 cells. In conclusion, the data indicate that 7,7,7-triphenyheptyl-UDP interacted competitively with the UDP binding site of UGT. The results also indicate that it is possible to design transition state analogue inhibitors with specificity for different UGT forms.     

Assessment of essential fatty acid and omega3-fatty acid status by measurement of erythrocyte 20:3omega9 (Mead acid), 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3

BACKGROUND: Early suspicion of essential fatty acid deficiency (EFAD) or omega3-deficiency may rather focus on polyunsaturated fatty acid (PUFA) or long-chain PUFA (LCP) analyses than clinical symptoms. We determined cut-off values for biochemical EFAD, omega3-and omega3/22:6omega3 [docosahexaenoic acid (DHA)]-deficiency by measurement of erythrocyte 20:3omega9 (Mead acid), 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3, respectively. METHODS: Cut-off values, based on 97.5 percentiles, derived from an apparently healthy omnivorous group (six Dominica breast-fed newborns, 32 breast-fed and 27 formula+LCP-fed Dutch low-birth-weight infants, 31 Jerusalem infants, 33 Dutch 3.5-year-old infants, 69 omnivorous Dutch adults and seven Dominica mothers) and an apparently healthy group with low dietary LCP intake (81 formula-fed Dutch low-birth-weight infants, 12 Dutch vegans). Cut-off values were evaluated by their application in an EFAD suspected group of 108, mostly malnourished, Pakistani children, three pediatric patients with chronic fat-malabsorption (abetal-ipoproteinemia, congenital jejunal and biliary atresia) and one patient with a peroxisomal beta-oxidation disorder. RESULTS: Erythrocyte 20:3omega9, 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3 proved age-dependent up to 0.2 years. Cut-off values for ages above 0.2 years were: 0.46mol% 20:3omega9 for EFAD, 0.068mol/mol 22:5omega6/20:4omega6 for omega3-deficiency, 0.22mol/mol 22:5omega6/22:6omega3 for omega3/DHA-marginality and 0.48mol/mol 22:5omega6/22:6omega3 for omega3/DHA-deficiency. Use of RBC 20:3omega9 and 22:5omega6/20:4omega6 cut-off values identified 20.4% of the Pakistani subjects as EFAD+omega3-deficient, 12.9% as EFAD+omega3-sufficient, 38.9% as EFA-sufficient+omega3-deficient and 27.8% as EFA-sufficient+omega3-sufficient. The patient with the peroxisomal disorder was classified as EFA-sufficient, omega3-sufficient (based on RBC 22:5omega6/20:4omega6) and omega3/DHA-deficient (based on RBC 22:5omega6/22:6omega3). The three other pediatric patients were classified as EFAD, omega3-deficient and omega3/DHA-deficient. CONCLUSION: Use of the combination of the present cut-off values for EFA, omega3 and omega3/DHA status assessment, as based on 97.5 percentiles, may serve for PUFA supplement intervention until better concepts have emerged.     

Selective utilization of omega 6 and omega 3 polyunsaturated fatty acids by human skin fibroblasts

Incorporation of exogenous [14C] arachidonate by human skin fibroblasts was found to be significantly greater than that of either [14C]linoleate or alpha-[14C] linolenate. Arachidonate was preferentially esterified in the PI + PS and PE classes of phospholipids. Over 40% of the incorporated [14C] arachidonate was chain elongated in 24 hours. Cells were also grown in lipid-free medium to enhance PUFA desaturation and elongation and the utilization of various omega 6 and omega 3 metabolites examined. Whereas [14C] linoleate partitioned approximately 50:50 between PL and TAG, eicosatrienoate (20:3 omega 6) was selectively sequestered in TAG. Arachidonate and docosatetraenoate (22:4 omega 6) were preferentially incorporated into phospholipids; the PI + PS fraction was most highly enriched with arachidonate. Modification of alpha-[14C] linolenate was more extensive than that of [14C] linoleate. Docosapentaenoate (22:5 omega 3) was the major omega 3 [14C] PUFA of PI + PS and PE. Eicosapentaeonate was not selectively incorporated into phospholipids; within phospholipids the 20:5 omega 3 was primarily in PC. These results indicate that human skin fibroblasts exhibit acyl specificity in the esterification of polyunsaturated fatty acids, including preferential utilization of arachidonate rather than other prostaglandin precursors in the PI + PS fraction.     

Long-chain omega 3 fatty acids, blood lipids and cardiovascular risk reduction

Increasing evidence suggests that omega 3 fatty acids derived from fish and fish oils may play a protective role in coronary heart disease and its many complications, through a variety of actions, including effects on lipids, blood pressure, cardiac and vascular function, prostanoids, coagulation and immunological responses. Interesting differences between the effects of highly purified eicosapentaenoic acid and docosahexaenoic acid are emerging, which may be relevant in the choice of omega 3 fatty acid for incorporation into food products. On the basis of our current knowledge, we believe it is justified to recommend, particularly to high-risk populations, an increased dietary intake of omega 3 fatty acids through the consumption of fish.     

Whole-body beta-oxidation of 18:2omega6 and 18:3omega3 in the pig varies markedly with weaning strategy and dietary 18:3omega3

Segregated early weaning (SEW) into a cleaner nursery increases food intake and growth in pigs, presumably because of reduced immune stimulation compared with conventionally reared, nonsegregated pigs (NSW). The aim of the present study was to evaluate the oxidation of linoleic acid (18:2omega6) and alpha-linolenic acid (18:3omega3) in SEW and NSW pigs. Pigs consumed a control or high 18:3omega3 diet (omega6 PUFA/omega3 PUFA; 21.3 vs. 2.5, respectively) and were weaned at either 14 days old into a SEW nursery or at 21 days old into a conventional NSW nursery. The major acute-phase protein of pigs but not haptoglobin increased in 35-day-old NSW pigs. NSW pigs had 15-25% lower carcass 18:2omega6 and 20-30% lower carcass 18:3omega3 (% composition) at 49 days old. Between 35- and 49-days-old, NSW pigs had a higher whole-body oxidation of 18:2omega6 (40-120%) and 18:3omega3 (30-80%). The high 18:3omega3 diet decreased the whole-body oxidation of 18:2omega6 by 73% and of 18:3omega3 by 63% in NSW pigs. We conclude that moderately cleaner housing SEW significantly decreases 18:2omega6 and 18:3omega3 oxidation in pigs.     

The role of omega6 to omega3 ratio in development and progression of age-related macular degeneration

The aim of this study is to investigate possible connection between omega-6/omega-3 fatty acid ratio and development and progression of Age-Related Macular Degeneration (ARMD). We examined 125 patients diagnosed with ARMD and divided into 5 groups of 25 patients according to CARMS (Clinical Age-Related Maculopathy Staging System). Control group consists of 51 patients with similar ages, without ARMD. All of them underwent stereobiomicroscopy, fundus photography and fluorescein angiography. Dietary fatty acids intake was measured using food frequency questionnaire (FFQ). The FFQ was based on previously validated questionnaire (DIETQ, Tinuviel Software, Warington, Ches, UK) and FFQ2 from Blue MountainEye Study. The data were analysed using food nutritient dana from McCance and Widdowson's Food Composition Tables, supplemented with a food fatty acid content database (Foodbase, London, UK). We noticed statistically significant difference between omega-6/omega-3 ratio in neovascular ARMD (stage 5) and all other groups including control group (p = 0.000020). The ratio in Stage 5 was about 11:1 like in Western diet. Stage 4-geographic atrophy (GA) has statistically significant difference in o-mega-6/omega-3 ratio compared with stage 1 (p = 0.000571), stage 2 (p = 0.000112) and stage3 (p = 0.000430). The ratio in first three groups is about 7-7.5:1 (greater then Mediteran-4-5:1, but lower then Western Diet-10-20:1). There is no statistically significant difference between first three stages (p > 0.05) and stage 4 and control group (p = 0.172388). Omega-6/omega-3 ratio is connected with development of neovascular ARMD. Decreased ratio protects against neovascular ARMD. On the contrary, GA seems to be connected with prolonged sunlight exposure (the ratio is about 6:1). It is good to know that changing nutrition habits someone can prevent development of severe neovascular form of ARMD because intravitreal anti-VEGF therapy limitations.     

Human interferon omega (omega) binds to the alpha/beta receptor.

It was proposed that human interferon omega (omega) binds to the interferon alpha/beta receptor but not to the interferon gamma receptor. However, since no studies were performed to provide direct evidence for this hypothesis, we carried out cross-linking experiments and saturation binding assays between a 32P-labeled human interferon-alpha (Hu-IFN-alpha) and unlabeled Hu-IFN-alpha A, -beta, -gamma, and -omega. These assays demonstrated that Hu-IFN-alpha A, -beta, and -omega, but not Hu-IFN-gamma, were able to block binding of 32P-labeled Hu-IFN-alpha A to human cells. These results indicate that Hu-IFN-omega binds to the alpha/beta receptor.     

Protein synthesis and degradation in isolated muscle. Effect of omega 3 and omega 6 fatty acids.

The ability of derivatives of the essential fatty acids linoleic acid (C18:2, omega 6) and alpha-linolenic acid (C18:3, omega 3) to stimulate rates of protein synthesis and degradation was investigated in isolated intact muscles from fasted rabbits. Both omega 6 derivatives examined, arachidonic acid (C20:4, omega 6) and dihomo-gamma-linolenic acid (C20:3, omega 6), when added at concentrations up to 1 microM, stimulated the rate of protein synthesis and the release of prostaglandin F2 alpha (PGF2 alpha). Metabolites of the omega 6 series, namely eicosapentaenoic acid (C20:5, omega 3) and docosahexaenoic acid (C22:6, omega 3), were without effect on the rate of protein synthesis and resulted in a decrease in the release of PGF2 alpha. None of the fatty acids had a significant effect on the rate of protein degradation. Although insulin (100 mu units/ml) also stimulated rates of protein synthesis when added alone, none of the omega 3 or omega 6 fatty acids, when added with insulin at concentrations of 0.2 microM, potentiated the effect of the hormone.     

Deuterium isotope effects and product studies for the oxidation of N(omega)-allyl-L-arginine and N(omega)-allyl-N(omega)-hydroxy-L-arginine by neuronal nitric oxide synthase

The nitric oxide synthases (NOS), which require heme, tetrahydrobiopterin, FMN, FAD, and NADPH, catalyze the O2-dependent conversion of L-arginine to L-citrulline and nitric oxide. N(omega)-Allyl-L-arginine, a mechanism-based inactivator of neuronal NOS, also is a substrate, producing L-arginine, acrolein, and H2O (Zhang, H. Q.; Dixon, R. P., Marletta, M. A.; Nikolic, D.; Van Breemen, R.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119, 10888). Two possible mechanisms for this turnover are proposed, one initiated by allyl C-H bond cleavage and the other by guanidino N H cleavage, and these mechanisms are investigated with the use of N(omega)-allyl-L-arginine (1), N(omega)-[1,1-(2)H2]allyl-L-arginine (7), N(omega)-allyl-N(omega)-hydroxy-L-arginine (2) and N(omega)-[1,1-(2)H2]allyl-N(omega)-hydroxy-L-arginine (8) as substrates. Significant isotope effects on the two kinetic parameters, kcat and kcat/Km, are observed in case of 1 and 7 during turnover, but not with 2 and 8. No kinetic isotope effects are observed for either compound in their role as inactivators. These results support a mechanism involving initial C-H bond cleavage of N(omega)-allyl-L-arginine followed by hydroxylation and breakdown to products.     

Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein.

Escherichia coli omega protein is found to form a complex with single-stranded DNA. The complex is stable in buoyant CsCl or Cs2SO4 density gradients. Addition of Mg(II) to the concentrated salt solutions, however, leads to the dissociation of the complex, even in the presence of EDTA in molar excess over Mg(II). The dissociated omega retains its enzymatic activity; the DNA recovered from the dissociated complex is indistinguishable from the original DNA. Exposure of the complex to alkali results in the cleavage of the DNA. This cleavage generates a 3'-hydroxyl DNA terminus, and the omega protein is found linked to the 5'-terminus, presumably covalently. Pronase digestion of the complex results initially in the removal of approximately 30% of the protein. A significant fraction of the residual complex is still stable in concentrated salt solutions, and can be dissociated by Mg(II). Extensive digestion with pronase results in the removal of the protein and the cleavage of the DNA chain.     

Preliminary crystallographic data for protease omega

Protease omega from Carica papaya L. has been purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12), with a = 7.42 +/- 0.02 nm, c = 7.79 +/- 0.02 nm with one molecule in the asymmetric unit. The crystals diffract to 0.19-nm resolution using synchrotron radiation.     

A catalogue of phi, psi and omega torsion angles for tetrapeptides in proteins

This work examined the degree of consistency, over multiple occurrences in several proteins, of conformations of tetrapeptides. A data base of 114 proteins was taken from the Brookhaven Data Bank. The number of occurrences of every tetrapeptide in these proteins was counted. For each tetrapeptide which occurred more than 6 times all phi, psi and omega angles were calculated and averaged over the occurrences. Due to the limitations of the data base there were only 24 tetrapeptides in this category. This does not provide sufficient information for the extensive prediction of structures which are not homologous to those already in the data base but the effectiveness of structure prediction can be expected to increase with the size of the data base.     

Mechanisms of chain folding in nucleic acids. The (omega, omega) plot and its correlation to the nucleotide geometry in yeast tRNAPhe1.

The (omega', omega) polot depicting the internucleotide P-O bond rotation angles in yeast phenylalanyl transfer RNA has established the interdependence of the phosphodiesters and the nucleotide geometries in the folding of the polynucleotide backbone. The plot distinguishes the regions characteristic of secondary helical structures and tertiary structural loops and bends. The folding of the polynucleotide chain is accomplished either solely by rotations around the P-O bonds or in concert with rotations around the nucleotide C4'-C5' bond with or without changes in the sugar ring pucker. In spite of differences in nucleotide sequence and intraloop tertiary interactions in the anticodon and pseudouridine loops, a characteristic repeating structural unit is found for the sugar-phosphate backbone of the tetranucleotide segment around the sharp turns.     

Interaction between DNA and an Escherichia coli protein omega

An E. coli protein, designated ω, has been purified at least 1000-fold. Treatment of a eovalently closed DNA duplex containing negative superhelical turns with ω results in the loss of most of the superhelical turns. The loss of superhelical turns follows a gradual course rather than a one-hit mechanism. This reaction does not require a cofactor. No other change in the physical properties of the DNA could be detected. The DNA remains covalently closed. Its ultraviolet absorption spectrum, circular dichroism, buoyant density in CsCl, sedimentation properties in neutral media containing varying amounts of ethidium and in an alkaline medium, and its susceptibility toward Neurospora endonuclease, are not significantly different from an untreated DNA containing the same number of superhelical turns. Thus it appears that ω is capable of introducing a “swivel” reversibly into a DNA. A plausible mechanism is postulated.     

Tocopherol esters inhibit human glutathione S-transferase omega

Human glutathione S-transferase omega 1-1 (hGSTO1-1) is a newly identified member of the glutathione S-transferase (GST) family of genes, which also contains alpha, mu, pi, sigma, theta, and zeta members. hGSTO1-1 catalyzes the reduction of arsenate, monomethylarsenate (MMA(V)), and dimethylarsenate (DMA(V)) and exhibits thioltransferase and dehydroascorbate reductase activities. Recent evidence has show that cytokine release inhibitory drugs, which specifically inhibit interleukin-1b (IL-1b), directly target hGSTO1-1. We found that (+)-alpha-tocopherol phosphate and (+)-alpha-tocopherol succinate inhibit hGSTO1-1 in a concentration-dependent manner with IC50 values of 2 microM and 4 microM, respectively. A Lineweaver-Burk plot demonstrated the uncompetitive nature of this inhibition. The molecular mechanism behind the inhibition of hGSTO1-1 by alpha-tocopherol esters (vitamin E) is important for understanding neurodegenerative diseases, which are also influenced by vitamin E.     

Effects of dietary omega3 and omega6 lipids and vitamin E on chemokine levels in autoimmune-prone MRL/MpJ-lpr/lpr mice

Elevated levels of chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), Monocyte Chemotactic Protein-1 (MCP-1), Macrophage Inflammatory Protein-1alpha (MIP-1alpha), and Macrophage Inflammatory Protein-1beta (MIP-1beta) have been found in rheumatoid arthritis (RA) and juvenile arthritis (JA), and they may be associated with the pathogenesis of these diseases. These chemokines are implicated in the migration of specific leukocytes into the joints. Omega-3 (omega3) fatty acid rich-fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study was designed to understand the effects of dietary lipids (omega-6 and omega-3 fatty acids) and vitamin E on the production of chemokines in autoimmune-prone MRL/lpr (a mouse model for RA) and congenic control MRL/++ mice. The MRL mice were fed for 4.5 months omega-6 and omega-3 diets that varied in lipid sources (corn oil; CO and fish oil; FO) and vitamin E levels (269 I.U./kg and 694 I.U./kg diet). Spleen cells were isolated and cultured aseptically in the presence of PHA for 48 h at 37 degrees C and the levels of chemokines (RANTES, JE/MCP-1 and MIP-1alpha) were determined in the cell-free supernatants. The levels of RANTES and JE/MCP-1 were significantly higher in MRL/lpr mice compared to MRL/++ mice. The FO had differential effect on RANTES and MCP-1 production by spleen cells. The production of RANTES and JE/MCP-1 by spleen cells in mice fed the FO diets was significantly lower than in mice fed the CO diets (p < 0.0001). The levels of vitamin E did not affect the production of RANTES and JE/MCP-1. The levels of vitamin E had a significant effect on MIP-1alpha as the spleen cells of mice fed diets containing 694 IU/kg diet of vitamin E produced significantly higher levels of MIP-1alpha compared to the group of mice fed the diets containing 269 IU of vitamin E (p < 0.0001). The data obtained from this study in MRL/lpr and MRL/++ mice suggest that FO diets containing omega-3 fatty acids are beneficial in decreasing the levels of certain pro-inflammatory chemokines (RANTES and MCP-1) thereby delaying the onset of and severity of autoimmune symptoms in MRL/lpr mouse model.