Interactions of Ly49 family receptors with MHC class I ligands in trans and cis.

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.

Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Analysis of the contribution of the beta-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae.

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Functional analysis of cis- and trans-acting elements of the Candida albicans CDR2 promoter with a novel promoter reporter system.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5'-CGGAWATCGGATATTTTTTT-3', and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P((CDR2))-HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.

cis- and trans-acting elements involved in reactivation of vaccinia virus early transcription.

We have previously shown that transcription from the vaccinia virus 7.5K early promoter is reactivated late in infection (J. Garcés, K. Masternak, B. Kunz, and R. Wittek, J. Virol. 67:5394-5401, 1993). To identify the sequence elements mediating reactivation, we constructed recombinant viruses harboring deletions, substitutions, or insertions in the 7.5K promoter or its flanking regions. The analysis of these viruses showed that sequences both upstream as well as downstream of the transcription initiation site contribute to reactivation of the 7.5K promoter. We tested whether reactivation could be explained by a high affinity of vaccinia virus early transcription factor to reactivated promoters. Bandshift experiments using purified protein showed that promoters which bind the factor with high affinity in general also have high early transcriptional activity. However, no correlation was found between affinity of the factor and reactivation. Interestingly, overexpression of recombinant early transcription factor in vaccinia virus-infected cells resulted in a shutdown of late transcription and in reactivation of promoters, which are normally not reactivated.

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin promoter in vitro.

Alterações estruturais in vivo dos isômeros todo-trans, 9-cis e 13-cis do beta-caroteno

Com o objetivo de verificar alterações estruturais nos isômeros todo-trans, 9- e 13-cis do beta-caroteno foi realizado um ensaio biológico baseado no modelo de esgotamento das reservas hepáticas de carotenóides em ratos. Animais depletados desses carotenóides receberam, durante quinze dias, os isômeros puros todo-trans, 9-cis e 13-cis do beta-caroteno. Ao final deste período, verificou-se a ocorrência de re-isomerização in vivo desses isômeros, a partir da quantificação dos mesmos depositados no fígado dos animais. Foi observada re-isomerização do 9-cis em todo-trans, do todo-trans em 9-cis, do 13-cis em 9-cis e todo-trans. O 13-cis foi mais susceptível à isomerização que o 9-cis, pois este último passou a todo-trans e nunca a 13-cis. Já o 13-cis, tanto pode se transformar em 9-cis quanto em todo-trans.

Efecto de la mezcla de ácido linoleico conjugado,cis-9, trans-11 y trans-10, cis-12, en la respuesta a la insulina en un modelo experimental diabetizado con dieta hipercalórica.

Tesis (Maestría en Ciencias en Nutrición) UANL, 2012.

The infrared vibration-rotation spectrum of trans and cis nitrous acid

Infrared spectra of the trans and the cis isomers of nitrous acid, both HONO and DONO, have been observed in the gas phase using a Fourier transform interferometer with a resolution of about 0.05 cm−1 from 4000 to 500 cm−1. Rotational analyses are reported on eleven of the fundamentals and some overtones.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.

Unexpectedly superior enantioselectivity for trans-stilbene cis-dihydroxylation over anchored triosmium carbonyl species in confined Al-MCM-41 channels

Superior enantioselectivity in the dihydroxylation of trans-stilbene catalysed by anchored triosmium carbonyl species without using a chiral modifier is observed inside sterically congested MCM-41 channels; this effect is more pronounced through the introduction of surface Al sites into the silicate.

Opposing effects of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid on blood lipids in healthy humans

Background: Conjugated linoleic acid (CLA) is reported to have weight-reducing and antiatherogenic properties when fed to laboratory animals. However, the effects of CLA on human health and, in particular, the effects of individual CLA isomers are unclear. Objective: This study investigated the effects of 3 doses of highly enriched cis-9,trans-11 (0.59, 1.19, and 2.38 g/d) or trans-10,cis-12 (0.63, 1.26, and 2.52 g/d) CLA preparations on body composition, blood lipid profile, and markers of insulin resistance in healthy men. Design: Healthy men consumed 1, 2, and 4 capsules sequentially, containing either 80% cis-9,trans-11 CLA or 80% trans-10,cis-12 CLA for consecutive 8-wk periods. This phase was followed by a 6-wk washout and a crossover to the other isomer. Results: Body composition was not significantly affected by either isomer of CLA. Mean plasma triacylglycerol concentration was higher during supplementation with trans-10,cis-12 CLA than during that with cis-9,trans-11 CLA, although there was no influence of dose. There were significant effects of both isomer and dose on plasma total cholesterol and LDL-cholesterol concentrations but not on HDL-cholesterol concentration. The ratios of LDL to HDL cholesterol and of total to HDL cholesterol were higher during supplementation with trans-10,cis-12 CLA than during that with cis-9,trans-11 CLA. CLA supplementation had no significant effect on plasma insulin concentration, homeostasis model for insulin resistance, or revised quantitative insulin sensitivity check index. Conclusion: Divergent effects of cis-9,trans-11 CLA and trans10,cis-12 CLA appear on the blood lipid profile in healthy humans: trans-10,cis-12 CLA increases LDL:HDL cholesterol and total:HDL cholesterol, whereas cis-9,trans-11 CLA decreases them.

Effects of cis-9,trans-11 and trans-1 0,cis-12 conjugated linoleic acid on immune cell function in healthy humans

Background: Animal studies have suggested that conjugated linoleic acid (CLA), a natural component of ruminant meat and dairy products, may confer beneficial effects on health. However, little information on the effects of CLA on immune function is available, especially in humans. Furthermore, the effects of individual isomers of CLA have not been adequately investigated. Objective: This study investigated the effects of supplementing the diet with 3 doses of highly enriched cis-9,trans-11 CLA (0.59, 1.19, and 2.38 g/d) or trans-10,cis-12 CLA (0.63, 1.26, and 2.52 g/d) on immune outcomes in healthy humans. Design: The study had a randomized, double-blind, crossover design. Healthy men consumed 1, 2, and 4 capsules sequentially that contained 80% of either cis-9,trans-11 CLA or trans-10,cis-12 CLA for consecutive 8-wk periods. This regimen was followed by a 6-wk washout and a crossover to the other isomer. Results: Both CLA isomers decreased mitogen-induced T lymphocyte activation in a dose-dependent manner. There was a significant negative correlation between mitogen-induced T lymphocyte activation and the proportions of both cis-9,trans-11 CLA and trans-10,cis-12 CLA in peripheral blood mononuclear cell lipids. However, CLA did not affect lymphocyte subpopulations or serum concentrations of C-reactive protein and did not have any consistent effects on ex vivo cytokine production. Conclusion: CLA supplementation results in a dose-dependent reduction in the mitogen-induced activation of T lymphocytes. The effects of cis-9,trans-l I CLA and trans-10,cis-12 CLA were similar, and there was a negative correlation between mitogen-induced T lymphocyte activation and the cis-9,trans-11 CLA and trans-10,cis-12 CLA contents of mononuclear cells.

Effects of dairy products naturally enriched with cis-9,trans-11 conjugated linoleic acid on the blood lipid profile in healthy middle-aged men

Background: Interest in the development of dairy products naturally enriched in conjugated linoleic acid (CLA) exists. However, feeding regimens that enhance the CLA content of milk also increase concentrations of trans-18:1 fatty acids. The implications for human health are not yet known. Objective: This study investigated the effects of consuming dairy products naturally enriched in cis-9,trans-11 CLA (and trans-11 18:1) on the blood lipid profile, the atherogenicity of LDL, and markers of inflammation and insulin resistance in healthy middle-aged men. Design: Healthy middle-aged men (n = 32) consumed ultra-heat-treated milk, butter, and cheese that provided 0.151 g/d (control) or 1.421 g/d (modified) cis-9,trans-11 CLA for 6 wk. This was followed by a 7-wk washout and a crossover to the other treatment. Results: Consumption of dairy products enriched with cis-9,trans-11 CLA and trans-11 18:1 did not significantly affect body weight, inflammatory markers, insulin, glucose, triacylglycerols, or total, LDL, and HDL cholesterol but resulted in a small increase in the ratio of LDL to HDL cholesterol. The modified dairy products changed LDL fatty acid composition but had no significant effect on LDL particle size or the susceptibility of LDL to oxidation. Overall, increased consumption of full-fat dairy products and naturally derived trans fatty acids did not cause significant changes in cardiovascular disease risk variables, as may be expected on the basis of current health recommendations. Conclusion: Dairy products naturally enriched with cis-9,trans-11 CLA and trans-11 18: 1 do not appear to have a significant effect on the blood lipid profile.