Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Orosomucoid (alpha-1 acid glycoprotein) phenotyping by use of immobilized pH gradients with 8 M urea and immunoblotting. A new variant encountered in a population study.

Orosomucoid (ORM) phenotyping has been performed on 329 unrelated Swiss subjects, using immobilized pH gradients with 8 M urea and 2% v/v 2-mercaptoethanol followed by immunoblotting. After desialylation the band patterns of ORM confirmed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles (ORM1*F1, ORM1*S and ORM1*F2). One rare and one new allele were detected. The rare variant, tentatively assigned to the second structural locus ORM2, is observed in a cathodal position and named ORM2 B1. The new variant, tentatively assigned to the first structural locus ORM1, is observed in a region located between ORM1 S and ORM1 F2, and named ORM1 F3. Moreover, the pI values of the ORM variants have been measured accurately with Immobiline Dry Plates (LKB): they were found to be within the pH range 4.93-5.14.

Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

1C.06: Ambulatory pulse pressure is negatively associated with excretions of urinary caffeine and its metabolites

OBJECTIVE: Systolic blood pressure (BP) has been associated with urinary caffeine and its metabolites such as paraxanthine and theophylline. Caffeine and caffeine metabolites could influence arterial pulse pressure (PP) via sympathomimetic effects, smooth muscle relaxation, and phosphodiesterase inhibition. The purpose of this analysis was to explore the association of ambulatory PP with urinary caffeine and its related metabolites in a large population-based sample. DESIGN AND METHOD: Families were randomly selected from the general population of three Swiss cities (2009-2013). Ambulatory BP monitoring was conducted using validated Diasys Integra devices. PP was defined as the difference between the systolic and diastolic ambulatory BP. Urinary caffeine, paraxanthine, theophylline, and theobromine excretions were measured in 24 h urine using ultra-high performance liquid chromatography tandem mass spectrometry. Urinary excretions were log-transformed to satisfy regression assumptions. We used linear mixed models to explore the associations of urinary caffeine and caffeine metabolite excretions with 24-hour, day- and night-time PP while adjusting for major confounders. RESULTS: The 836 participants (48.9% men) included in this analysis had mean (±SD) age of 47.8 (±17.5), and mean 24-hour systolic and diastolic BP of 120.1 mmHg (±13.9) and 78.0 (±8.6). Except theobromine, log transformed urinary caffeine and caffeine metabolite excretions were associated negatively with 24-hour, daytime and night-time ambulatory PP. 24-hour, daytime, and night-time ambulatory PP decreased by -0.804 mmHg (SE, 0.209), -0.749 (0.215), and -0.968 (0.243) (all P values <0.005), for each doubling excretion of caffeine. Strong negative associations with night-time ambulatory PP were observed for paraxanthine and theophylline.(Figure is included in full-text article.) CONCLUSIONS: : The negative associations of PP with caffeine, paraxanthine, and theophylline excretions suggest that caffeine and its metabolites do lower BP, possibly by modifying arterial stiffness.

Moderately oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on the oxidative stability of rabbit plasma, liver, and meat

The aim of this study was to assess the alterations in plasma, liver, and meat oxidative stability and R-tocopherol content when moderately oxidized sunflower oils were added to feeds and when feeds were supplemented with R-tocopheryl acetate (100 mg/kg) and Zn (200 mg/kg). The effects of cooking the meat and its subsequent refrigeration were also studied. When the content of primary oxidation compounds of the oil was high, rabbit plasma, liver, and meat R-tocopherol content was reduced and meat susceptibility to oxidation increased. The addition of oil with a high content of secondary oxidation compounds (oil heated at 140 'C, 31 h) to feed also led to an increase in meat susceptibi- lity to oxidation, although it presented an R-tocopherol content similar to that of nonheated oil. Feed supplementation with R-tocopheryl acetate increased tissue R-tocopherol content and improved the oxidative stability of liver and meat. However, in the latter, it was less effective when oil heated at 55 'C was added.

Oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on rabbit plasma, liver and meat fatty acid composition and meat Zn, Cu, Fe and Se content

The effects of the addition of heated oils to feeds (3%, w/w) and the dietary supplementation with a-tocopheryl acetate (TA; 100 mg/kg) and Zn (200 mg/kg) on rabbit tissue fatty acid (FA) composition and on the Zn, Cu, Fe and Se content in meat were assessed. Heating unrefined sunflower oil (SO) at 558C for 245 h increased its content in primary oxidation products and reduced its a-tocopherol content. However, this did not significantly affect tissue FA composition. Heating SO at 1408C for 31 h increased its content in secondary oxidation products and in some FA isomers asc9,t11-CLA and di-trans CLA. This led to increases in di-trans CLA in liver and in t9,c12-18:2 in meat. The c9,t11-CLA was the most incorporated CLA isomer in tissues. The dietary supplementation with a-TA did not affect the FA composition of plasma, liver or meat. The cooking of vacuum-packed rabbit meat at 788C for 5 min reduced significantly but slightly its polyunsaturated FA content. The dietary supplementation with Zn did not modify the content of Zn, Fe or Se in meat, but it reduced its Cu content. On the other hand, it increased the content of some FAs in meat when SO heated at 1408C for 31 h was added to feeds.