PPARγ2 gene Pro12Ala and PPARα gene Leu162Val single nucleotide polymorphisms interact with dietary intake of fat in determination of plasma lipid concentrations

Background/Aims: The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of lipid metabolism, activated by unsaturated fatty acids. We investigated independent and interactive effects of PPARγ2 gene PPARG Pro12Ala (rs1801282) andPPARαgene PPARA Leu162Val (rs1800206) genotypes with dietary intake of fatty acids on concentrations of plasma lipids in subjects of whom 47.5% had metabolic syndrome. Methods: The RISCK study is a parallel design, randomised controlled trial. Plasma lipids were quantified at baseline after a 4-week high saturated fatty acids diet and after three parallel 24-week interventions with reference (high saturated fatty acids), high monounsaturated fatty acids and low-fat diets. Single nucleotide polymorphisms were genotyped in 466 subjects. Results: At baseline, the PPARG Ala12allele was associated with increased plasma total cholesterol (n = 378; p = 0.04), LDL cholesterol (p = 0.05) and apoB (p =0.05) after adjustment for age, gender and ethnicity. At baseline, PPARA Leu162Val × PPARG Pro12Ala genotype interaction did not significantly influence plasma lipid concentrations. After dietary intervention, gene-gene interaction significantly influenced LDL cholesterol (p =0.0002) and small dense LDL as a proportion of LDL (p = 0.005) after adjustments. Conclusions: Interaction between PPARG Pro12Ala and PPARA Leu162Valgenotypes may influence plasma LDL cholesterol concentration and the proportion as small dense LDL after a high monounsaturated fatty acids diet.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Elevated atmospheric carbon dioxide impairs the performance of root-feeding vine weevils by modifying root growth and secondary metabolites

Predicting how insect crop pests will respond to global climate change is an important part of increasing crop production for future food security, and will increasingly rely on empirically based evidence. The effects of atmospheric composition, especially elevated carbon dioxide (eCO(2)), on insect herbivores have been well studied, but this research has focussed almost exclusively on aboveground insects. However, responses of root-feeding insects to eCO(2) are unlikely to mirror these trends because of fundamental differences between aboveground and belowground habitats. Moreover, changes in secondary metabolites and defensive responses to insect attack under eCO(2) conditions are largely unexplored for root herbivore interactions. This study investigated how eCO(2) (700 mu mol mol-1) affected a root-feeding herbivore via changes to plant growth and concentrations of carbon (C), nitrogen (N) and phenolics. This study used the root-feeding vine weevil, Otiorhynchus sulcatus and the perennial crop, Ribes nigrum. Weevil populations decreased by 33% and body mass decreased by 23% (from 7.2 to 5.4 mg) in eCO(2). Root biomass decreased by 16% in eCO(2), which was strongly correlated with weevil performance. While root N concentrations fell by 8%, there were no significant effects of eCO(2) on root C and N concentrations. Weevils caused a sink in plants, resulting in 8-12% decreases in leaf C concentration following herbivory. There was an interactive effect of CO(2) and root herbivory on root phenolic concentrations, whereby weevils induced an increase at ambient CO(2), suggestive of defensive response, but caused a decrease under eCO(2). Contrary to predictions, there was a positive relationship between root phenolics and weevil performance. We conclude that impaired root-growth underpinned the negative effects of eCO(2) on vine weevils and speculate that the plant's failure to mount a defensive response at eCO(2) may have intensified these negative effects.

The Glu298Asp single nucleotide polymorphism in the endothelial nitric oxide synthase gene differentially effects the vascular response to acute consumption of fruit and vegetable puree-based drinks

Scope Diets low in fruits and vegetables (FV) are responsible for 2.7 million deaths from cardiovascular diseases (CVD) and certain cancers annually. Many FV and their juices contain flavonoids, some of which increase endothelial nitric oxide synthase (eNOS) activity. A single nucleotide polymorphism in the eNOS gene, where thymine (T) replaces guanine (G) at position 894 predicting substitution of glutamate for aspartate at codon 298 (Glu298Asp), has been associated with increased CVD risk due to effects on nitric oxide synthesis and subsequently vascular reactivity. Individuals can be homozygous for guanine (GG), thymine (TT) or heterozygous (GT). Methods and results We investigated the effects of acute ingestion of a FV-puree-based-drink (FVPD) on vasodilation and antioxidant status in subjects retrospectively genotyped for this polymorphism. Healthy volunteers (n = 24; 11 GG, 11 GT, 2 TT) aged 30–70 were recruited to a randomized, controlled, crossover, acute study. We showed that acute consumption of 400 mL FVPD differentially affected individuals depending on their genotype. There was a significant genotype interaction for endothelium-dependent vasodilation measured by laser Doppler imaging with iontophoresis (P < 0.05) and ex vivo low-density lipoproteins (LDL) oxidation (P = 0.002). GG subjects had increased endothelium-dependent vasodilation 180 min (P = 0.028) and reduced ex vivo LDL oxidation (P = 0.013) after 60 min after FVPD compared with control, no differences were observed in GT subjects. Conclusion eNOS Glu298Asp genotype differentially affects vasodilation and ex vivo LDL oxidation after consumption of FV in the form of a puree-based drink.

In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites

With the aim of investigating the potential of flavan-3-ols to influence the growth of intestinal bacterial groups, we have carried out the in vitro fermentation, with human faecal microbiota, of two purified fractions from grape seed extract (GSE): GSE-M (70% monomers and 28% procyanidins) and GSE-O (21% monomers and 78 % procyanidins). Samples were collected at 0, 5, 10, 24, 30 and 48 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and for analysis of phenolic metabolites. Both GSE-M and GSE-O fractions promoted growth of Lactobacillus/Enterococcus and decrease in the Clostridium histolyticum group during fermentation, although the effects were only statistically significant with GSE-M for Lactobacillus/Enterococcus (at 5 and 10 h of fermentation) and GSE-O for C. histolyticum (at 10 h of fermentation). Main changes in polyphenol catabolism also occurred during the first 10 h of fermentation, however no significant correlation coefficients (P>0.05) were found between changes in microbial populations and precursor flavan-3-ols or microbial metabolites. Together these data suggest that the flavan-3-ol profile of a particular food source could affect the microbiota composition and its catabolic activity, inducing changes that could in turn affect the bioavailability and potential bioactivity of these compounds.

The impact of obesity-related single nucleotide polymorphisms on appetite and energy intake: a pilot study

An increasing number of studies have reported a heritable component for the regulation of energy intake and eating behaviour, although the individual polymorphisms and their ‘effect size’ are not fully elucidated. The aim of the present study was to examine the relationship between specific SNP and appetite responses and energy intake in overweight men. In a randomised cross-over trial, forty overweight men (age 32 (sd 09) years; BMI 27 (sd 2) kg/m2) attended four sessions 1 week apart and received three isoenergetic and isovolumetric servings of dairy snacks or water (control) in random order. Appetite ratings were determined using visual analogue scales and energy intake at an ad libitum lunch was assessed 90 min after the dairy snacks. Individuals were genotyped for SNP in the fat mass and obesity-associated (FTO), leptin (LEP), leptin receptor (LEPR) genes and a variant near the melanocortin-4 receptor (MC4R) locus. The postprandial fullness rating over the full experiment following intake of the different snacks was 17·2 % (P= 0·026) lower in A carriers compared with TT homozygotes for rs9939609 (FTO, dominant) and 18·6 % (P= 0·020) lower in G carriers compared with AA homozygotes for rs7799039 (LEP, dominant). These observations indicate that FTO and LEP polymorphisms are related to the variation in the feeling of fullness and may play a role in the regulation of food intake. Further studies are required to confirm these initial observations and investigate the ‘penetrance’ of these genotypes in additional population subgroups.

APOA5 genotype influences the association between 25-hydroxyvitamin D and high density lipoprotein cholesterol

OBJECTIVE: Circulating levels of 25-hydroxyvitamin D (25OHD) are positively associated with high density lipoprotein (HDL) cholesterol. We sought to replicate a previously reported interaction between APOA5 genotype and vitamin D, and to examine whether HDL-associated genetic loci modify the association between serum 25OHD and HDL cholesterol. METHODS: We examined whether 42 single nucleotide polymorphisms (SNPs) modify the association between serum 25OHD and HDL cholesterol in the 1958 British Birth cohort (aged 45 years, n = 4978). RESULTS: We identified a borderline interaction between the SNP rs12272004 (near the APOA5) and serum 25OHD on HDL cholesterol (P(interaction) = 0.05). The interaction was particularly prominent among the samples collected during winter (P(interaction) = 0.001). None of the other loci showed an interaction with serum 25OHD concentrations on HDL cholesterol. CONCLUSIONS: Our study in 4978 British Whites provides further support that APOA5 genotype modifies the association between vitamin D metabolites and HDL cholesterol.

Analyses of single nucleotide polymorphisms in selected nutrient-sensitive genes in weight-regain prevention: the DIOGENES study

BACKGROUND: Differences in the interindividual response to dietary intervention could be modified by genetic variation in nutrient-sensitive genes. OBJECTIVE: This study examined single nucleotide polymorphisms (SNPs) in presumed nutrient-sensitive candidate genes for obesity and obesity-related diseases for main and dietary interaction effects on weight, waist circumference, and fat mass regain over 6 mo. DESIGN: In total, 742 participants who had lost ≥ 8% of their initial body weight were randomly assigned to follow 1 of 5 different ad libitum diets with different glycemic indexes and contents of dietary protein. The SNP main and SNP-diet interaction effects were analyzed by using linear regression models, corrected for multiple testing by using Bonferroni correction and evaluated by using quantile-quantile (Q-Q) plots. RESULTS: After correction for multiple testing, none of the SNPs were significantly associated with weight, waist circumference, or fat mass regain. Q-Q plots showed that ALOX5AP rs4769873 showed a higher observed than predicted P value for the association with less waist circumference regain over 6 mo (-3.1 cm/allele; 95% CI: -4.6, -1.6; P/Bonferroni-corrected P = 0.000039/0.076), independently of diet. Additional associations were identified by using Q-Q plots for SNPs in ALOX5AP, TNF, and KCNJ11 for main effects; in LPL and TUB for glycemic index interaction effects on waist circumference regain; in GHRL, CCK, MLXIPL, and LEPR on weight; in PPARC1A, PCK2, ALOX5AP, PYY, and ADRB3 on waist circumference; and in PPARD, FABP1, PLAUR, and LPIN1 on fat mass regain for dietary protein interaction. CONCLUSION: The observed effects of SNP-diet interactions on weight, waist, and fat mass regain suggest that genetic variation in nutrient-sensitive genes can modify the response to diet. This trial was registered at clinicaltrials.gov as NCT00390637.

Expression of the Rap1 guanine nucleotide exchange factor, MR-GEF, is altered in individuals with bipolar disorder

In the rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1 and Dlx2. The Rap-1 guanine nucleotide exchange factor, MR-GEF, is turned on by many of these developing GABAergic neurons. Expression of both Dlx1/2 and MR-GEF is retained in both adult mouse and human forebrain where, in human, decreased Dlx1 expression has been associated with psychosis. Using in situ hybridization studies we show that MR-GEF expression is significantly down-regulated in the forebrain of Dlx1/2 double mutant mice suggesting that MR-GEF and Dlx1/2 form part of a common signalling pathway during GABAergic neuronal development. We therefore compared MR-GEF expression by in situ hybridization in individuals with major psychiatric disorders (schizophrenia, bipolar disorder, major depression) and control individuals. We observed a significant positive correlation between layers II and IV of the dorso-lateral prefrontal cortex (DLPFC) in the percentage of MR-GEF expressing neurons in individuals with bipolar disorder, but not in individuals with schizophrenia, major depressive disorder or in controls. Since MR-GEF encodes a Rap1 GEF able to activate G-protein signalling, we suggest that changes in MR-GEF expression could potentially influence neurotransmission.

Expression of the guanine nucleotide exchange factor, mr-gef, is regulated during the differentiation of specific subsets of telencephalic neurons

We have performed a screen combining subtractive hybridization with PCR to isolate genes that are regulated when neuroepithelial (NE) cells differentiate into neurons. From this screen, we have isolated a number of known genes that have not previously been associated with neurogenesis, together with several novel genes. Here we report that one of these genes, encoding a guanine nucleotide exchange factor (GEF), is regulated during the differentiation of distinct neuronal populations. We have cloned both rat and mouse GEF genes and shown that they are orthologs of the human gene, MR-GEF, which encodes a GEF that specifically activates the small GTPase, Rap1. We have therefore named the rat gene rat mr-gef (rmr-gef) and the mouse gene mouse mr-gef (mmr-gef). Here, we will collectively refer to these two rodent genes as mr-gef. Expression studies show that mr-gef is expressed by young neurons of the developing rodent CNS but not by progenitor cells in the ventricular zone (VZ). The expression pattern of mr-gef during early telencephalic neurogenesis is strikingly similar to that of GABA and the LIM homeobox gene Lhx6, a transcription factor expressed by GABAergic interneurons generated in the ventral telencephalon, some of which migrate into the cortex during development. These observations suggest that mr-gef encodes a protein that is part of a signaling pathway involved in telencephalic neurogenesis; particularly in the development of GABAergic interneurons.

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-Kinase and cyclic nucleotide signaling

OBJECTIVE: Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. APPROACH AND RESULTS: Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. CONCLUSIONS: This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis. '

Top single nucleotide polymorphisms affecting carbohydrate metabolism in Metabolic Syndrome: from the LIPGENE study

Rationale:Metabolic Syndrome (MetS) is a high prevalence condition characterized by altered energy metabolism, insulin resistance and elevated cardiovascular risk.Objectives:Although many individual single nucleotide polymorphisms (SNPs) have been linked to certain MetS features, there are few studies analyzing the influence of SNPs on carbohydrate metabolism in MetS.Methods:904 SNPs (tag SNPs and functional SNPs) were tested for influence in eight fasting and dynamic markers of carbohydrate metabolism, performing an intravenous glucose tolerance test in 450 participants of the LIPGENE study.Findings:From 382 initial gene-phenotype associations between SNPs and any phenotypic variables, 61 (a 16 % of the pre-selected) remained significant after Bootstrapping. Top SNPs affecting glucose metabolism variables were as follows: fasting glucose: rs26125 (PPARGC1B); fasting insulin: rs4759277 (LRP1); C peptide: rs4759277 (LRP1); HOMA-IR: rs4759277 (LRP1); QUICKI: rs184003 (AGER); SI: rs7301876 (ABCC9), AIRg: rs290481 (TCF7L2) and DI: rs12691 (CEBPA).Conclusions:We describe here the top SNPs linked to phenotypic features in carbohydrate metabolism among aproximately 1000 candidate gene variations in fasting and postprandial samples of 450 patients with MetS from the LIPGENE study.

Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.

Single nucleotide polymorphism rs6716901 in SLC25A12 gene is associated with Asperger syndrome

BACKGROUND: Autism Spectrum Conditions (ASC) are a group of developmental conditions which affect communication, social interactions and behaviour. Mitochondrial oxidative dysfunction has been suggested as a mechanism of autism based on the results of multiple genetic association and expression studies. SLC25A12 is a gene encoding a calcium-binding carrier protein that localizes to the mitochondria and is involved in the exchange of aspartate for glutamate in the inner membrane of the mitochondria regulating the cytosolic redox state. rs2056202 SNP in this gene has previously been associated with ASC. SNPs rs6716901 and rs3765166 analysed in this study have not been previously explored in association with AS. METHODS: We genotyped three SNPs (rs2056202, rs3765166, and rs6716901) in SLC25A12 in n?=?117 individuals with Asperger syndrome (AS) and n?=?426 controls, all of Caucasian ancestry. RESULTS: rs6716901 showed significant association with AS (P?=?0.008) after correcting for multiple testing. We did not replicate the previously identified association between rs2056202 and AS in our sample. Similarly, rs3765166 (P?=?0.11) showed no significant association with AS. CONCLUSION: The present study, in combination with previous studies, provides evidence for SLC25A12 as involved in the etiology of AS. Further cellular and molecular studies are required to elucidate the role of this gene in ASC.