Thr54 allele carriers of the Ala54Thr variant of FABP2 gene have associations with metabolic syndrome and hypertriglyceridemia in urban South Indians

The intestinal fatty acid-binding protein gene is proposed as a candidate gene for diabetes because the protein it codes is involved in fatty acid absorption and metabolism. This study investigates the association of the Ala54Thr variant of the intestinal fatty acid-binding protein gene on type 2 diabetes mellitus and other related metabolic traits in Asian Indians. Ala54Thr polymorphism was genotyped by using polymerase chain reaction-restriction fragment length polymorphism in unrelated 773 type 2 diabetic and 899 normal glucose-tolerant (NGT) subjects, randomly chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in South India. The Ala54Thr polymorphism was not associated with type 2 diabetes mellitus or obesity. However, genotype-phenotype study revealed that the NGT subjects carrying the Thr54 allele had significantly higher 2-hour plasma glucose (P = .007), glycated hemoglobin (P = .004), 2-hour insulin (P = .027), and fasting low-density lipoprotein cholesterol (P = .032) levels compared with those with the Ala54 allele. Normal glucose-tolerant subjects with Ala54Thr and Thr54Thr genotypes had significantly higher fasting serum triglyceride levels (P = .003) compared with those with Ala54Ala. The subjects were stratified into those with hypertriglyceridemia (serum triglyceride levels >or=150 mg/dL) and those without. The odds ratio for hypertriglyceridemia for the individuals carrying the Ala54Thr genotype was 1.491 (95% confidence interval [CI], 1.22-1.83, P < .0001), and for those carrying the Thr54Thr genotype, it was 1.888 (95% CI, 1.34-2.67; P < .0001). Subjects were also stratified into those with metabolic syndrome (MS) and those without, according to modified Adult Treatment Panel III guidelines. The odds ratio (adjusted for age and sex) for MS for the individuals carrying the Ala54Thr genotype was 1.240 (95% CI, 1.02-1.51; P = .03), whereas for those carrying the Thr54Thr genotype, it was 1.812 (95% CI, 1.28-2.57; P = .001). Carriers of the Thr54 allele have associations with MS and hypertriglyceridemia in this urban South Indian population.

Phenolic acid intake, delivered via moderate champagne wine consumption, improves spatial working memory via the modulation of hippocampal and cortical protein expression/activation.

Aims: While much data exist for the effects of flavonoid-rich foods on spatial memory in rodents, there are no such data for foods/beverages predominantly containing hydroxycinnamates and phenolic acids. To address this, we investigated the effects of moderate Champagne wine intake, which is rich in these components, on spatial memory and related mechanisms relative to the alcohol- and energy-matched controls. Results: In contrast to the isocaloric and alcohol-matched controls, supplementation with Champagne wine (1.78 ml/kg BW, alcohol 12.5% vol.) for 6 weeks led to an improvement in spatial working memory in aged rodents. Targeted protein arrays indicated that these behavioral effects were paralleled by the differential expression of a number of hippocampal and cortical proteins (relative to the isocaloric control group), including those involved in signal transduction, neuroplasticity, apoptosis, and cell cycle regulation. Western immunoblotting confirmed the differential modulation of brain-derived neurotrophic factor, cAMP response-element-binding protein (CREB), p38, dystrophin, 2',3'-cyclic-nucleotide 3'-phosphodiesterase, mammalian target of rapamycin (mTOR), and Bcl-xL in response to Champagne supplementation compared to the control drink, and the modulation of mTOR, Bcl-xL, and CREB in response to alcohol supplementation. Innovation: Our data suggest that smaller phenolics such as gallic acid, protocatechuic acid, tyrosol, caftaric acid, and caffeic acid, in addition to flavonoids, are capable of exerting improvements in spatial memory via the modulation in hippocampal signaling and protein expression. Conclusion: Changes in spatial working memory induced by the Champagne supplementation are linked to the effects of absorbed phenolics on cytoskeletal proteins, neurotrophin expression, and the effects of alcohol on the regulation of apoptotic events in the hippocampus and cortex. Antioxid. Redox Signal. 00, 000-000.

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

Dietary, physiological, genetic and pathological influences on postprandial lipid metabolism

Most of diurnal time is spent in a postprandial state due to successive meal intakes during the day. As long as the meals contain enough fat, a transient increase in triacylglycerolaemia and a change in lipoprotein pattern occurs. The extent and kinetics of such postprandial changes are highly variable and are modulated by numerous factors. This review focuses on factors affecting postprandial lipoprotein metabolism and genes, their variability and their relationship with intermediate phenotypes and risk of CHD. Postprandial lipoprotein metabolism is modulated by background dietary pattern as well as meal composition (fat amount and type, carbohydrate, protein, fibre, alcohol) and several lifestyle conditions (physical activity, tobacco use), physiological factors (age, gender, menopausal status) and pathological conditions (obesity, insulin resistance, diabetes mellitus). The roles of many genes have been explored in order to establish the possible implications of their variability in lipid metabolism and CHD risk. The postprandial lipid response has been shown to be modified by polymorphisms within the genes for apo A-I, A-IV, AN, E, B, C-I and C-III, lipoprotein lipase, hepatic lipase, fatty acid binding and transport proteins, microsomal trigyceride transfer protein and scavenger receptor class B type I. Overall, the variability in postprandial response is important and complex, and the interactions between nutrients or dietary or meal compositions and gene variants need further investigation. The extent of present knowledge and needs for future studies are discussed in light of ongoing developments in nutrigenetics.

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal 'Peptidase_M75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr_3370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M(W) 27,772Da). Circular dichroism spectroscopy of EfeM indicated a mainly alpha-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca++ stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2α. Since TCTP has been characterized as an antiapoptotic and Ca++-binding protein, we asked whether it is involved in protecting cells from Ca++-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2α phosphorylation.

Modulation of pro-survival Akt/protein kinase B and ERK1/2 signaling cascades by quercetin and its in vivo metabolites underlie their action on neuronal viability

Much recent interest has focused on the potential of flavonoids to interact with intracellular signaling pathways such as with the mitogen-activated protein kinase cascade. We have investigated whether the observed strong neurotoxic potential of quercetin in primary cortical neurons may occur via specific and sensitive interactions within neuronal mitogen-activated protein kinase and Akt/protein kinase B (PKB) signaling cascades, both implicated in neuronal apoptosis. Quercetin induced potent inhibition of both Akt/PKB and ERK phosphorylation, resulting in reduced phosphorylation of BAD and a strong activation of caspase-3. High quercetin concentrations (30 microM) led to sustained loss of Akt phosphorylation and subsequent Akt cleavage by caspase-3, whereas at lower concentrations (<10 microM) the inhibition of Akt phosphorylation was transient and eventually returned to basal levels. Lower levels of quercetin also induced strong activation of the pro-survival transcription factor cAMP-responsive element-binding protein, although this did not prevent neuronal damage. O-Methylated quercetin metabolites inhibited Akt/PKB to lesser extent and did not induce such strong activation of caspase-3, which was reflected in the lower amount of damage they inflicted on neurons. In contrast, neither quercetin nor its O-methylated metabolites had any measurable effect on c-Jun N-terminal kinase phosphorylation. The glucuronide of quercetin was not toxic and did not evoke any alterations in neuronal signaling, probably reflecting its inability to enter neurons. Together these data suggest that quercetin and to a lesser extent its O-methylated metabolites may induce neuronal death via a mechanism involving an inhibition of neuronal survival signaling through the inhibition of both Akt/PKB and ERK rather than by an activation of the c-Jun N-terminal kinase-mediated death pathway.

A gene variation (rs12691) in the CCAT/enhancer building α modulates glucose metabolism in metabolic syndrome

Background and aims CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor involved in adipogenesis and energy homeostasis. Caloric restriction reduces CEBPA protein expression in patients with metabolic syndrome (MetS). A previous report linked rs12691 SNP in CEBPA to altered concentration of fasting triglycerides. Our objective was to assess the effects of rs12691 in glucose metabolism in Metabolic Syndrome (MetS) patients. Methods and results Glucose metabolism was assessed by static (glucose, insulin, adiponectin, leptin and resistin plasma concentrations) and dynamic (disposition index, insulin sensitivity index, HOMA-IR and acute insulin response to glucose) indices, performed at baseline and after 12 weeks of 4 dietary interventions (high saturated fatty acid (SFA), high monounsaturated fatty acid (MUFA), low-fat and low-fat-high-n3 polyunsaturated fatty acid (PUFA)) in 486 subjects with MetS. Carriers of the minor A allele of rs12691 had altered disposition index (p = 0.0003), lower acute insulin response (p = 0.005) and a lower insulin sensitivity index (p = 0.025) indicating a lower insulin sensitivity and a lower insulin secretion, at baseline and at the end of the diets. Furthermore, A allele carriers displayed lower HDL concentration. Conclusion The presence of the A allele of rs12691 influences glucose metabolism of MetS patients. Clinical Trials Registry number NCT00429195.

Prediction and functional analysis of native disorder in proteins from the Three Kingdoms of Life

An automatic method for recognizing natively disordered regions from amino acid sequence is described and benchmarked against predictors that were assessed at the latest critical assessment of techniques for protein structure prediction (CASP) experiment. The method attains a Wilcoxon score of 90.0, which represents a statistically significant improvement on the methods evaluated on the same targets at CASP. The classifier, DISOPRED2, was used to estimate the frequency of native disorder in several representative genomes from the three kingdoms of life. Putative, long (>30 residue) disordered segments are found to occur in 2.0% of archaean, 4.2% of eubacterial and 33.0% of eukaryotic proteins. The function of proteins with long predicted regions of disorder was investigated using the gene ontology annotations supplied with the Saccharomyces genome database. The analysis of the yeast proteome suggests that proteins containing disorder are often located in the cell nucleus and are involved in the regulation of transcription and cell signalling. The results also indicate that native disorder is associated with the molecular functions of kinase activity and nucleic acid binding.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.

Small guanine nucleotide-binding proteins and myocardial hypertrophy.

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.