Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae

The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly theJ _max value of the high-affinity system of the mutant was 25–35 % of the correspondingJ _max value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.     

Proton extrusion inSaccharomyces cerevisiae mutants in very dilute suspensions

Substrate-induced H⁺ extrusion was studied in dilute (0.04–0.5 mg dry mass per mL) unbuffered suspensions ofS. cerevisiae. Wild-type strains 196-2 and K and 196-2-derived mutants altered in hexose transport, glucosephosphate isomerase, mannosephosphate isomerase, pyruvate kinase, and arho petite mutant were characterized as to growth, biochemical and H⁺-pumping properties. Their H⁺ extrusion differed, depending on strain, growth conditions, and the H⁺-efflux-inducing substrate; the efficiency of the process depended critically on the balance between substrate uptake, its dissimilation, attendant mobilization of energy sources and build-up of acidity sources in the cell, and the energy supply to H⁺excreting systems.     

Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Comprehensive genotyping of the USA national maize inbred seed bank

Background

Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world.

Results

The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits.

Conclusions

The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity.     

Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

Chronic Administration of Proanthocyanidins or Docosahexaenoic Acid Reversess the Increase of miR-33a and miR-122 in Dyslipidemic Obese Rats

miR-33 and miR-122 are major regulators of lipid metabolism in the liver, and their deregulation has been linked to the development of metabolic diseases such as obesity and metabolic syndrome. However, the biological importance of these miRNAs has been defined using genetic models. The aim of this study was to evaluate whether the levels of miR-122 and miR-33a in rat liver correlate with lipemia in nutritional models. For this purpose, we analyzed the levels of miRNA-33a and miR-122 in the livers of dyslipidemic cafeteria diet-fed rats and of cafeteria diet-fed rats supplemented with proanthocyanidins and/or ω-3 PUFAs because these two dietary components are well-known to counteract dyslipidemia. The results showed that the dyslipidemia induced in rats that were fed a cafeteria diet resulted in the upregulation of miR-33a and miR-122 in the liver, whereas the presence of proanthocyanidins and/or ω-3 PUFAs counteracted the increase of these two miRNAs. However, srebp2, the host gene of miR-33a, was significantly repressed by ω-3 PUFAs but not by proanthocyanidins. Liver mRNA levels of the miR-122 and miR-33a target genes, fas and pparβ/δ, cpt1a and abca1, respectively, were consistent with the expression of these two miRNAs under each condition. Moreover, the miR-33a and abca1 levels were also analyzed in PBMCs. Interestingly, the miR-33a levels evaluated in PBMCs under each condition were similar to the liver levels but enhanced. This demonstrates that miR-33a is expressed in PBMCs and that these cells can be used as a non-invasive way to reflect the expression of this miRNA in the liver. These findings cast new light on the regulation of miR-33a and miR-122 in a dyslipidemic model of obese rats and the way these miRNAs are modulated by dietary components in the liver and in PBMCs.     

Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be shed in the semen of infected boars. To determine whether the reproductive tissues could be a persistent source of virus and the possible origin of PRRSV found in semen of infected boars, 20 PRRSV-seronegative boars were intranasally inoculated with 5 x 10(6) median tissue culture infective doses (TCID50) of PRRSV and necropsied at different times post-inoculation (p.i.) from Day 2 to Day 37 p.i. Blood samples were collected before experimental inoculation, at necropsy and at different times p.i. At necropsy, epididymal semen and reproductive tissues were collected and the presence of the virus determined by virus isolation. The infection of the boars was demonstrated by the isolation of the virus from the sera of all inoculated boars and by seroconversion. PRRSV was detected in serum samples from Day 2 to Day 23 p.i., although the viremic period was largely dependent on the individual response to infection. Viral replication was proven within different reproductive tissues from Day 2 to Day 23 p.i., being most consistently found in the epididymus. In addition, PRRSV was isolated in semen from Day 4 to Day 10 p.i. The correlation of a diminished viremia and the inability to isolate PRRSV from semen or reproductive tissues may be due to one of two possibilities. First, viremia is responsible for most of the virus isolated from reproductive tissues due to the movement of PRRSV-infected cells out of the blood and into the tissues. Second, viremia may initially seed the reproductive tissues with PRRSV, and then the virus is produced into the reproductive tract and shed into semen at low levels.     

Lipogenesis Is Decreased by Grape Seed Proanthocyanidins According to Liver Proteomics of Rats Fed a High Fat Diet

Bioactive proanthocyanidins have been reported to have several beneficial effects on health in relation to metabolic syndrome, type 2 diabetes, and cardiovascular disease. We studied the effect of grape seed proanthocyanidin extract (GSPE) in rats fed a high fat diet (HFD). This is the first study of the effects of flavonoids on the liver proteome of rats suffering from metabolic syndrome. Three groups of rats were fed over a period of 13 weeks either a chow diet (control), an HFD, or a high fat diet supplemented for the last 10 days with GSPE (HFD + GSPE). The liver proteome was fractionated, using a Triton X-114-based two-phase separation, into soluble and membrane protein fractions so that total proteome coverage was considerably improved. The data from isobaric tag for relative and absolute quantitation (iTRAQ)-based nano-LC-MS/MS analysis revealed 90 proteins with a significant (p < 0.05) minimal expression difference of 20% due to metabolic syndrome (HFD versus control) and 75 proteins due to GSPE treatment (HFD + GSPE versus HFD). The same animals have previously been studied (Quesada, H., del Bas, J. M., Pajuelo, D., Díaz, S., Fernandez-Larrea, J., Pinent, M., Arola, L., Salvadó, M. J., and Bladé, C. (2009) Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver. Int. J. Obes. 33, 1007–1012), and GSPE was shown to correct dyslipidemia observed in HFD-fed rats probably through the repression of hepatic lipogenesis. Our data corroborate those findings with an extensive list of proteins describing the induction of hepatic glycogenesis, glycolysis, and fatty acid and triglyceride synthesis in HFD, whereas the opposite pattern was observed to a large extent in GSPE-treated animals. GSPE was shown to have a wider effect than previously thought, and putative targets of GSPE involved in the reversal of the symptoms of metabolic syndrome were revealed. Some of these novel candidate proteins such as GFPT1, CD36, PLAA (phospholipase A₂-activating protein), METTL7B, SLC30A1, several G signaling proteins, and the sulfide-metabolizing ETHE1 and SQRDL (sulfide-quinone reductase-like) might be considered as drug targets for the treatment of metabolic syndrome.An increase in high calorie diets and a sedentary lifestyle are considered the key factors in explaining the epidemic rise in obesity in developed countries (1). Obese patients, especially those with abdominal obesity due to visceral adipose tissue accumulation, run a higher risk of impaired glucose tolerance, which frequently evolves into insulin resistance (2). Obesity and insulin resistance are frequently associated with hypertension, proatherogenic dyslipidemia, chronic inflammation, a prothrombotic state, and recently also fatty liver (3), conditions that together make up what is known as metabolic syndrome and lead to an increased risk of developing cardiovascular disease (CVD)¹ and type 2 diabetes (4). Conversely, some dietary patterns and specific food components have been associated with a lower prevalence of obesity, type 2 diabetes, and CVD. In this sense, the traditional Mediterranean diet (characterized by a high fiber content, low glycemic index carbohydrates, unsaturated fats, vitamins, and antioxidant polyphenols) has been linked to a lower incidence of CVD, obesity, and type 2 diabetes (5–8). Moreover, the French population presents a very low prevalence of death due to CVD despite consuming a diet rich in saturated fats and cholesterol. This phenomenon, known as “the French paradox” (9), has been ascribed to the moderate consumption of red wine and specifically to its content of polyphenols (10–12).Polyphenols include flavonoids of which flavan-3-ols and their oligomeric forms (proanthocyanidins) have been reported to exhibit several beneficial health effects by acting as antioxidant, anticarcinogen, cardioprotective, antimicrobial, antiviral, and neuroprotective agents (for a review, see Ref. 13). Specifically, grape and wine proanthocyanidins have a cardioprotective effect through increasing plasma high density lipoprotein cholesterol, decreasing low density lipoprotein-derived atherosclerotic foam cell lesions, attenuating oxidant formation by quenching harmful radicals, increasing endothelium-dependent vasorelaxation, etc. (13). In this context, our group has been working for years on the effect of a grape seed proanthocyanidin extract (GSPE) (containing monomers and oligomers of flavan-3-ols) in relation to metabolic syndrome. In previous works, we have found that GSPE prevents oxidative injury (14), has an insulinomimetic effect on adipocytes and adipose tissue (15), modulates glucose homeostasis (16), decreases plasma levels of triglycerides (TGs) and apolipoprotein B in normolipidemic rats (17), and acts as an in vitro (18, 19) and in vivo (20) anti-inflammatory. We have also shown that GSPE decreases postprandial plasma TG and apolipoprotein B in mice through a hepatic induction of a farnesoid X receptor (FXR) and the small heterodimer partner (SHP) that in turn down-regulates SREBP1c and other lipogenic genes in the liver (21, 22). Furthermore, we have demonstrated that the molecules responsible for the reduced TG synthesis in HepG2 cells treated with GSPE are the sum of a proanthocyanidins trimer and a dimer gallate because they reproduce the GSPE effect (23).The effect of GSPE on metabolic syndrome has been studied in our laboratory by feeding rats a “cafeteria diet.” This diet is an experimental model of a western high sugar and high fat diet extensively used to produce obesity in rats because its palatability induces the animals to increase their energy intake (24). In a recent study conducted by our group (25) as well as this study, the rats were fed a high fat diet (HFD) (cafeteria diet) for 13 weeks, and one group of the animals was treated with a daily dose of GSPE (25 mg/kg of body weight) for the last 10 days (HFD + GSPE). In that study, HFD was shown to cause the animals to be overweight and to suffer from fatty liver, dyslipidemia, and hepatic overexpression of key genes involved in lipogenesis and VLDL assembly, whereas GSPE treatment corrected dyslipidemia and down-regulated some of the genes up-regulated by HFD (25).To better investigate the mechanism behind the changes observed in HFD- and HFD + GSPE-fed rats, we analyzed protein expression in the liver. Because GSPE treatment and obesity have multiple effects, a proteome-wide approach is needed to map proteins from different pathways. Proteomics studies related to obesity, metabolic syndrome, fatty liver, or insulin resistance have previously been performed on the liver (26–32). Two such studies looked into the effects of flavonoids in mouse livers (33, 34), but to our knowledge, this is the first hepatic proteome analysis of the effect of flavonoids in rats suffering from metabolic syndrome. To improve the proteome coverage of the complex liver samples, we performed a proteome fractionation according to protein solubility using a two-phase detergent protocol (35). This strategy was advantageous because it captured membrane proteins that otherwise would have been difficult to detect. The resulting soluble and membrane protein fractions were digested, iTRAQ-labeled, fractionated according to isoelectric point, and analyzed by nano-LC-MS/MS. The proteomics study presented here reports a differential expression due to HFD or HFD + GSPE for approximately 140 proteins, indicating that both conditions were potent modifiers of the liver proteome. We have focused on the sugar and lipid metabolism data, which confirmed the repression of hepatic lipogenesis in HFD + GSPE rats. Additionally, new proteins have been revealed as putative GSPE targets.