Gene Expression Of Related Enzymes Research Articles

Storage Temperature Affects the Accumulation of Phenolics in Fresh-Cut Melon by Accelerating the Sucrose Decomposition

To accurately evaluate the role of storage temperature in improving the quality of fresh-cut fruits and vegetables, the effects of two storage temperatures (5 °C and 15 °C) on the phenylpropanoid pathway and sucrose metabolism in fresh-cut melon (cv. Yugu) cubes were determined. A higher temperature (15 °C) expedited sucrose decomposition in the melon cubes at the early stage of storage, resulting in higher levels of glucose and fructose. This effect was corroborated by increased activities of acid invertase (AI), neutral invertase (NI), and sucrose synthase cleavage (SS-c), along with higher expressions of CmAI1/2, CmNI1/2, and CmSS1/2 in the melon cubes at 15 °C. Additionally, the higher activity and gene expression of hexokinase in melon cubes at 15 °C led to an increase in the utilization rate of sugars toward downstream metabolic pathways. Moreover, the melon cube storage at 15 °C elevated the activities and gene expressions of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumaric acid: CoA ligase (4CL), thereby increasing the synthesis of phenolics. Sucrose showed a significant negative correlation with PAL, C4H, and 4CL, as well as with CmPAL5/7 and CmC4H1/3. However, hexokinase displayed a significant positive correlation with PAL, C4H, and 4CL, as well as with CmPAL1, CmPAL3-9, CmC4Hs, and Cm4CLs. These findings demonstrate that a higher-temperature storage of melon cubes can accelerate the phenylpropanoid pathway and sucrose metabolism by regulating the activity and gene expression of related enzymes, thereby inducing phenolic accumulation. These results also indicate that lower-temperature storage is not conducive to the conversion of sugars into phenolics in fresh-cut melon. Therefore, the temperature can be appropriately and briefly raised in the production and preservation process of fresh-cut melon to obtain higher levels of phenolics.

Regulation on copper-tolerance in Citrus sinensis seedlings by boron addition: Insights from root exudates, related metabolism, and gene expression

Boron (B) can alleviate Citrus copper (Cu)-toxicity. However, the underlying mechanism by which B mitigates Cu-toxicity is unclear. ‘Xuegan’ (Citrus sinensis) seedlings were exposed to 0.5 (control) or 350 (Cu-toxicity) µM Cu and 2.5 or 25 µM B for 24 weeks. Thereafter, we investigated the secretion of low molecular weight compounds [LMWCs; citrate, malate, total soluble sugars (TSS), total phenolics (TP), and total free amino acids (TFAA)] by excised roots and their concentrations in roots and leaves, as well as related enzyme gene expression and activities in roots and leaves. Cu-stress stimulated root release of malate and TFAA, which might contribute to citrus Cu-tolerance. However, B-mediated-mitigation of Cu-stress could not be explained in this way, since B addition failed to further stimulate malate and TFAA secretion. Indeed, B addition decreased Cu-stimulated-secretion of malate. Further analysis suggested that Cu-induced-exudation of malate and TFAA was not regulated by their levels in roots. By contrast, B addition increased malate, citrate, and TFAA concentrations in Cu-toxic roots. Cu-toxicity increased TP concentration in 25 μM B-treated leaves, but not in 2.5 μM B-treated leaves. Our findings suggested that the internal detoxification of Cu by LMWCs played a role in B-mediated-alleviation of Cu-toxicity.

Impaired cardiac glycolysis and glycogen depletion are linked to poor myocardial outcomes in juvenile male swine with metabolic syndrome and ischemia.

Obesity continues to rise in the juveniles and obese children are more likely to develop metabolic syndrome (MetS) and related cardiovascular disease. Unfortunately, effective prevention and long-term treatment options remain limited. We determined the juvenile cardiac response to MetS in a swine model. Juvenile male swine were fed either an obesogenic diet, to induce MetS, or a lean diet, as a control (LD). Myocardial ischemia was induced with surgically placed ameroid constrictor on the left circumflex artery. Physiological data were recorded and at 22 weeks of age the animals underwent a terminal harvest procedure and myocardial tissue was extracted for total metabolic and proteomic LC/MS-MS, RNA-seq analysis, and data underwent nonnegative matrix factorization for metabolic signatures. Significantly altered in MetS versus. LD were the glycolysis-related metabolites and enzymes. In MetS compared with LD Glycogen synthase 1 (GYS1)-glycogen phosphorylases (PYGM/PYGL) expression disbalance resulted in a loss of myocardial glycogen. Our findings are consistent with the concept that transcriptionally driven myocardial changes in glycogen and glucose metabolism-related enzymes lead to a deficiency of their metabolite products in MetS. This abnormal energy metabolism provides insight into the pathogenesis of the juvenile heart in MetS. This study reveals that MetS and ischemia diminishes ATP availability in the myocardium via altering the glucose-G6P-pyruvate axis at the level of metabolites and gene expression of related enzymes. The observed severe glycogen depletion in MetS coincides with disbalance in expression of GYS1 and both PYGM and PYGL. This altered energy substrate metabolism is a potential target of pharmacological agents for improving juvenile myocardial function in MetS and ischemia.

Hot air and UV-C treatments promote anthocyanin accumulation in peach fruit through their regulations of sugars and organic acids

In our present study, we aimed to explore the effects of hot air and UV-C on anthocyanins and the interaction among anthocyanin, sucrose and organic acids in peaches during postharvest storage. Peaches were treated with hot air or UV-C and stored at 1 °C for 35 days. The results showed that both treatments significantly enhanced the accumulation of anthocyanins and suppressed the degradation of sucrose, citric and malic acids. An in vitro test verified that sucrose, citric and malic acid penetrated the tissue and then induced the biosynthesis of anthocyanins by up regulating anthocyanin-related enzymes. In addition, hot air and UV-C directly enhanced the activities and gene expression of related enzymes to promote the accumulation of anthocyanins. PAL, ANS and UFGT played crucial roles in the biosynthesis of anthocyanins in peach fruit after harvest, and these three enzymes can be stimulated by HA, UV-C, sucrose, citric and malic acid.

Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.

Quantitative Gene Expression of Peroxidase, Polyphenoloxidase and Catalase as Molecular Markers for Resistance against <i>Ralstonia solanacearum</i>

Brown rot or bacterial wilt of potato caused by Ralstonia solanacearum, is an economically important disease. Potato, cv. Nicola, was found to be relatively highly resistant to the infection with R. solanacearum and showed 15.12% wilt disease index, meantime, cv. Kara showed intermediate resistance with 37.40% disease index while, cv. Spunta was susceptible and showed 80.33% disease index. The role of defense-related enzymes in imparting resistance in potato against R. solanacearum was investigated by quantifying enzymes activity and gene expression of three defense- related enzymes, peroxidase, polyphenol oxidase and catalase. Peroxidase showed maximum activity 0.488 min-1&middotg-1 early at 12 h after pathogen inoculation in the cv. Nicola, whereas in susceptible cultivar Spunta showed lower activity of maximum 0.226 min-1&middotg-1 later at 48 h after inoculation. While, the moderately resistant cultivar Kara showed intermediate activity for the peak and its time. Meanwhile, polyphenol oxidase showed similar trends to that of peroxidase. On the contrary, catalase showed the highest activity values in the susceptible, cv. Spunta, while, in relatively highly resistant (cv. Nicola) and the moderately resistant (cv. Kara) showed lower values of activity and up to 96 h after inoculation. Meanwhile, gene expression of related enzymes the RT-PCR was used. At zero time, the relatively highly resistant potato cultivar, Nicola, showed the highest values of gene expression for both Peroxidase (POD) and Poly Phenol Oxidase (PPO). While, the susceptible potato cultivar, Spunta showed the lowest values. On the contrary, Catalase (CAT) gene expression was the highest in the susceptible, cv. Spunta, and was the lowest in the relatively highly resistant, cv. Nicola, while, was of intermediate values in the intermediate resistance, cv. Kara. Results show that peroxidase and polyphenol oxidase activities can be used as biochemical markers to reveal the resistance and susceptibility nature of potato cultivars against bacterial wilt disease of potato caused by R. solanacaerum.

Targeted Integration of RNA-Seq and Metabolite Data to Elucidate Curcuminoid Biosynthesis in Four Curcuma Species.

Curcuminoids, namely curcumin and its analogs, are secondary metabolites that act as the primary active constituents of turmeric (Curcuma longa). The contents of these curcuminoids vary among species in the genus Curcuma. For this reason, we compared two wild strains and two cultivars to understand the differences in the synthesis of curcuminoids. Because the fluxes of metabolic reactions depend on the amounts of their substrate and the activity of the catalysts, we analyzed the metabolite concentrations and gene expression of related enzymes. We developed a method based on RNA sequencing (RNA-Seq) analysis that focuses on a specific set of genes to detect expression differences between species in detail. We developed a 'selection-first' method for RNA-Seq analysis in which short reads are mapped to selected enzymes in the target biosynthetic pathways in order to reduce the effect of mapping errors. Using this method, we found that the difference in the contents of curcuminoids among the species, as measured by gas chromatography-mass spectrometry, could be explained by the changes in the expression of genes encoding diketide-CoA synthase, and curcumin synthase at the branching point of the curcuminoid biosynthesis pathway.

1,25-Dihydroxyvitamin D regulation of glucose metabolism in Harvey-ras transformed MCF10A human breast epithelial cells

This study was designed to investigate the impact of 1,25-dihydroxyvitamin D (1,25(OH)2D) on glucose metabolism during early cancer progression. Untransformed and ras-oncogene transfected (ras) MCF10A human breast epithelial cells were employed to model early breast cancer progression. 1,25(OH)2D modified the response of the ras cells to glucose restriction, suggesting 1,25(OH)2D may reduce the ras cell glucose addiction noted in cancer cells. To understand the 1,25(OH)2D regulation of glucose metabolism, following four-day 1,25(OH)2D treatment, metabolite fluxes at the cell membrane were measured by a nanoprobe biosensor, [13C6]glucose flux by 13C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of related enzymes was assessed. Treatment with 1,25(OH)2D reduced glycolysis as flux of glucose to 3-phosphoglycerate was reduced by 15% (P=0.017) and 32% (P<0.003) in MCF10A and ras cells respectively. In the ras cells, 1,25(OH)2D reduced lactate dehydrogenase activity by 15% (P<0.05) with a concomitant 10% reduction in the flux of glucose to lactate (P=0.006), and reduction in the level of intracellular lactate by 55% (P=0.029). Treatment with 1,25(OH)2D reduced flux of glucose to acetyl-coA 24% (P=0.002) and 41% (P<0.001), and flux to oxaloacetate 33% (P=0.003) and 34% (P=0.027) in the MCF10A and ras cells, respectively, suggesting a reduction in tricarboxylic acid (TCA) cycle activity. The results suggest a novel mechanism involving the regulation of glucose metabolism by which 1,25(OH)2D may prevent breast cancer progression.

Divergent effects of estradiol on gene expression of catecholamine biosynthetic enzymes

Within the catecholaminergic systems, there are contradictory findings regarding ability of estradiol to regulate expression of genes related to catecholamine biosynthesis. Several parameters important for effects of estradiol on the catecholamine (CA) related enzyme gene expression were examined in two CA regions. Ovariectomized (OVX) female rats were given prolonged estradiol treatments, either in a pulsatile fashion by injections or continuously by pellets. The mode affected the response of tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH) mRNAs differentially in the nucleus of solitary tract (NTS) and the locus coeruleus (LC). In rostral–medial NTS, TH mRNA levels were increased with injections, but declined in rats administered estradiol by pellets. In LC, a significant change was only observed in GTPCH with injections. These differences may reflect activation of different estrogen receptors (ER). The response to estradiol in the presence of ERα and ER β was examined in PC12 cell culture. Estradiol directly regulated promoter activity of TH, GTPCH and dopamine β-hydroxylase (DBH) genes. With ERα, 17 β-estradiol elevated TH promoter activity, while there was a decline with ERβ. In contrast, both DBH and GTPCH promoters were enhanced by 17 β-estradiol over a wide range of concentrations with either ER subtype. Thus, mode of administration, location examined and ER subtype expressed are important considerations in the overall response of catecholamine related enzymes to estradiol.

7β-Hydroxy-epiandrosterone-mediated regulation of the prostaglandin synthesis pathway in human peripheral blood monocytes

7α-Hydroxy-DHEA, 7β-hydroxy-DHEA and 7β-hydroxy-EpiA are native metabolites of dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA). Since numerous steroids are reported to interfere with inflammatory and immune processes, our objective was to test the effects of these hydroxysteroids on prostaglandin (PG) production and related enzyme gene expression. Human peripheral blood monocytes were cultured for 4 and 24 h in the presence of each of the steroids (1–100 nM), with and without addition of TNF-α (10 ng/mL). Levels of PGE 2, PGD 2 and 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2) were measured in the incubation medium, and cell content of cyclooxygenase (COX-2), and PGE and PGD synthases (m-PGES1, H-PGDS, L-PGDS), and peroxisome proliferator activated receptor (PPAR-γ) was assessed by quantitative RT-PCR and Western blots. Addition of TNF-α resulted in elevated PG production and increased COX-2 and m-PGES1 levels. Among the three steroids tested, only 7β-hydroxy-EpiA decreased COX-2, m-PGES1 and PPAR-γ expression while markedly decreasing PGE 2 and increasing 15d-PGJ 2 production. These results suggest that 7β-hydroxy-EpiA is a native trigger of cellular protection through simultaneous activation of 15d-PGJ 2 and depression of PGE 2 synthesis, and that these effects may be mediated by activation of a putative receptor, specific for 7β-hydroxy-EpiA.