Expression Of Related Enzymes Research Articles

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.

Effects of zinc deficiency and zinc supplementation on homocysteine levels and related enzyme expression in rats

Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n=8/group) for 3 weeks: Zn deficient (ZD; <1mg Zn/kg); Zn control (ZC; 30mg Zn/kg); Zn supplemented (ZS; 300mg Zn/kg); pair fed (PF; 30mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P<0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P<0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P<0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P<0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency.

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.

Dietary effect of royal jelly supplementation on epidermal levels of hydration, filaggrins, free amino acids and the related enzyme expression in UV irradiated hairless mice

Dietary effect of royal jelly supplementation on epidermal levels of hydration, filaggrins, free amino acids and the related enzyme expression in UV irradiated hairless mice

PP073. Gestational hypertension: A study on placental expression of endothelial and inducible nitric oxide synthase and no metabolism

Hypertension is one of the most common medical disorders in pregnancy and a major cause of maternal and perinatal morbidity and death. In primates adequate development of the embryo, and later of the fetus, depends on a successful hemomonochorial placentation. Nitric oxide (NO) a low molecular weight mediator, induces vasodilatation, inhibits platelet aggregation, and prevents the adhesion of platelets to endothelial cells. Till date, no data are available regarding gestational hypertension (GH) placenta and no metabolism and related enzyme expression and activity. The present study aimed to evaluate eNOS and iNOS expression in the placentas of both normal and GH patients, by means of Real-Time quantitative PCR, measure placental nitric oxide and peroxynitrite levels in the same group of subjects, and correlate such findings with HELLP group already published. Fifteen patients with gestational hypertension and thirty healthy pregnant controls comparable for maternal and gestational age were enrolled in the study. Placental tissue was taken immediately after delivery. eNOS and iNOS mRNA levels were evaluated Real-Time quantitative PCR, whereas nitric oxide and peroxynitrite production was measured by a commercially available kit. Placental eNOS and iNOS mRNA levels were significantly reduced in GH (2,02-fold reduction and 2,33-fold reduction, respectively) when compared to controls. Conversely, NO and ONOO(-) production were significantly higher in GH group compared to control group (31.56±4.15nmol NO/mg prot vs. 23.98±5.14nmol NO/mg prot and 68.49±8.57 arbitrary fluorescence units vs 17.31±2.25 arbitrary fluorescence units; p<0, 05). Such results were compared to HELLP group obtained in an already published study. As from results herein reported, we can hypothesize that complex mechanisms involving NO pathways cause a placental vasculature damage. However, it is not easy to understand if these changes could be interpreted as causes or consequences of this pathologic state.

Monoamine Oxidase A Down-Regulation Contributes to High Metanephrine Concentration in Pheochromocytoma

The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. A multicenter comparative study was conducted. The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Regulation of Fasting Fuel Metabolism by Toll-Like Receptor 4

OBJECTIVEToll-like receptor 4 (TLR4) has been reported to induce insulin resistance through inflammation in high-fat–fed mice. However, the physiological role of TLR4 in metabolism is unknown. Here, we investigated the involvement of TLR4 in fasting metabolism.RESEARCH DESIGN AND METHODSWild-type and TLR4 deficient (TLR4−/−) mice were either fed or fasted for 24 h. Glucose and lipid levels in circulation and tissues were measured. Glucose and lipid metabolism in tissues, as well as the expression of related enzymes, was examined.RESULTSMice lacking TLR4 displayed aggravated fasting hypoglycemia, along with normal hepatic gluconeogenesis, but reversed activity of pyruvate dehydrogenase complex (PDC) in skeletal muscle, which might account for the fasting hypoglycemia. TLR4−/− mice also exhibited higher lipid levels in circulation and skeletal muscle after fasting and reversed expression of lipogenic enzymes in skeletal muscle but not liver and adipose tissue. Adipose tissue lipolysis is normal and muscle fatty acid oxidation is increased in TLR4−/− mice after fasting. Inhibition of fatty acid synthesis in TLR4−/− mice abolished hyperlipidemia, hypoglycemia, and PDC activity increase, suggesting that TLR4-dependent inhibition of muscle lipogenesis may contribute to glucose and lipid homeostasis during fasting. Further studies showed that TLR4 deficiency had no effect on insulin signaling and muscle proinflammatory cytokine production in response to fasting.CONCLUSIONSThese data suggest that TLR4 plays a critical role in glucose and lipid metabolism independent of insulin during fasting and identify a novel physiological role for TLR4 in fuel homeostasis.

N- and O-Glycans Modulate Galectin-1 Binding, CD45 Signaling, and T Cell Death

Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

Gender affects liver desaturase expression in a rat model of n−3 fatty acid repletion

Dietary n−3 polyunsaturated fatty acids (PUFA) are major components of cell membranes and have beneficial effects on human health. Docosahexaenoic acid (DHA; 22:6 n−3) is the most biologically important n−3 PUFA and can be synthesized from its dietary essential precursor, α-linolenic acid (ALA; 18:3 n−3). Gender differences in the efficiency of DHA bioconversion have been reported, but underlying molecular mechanisms are unknown. We compared the capacity for DHA synthesis from ALA and the expression of related enzymes in the liver and cerebral cortex between male and female rats. Wistar rats, born with a low-DHA status, were supplied with a suboptimal amount of ALA from weaning to 8 weeks of age. Fatty acid composition was determined by gas chromatography, the mRNA expression of different genes involved in PUFA metabolism was determined by RT-PCR (low-density array) and the expression of proteins was determined by Western blot analysis. At 8 weeks, DHA content was higher (+20 to +40%) in each phospholipid class of female livers compared to male livers. The “Δ4,” Δ5 and Δ6 desaturation indexes were 1.2–3 times higher in females than in males. The mRNA expression of Δ5- and Δ6-desaturase genes was 3.8 and 2.5 times greater, respectively, and the Δ5-desaturase protein was higher in female livers (+50%). No gender difference was observed in the cerebral cortex. We conclude that female rats replete their DHA status more readily than males, probably due to a higher expression of liver desaturases. Our results support the hypothesis on hormonal regulation of PUFA metabolism, which should be taken into account for specific nutritional recommendations.

Decreased endopelvic fascia elastin content in uterine prolapse

Genital prolapse is a debilitating manifestation of pelvic floor dysfunction. The cause of this condition has not been elucidated. The purpose of this study was to determine elastin content and RNA expression of related enzymes of elastin synthesis in uterosacral ligament biopsies from women with severe prolapse, and controls with normal pelvic support. Biopsies were taken from the uterosacral ligament tissue of 31 women with Grade III or greater prolapse and 29 women with normal pelvic support. Elastin content was assessed by measuring desmosine using radioimmunoassay, and quantitative real time PCR was performed to quantify mRNA levels of lysyl oxidase (LOX), lysyl oxidase like-1 (LOXL1), LOXL2 and fibulin-5 (FIB-5). The mean desmosine concentration found in uterosacral ligaments of women with prolapse (n =26) was 103.3+/-59.3 pmolD/mgP compared to controls (n =29) 120.5+/-47.4 pmolD/mgP (p =0.1943). In the subgroup of subjects with complete procidentia (n =8), mean desmosine concentration was 50.6+/-25.8 and 127.1+/-42.2 pmolD/mgP in age-matched controls (n =12) (p <0.05). In tissue from subjects with more than 2 vaginal deliveries (n =18), the mean desmosine concentration was 99.9+/-60.7 and 133.0+/-44.0 pmolD/mgP in controls (n =17) (p <0.05). Expression of LOX, LOXL1 and LOXL2 decreased 8.2-fold+/-3.4, 5.0-fold+/-1.7 and 15.2-fold+/-5.2, respectively (mean+/-SD) in cases versus controls (p<0.05). Expression of FIB-5 was increased 3.1-fold+/-0.7 compared to controls (p<0.05). Significantly decreased desmosine content was measured in the uterosacral ligament tissue from women with prolapse versus controls in women with parity >2 and in women with complete procidentia. Suppression of mRNA for LOX and two LOX isoenzymes was correspondingly present. These results suggest that altered elastin metabolism is present in women with uterine prolapse.

A High‐Fat Diet Has a Tissue‐Specific Effect on Adiponectin and Related Enzyme Expression

This study was designed to test whether adiponectin plays a role in diet-induced obesity and insulin resistance and acts as a mediator to induce or inhibit specific metabolic pathways involved in lipid metabolism Forty C57BL/6J male mice were fed either a high-fat (HF) or control diet for 4 months, and adiponectin, its receptors, and enzyme expression in liver and muscle tissue were measured. Mice fed the HF diet exhibited significantly greater weight gain, abnormal oral glucose tolerance test curves, and elevated homeostasis model assessment of insulin resistance (5.3 +/- 0.89 vs. 2.8 +/- 0.39). A significant reduction of adiponectin RNA expression (51%) and protein levels (15%) was observed in the adipose tissue of HF animals; however, serum adiponectin levels did not differ between groups (7.12 +/- 0.34 mug/mL vs. 6.44 +/- 0.38 microg/mL). Expression of hepatic mRNA of AdipoR1 and AdipoR2 was reduced by 15% and 25%, respectively, in animals fed the HF diet. In contrast, receptor mRNA expression of AdipoR1 and AdipoR2 increased by 25% and 30%, respectively, in muscle tissue. No effect was found on hepatic adenosine monophosphate-activated protein kinase expression; however, a significant reduction of phosphoadenosine monophosphate kinase levels in muscles was observed. Hepatic acetyl-coenzyme A carboxylase was similar between groups, but in muscles, the inactive form phosphoacetyl-coenzyme A carboxylase was significantly reduced (p < 0.05). The HF diet led to decreased insulin sensitivity accompanied by impaired activity of adiponectin-related enzymes in skeletal muscles but not in the liver. These results suggest that the HF diet has a tissue-specific effect on adiponectin and associated enzyme expression.