Severe Liver Cirrhosis Markedly Reduces AhR-Mediated Induction of Cytochrome P450 in Rats by Decreasing the Transcription of Target Genes

PubMed Central

Floreani, Maura; De Martin, Sara; Gabbia, Daniela; Barbierato, Massimo; Nassi, Alberto; Mescoli, Claudia; Orlando, Rocco; Bova, Sergio; Angeli, Paolo; Gola, Elisabetta; Sticca, Antonietta; Palatini, Pietro

2013-01-01

Although the induction of cytochrome P450 (CYP) has long been investigated in patients with cirrhosis, the question whether liver dysfunction impairs the response to CYP inducers still remains unresolved. Moreover, the mechanism underlying the possible effect of cirrhosis on induction has not been investigated. Since ethical constraints do not permit methodologically rigorous studies in humans, this question was addressed by investigating the effect of the prototypical inducer benzo[a]pyrene (BP) on CYP1A1 and CYP1A2 in cirrhotic rats stratified according to the severity of liver dysfunction. We simultaneously assessed mRNA level, protein expression and enzymatic activity of the CYP1A enzymes, as well as mRNA and protein expressions of the aryl hydrocarbon receptor (AhR), which mediates the BP effect. Basal mRNA and protein expressions of CYP1A1 were virtually absent in both healthy and cirrhotic rats. On the contrary, CYP1A2 mRNA, protein and enzyme activity were constitutively present in healthy rats and decreased significantly as liver function worsened. BP treatment markedly increased the concentrations of mRNA and immunodetectable protein, and the enzymatic activities of both CYP1A enzymes to similar levels in healthy and non-ascitic cirrhotic rats. Induced mRNA levels, protein expressions and enzymatic activities of both CYPs were much lower in ascitic rats and were proportionally reduced. Both constitutive and induced protein expressions of AhR were significantly lower in ascitic than in healthy rats. These results indicate that the inducibility of CYP1A enzymes is well preserved in compensated cirrhosis, whereas it is markedly reduced when liver dysfunction becomes severe. Induction appears to be impaired at the transcriptional level, due to the reduced expression of AhR, which controls the transcription of CYP1A genes. PMID:23626760

Gene expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress.

PubMed

Guo, Hui; Xian, Jian-An; Li, Bin; Ye, Chao-Xia; Wang, An-Li; Miao, Yu-Tao; Liao, Shao-An

2013-05-01

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PubMed Central

Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers. PMID:28323908

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed Central

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-01-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

Differential utilization of decapping enzymes in mammalian mRNA decay pathways

PubMed Central

Li, You; Song, Mangen; Kiledjian, Megerditch

2011-01-01

mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells. PMID:21224379

Hepatic Xenobiotic Metabolizing Enzyme Gene Expression Through the Life Stages of the Mouse

EPA Science Inventory

BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been ca...

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

mRNA secondary structure engineering of Thermobifida fusca endoglucanase (Cel6A) for enhanced expression in Escherichia coli.

PubMed

Ali, Imran; Asghar, Rehana; Ahmed, Sajjad; Sajjad, Muhammad; Tariq, Muhammad; Waheed Akhtar, M

2015-03-01

The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5'-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = -2.7 kcal mol(-1)was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = -7.8 kcal mol(-1) and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg(-1) on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg(-1)), alkali treated bagass (1.7 Umg(-1)), filter paper (1.2 Umg(-1)) and BMCC (0.3 Umg(-1)). Metal ions affect endoglucanase activity in different ways. Only Fe(2+) exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2(+) (91.8 %).

The effect of ZnO nanoparticles on liver function in rats

PubMed Central

Tang, Hua-Qiao; Xu, Min; Rong, Qian; Jin, Ru-Wen; Liu, Qi-Ji; Li, Ying-Lun

2016-01-01

Zinc oxide (ZnO) is widely incorporated as a food additive in animal diets. In order to optimize the beneficial effects of ZnO and minimize any resultant environmental pollution, ZnO nanoparticles are often used for delivery of the zinc. However, the possible toxic effects of ZnO nanoparticles, including effects on cytochrome P450 (CYP450) enzymes, have not been evaluated. In this study, we investigated the effect of ZnO nanoparticles, in doses used in animal feeds, on CYP450 enzymes, liver and intestinal enzymes, liver and kidney histopathology, and hematologic indices in rats. We found that liver and kidney injury occurred when the concentrations of ZnO nanoparticles in feed were 300–600 mg/kg. Also, liver mRNA expression for constitutive androstane receptor was suppressed and mRNA expression for pregnane X receptor was induced when feed containing ZnO nanoparticles was given at a concentration of 600 mg/kg. Although the expression of mRNA for CYP 2C11 and 3A2 enzymes was induced by ZnO nanoparticles, the activities of CYP 2C11 and 3A2 were suppressed. While liver CYP 1A2 mRNA expression was suppressed, CYP 1A2 activity remained unchanged at all ZnO nanoparticle doses. Therefore, it has been concluded that ZnO nanoparticles, in the doses customarily added to animal feed, changed the indices of hematology and blood chemistry, altered the expression and activity of hepatic CYP enzymes, and induced pathological changes in liver and kidney tissues of rats. These findings suggest that greater attention needs to be paid to the toxic effects of ZnO nanoparticles in animal feed, with the possibility that the doses of ZnO should be reduced. PMID:27621621

AKR1C1 and SRD5A1 messenger RNA expression at term in the human myometrium and chorioamniotic membranes.

PubMed

Lee, Richard H; Stanczyk, Frank Z; Stolz, Andrew; Ji, Qing; Yang, Gloria; Goodwin, T Murphy

2008-10-01

We sought to determine relative mRNA expression of AKR1C1 and SRD5A1, which respectively encode for the key progesterone metabolizing enzymes, 20alpha-hydroxysteroid dehydrogenase and 5alpha-reductase type 1, in the myometrium and chorioamniotic membranes during human spontaneous or induced labor and nonlabor. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to compare relative mRNA expression of AKR1C1 and SRD5A1 in the myometrium and chorioamniotic membranes from 20 subjects during three different states of labor: not in labor ( N = 10), spontaneous labor ( N = 5), or induced labor ( N = 5). Labor was defined as regular uterine contractions that resulted in cervical dilation. Myometrial AKR1C1 mRNA expression was significantly greater in spontaneously laboring subjects compared with those not in labor (2.4-fold [1.97 to 2.98], P = 0.02). There was no difference in myometrial AKR1C1 mRNA expression between those with induced labor compared with those not in labor. Regardless of labor status, no differences were observed in the chorioamniotic membrane AKR1C1 mRNA expression between the groups. SRD5A1 mRNA expression was significantly lower in the membranes of both laboring groups when compared with those not in labor (spontaneous: 0.10-fold [0.06 to 0.18], P = 0.007; induced: 0.09-fold [0.03 to 0.25], P = 0.013). Regardless of labor status, there was no difference in SRD5A1 mRNA expression in the myometrium. Our study demonstrated tissue-specific changes in progesterone metabolizing enzyme mRNA expression in human intrauterine tissue at term associated with labor status. These observed changes in mRNA expression may have important implications for progesterone metabolism at those specific sites and thereby may differentially regulate the tissue-specific progesterone concentration and/or the level of specific progesterone metabolites.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

PubMed

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites. Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.

PubMed

Urbatzka, R; Lutz, I; Kloas, W

2007-01-01

The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.

Molecular interactions of natural and synthetic steroids in female hamsters' flank organs.

PubMed

Cabeza, Marisa; Naranjo, Barak; Heuze, Yvonne; Sánchez, Araceli; Hernández, Mercedes; Sainz, Teresita; Bratoeff, Eugene

2012-05-01

The initial step of steroidal action on target cells is gene activation; therefore, the quantification of mRNA is a direct method for comparing the role of different steroids in the skin. This study demonstrated the role of several steroids on the mRNA expression encoding for different enzymes involved in the lipid metabolism in hamsters' flank organs, which are a pilosebaceous complex. To determine the effect of treatments with testosterone (T) progesterone (P), levonorgestrel (LNG), 17α-p-chlorobenzoyloxy-6-chloropregn-4,6-diene-3,20-dione (5) and 17α-p-chlorobenzoyloxy-4,6-pregnadiene-3,20-dione (6); T and/or LNG; T and 5 or 6; P and/or 5 or 6 on the expression of mRNA encoding for lipid enzymes, the steroids were applied to the glands; later, the mRNAs expression for the enzymes was determined by PCR. The binding of 5 and 6 to the progesterone receptor (PR) was also evaluated. Treatments with T, LNG, T+LNG, P, T+P, 5, T+5, T+6, P, P+5 and P+6 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase as compared to the controls. However, squalene synthase was increased with all treatments except with T+5 and 6; 6 did not significantly increase the expression for GPAT or HMG-CoA-S, however it increased the concentration of HMG-CoA-R enzyme. 5 and 6 bind to the PR, thus indicating that the effect of these steroids on the mRNA expression could be the result of their binding. The lipid metabolism is regulated by several steroids thought different mechanism of action, in flank organs. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Expression of ceramide-metabolising enzymes in subcutaneous and intra-abdominal human adipose tissue

PubMed Central

2012-01-01

Background Inflammation and increased ceramide concentrations characterise adipose tissue of obese women with high liver fat content compared to equally obese women with normal liver fat content. The present study characterises enzymes involved in ceramide metabolism in subcutaneous and intra-abdominal adipose tissue. Methods Pathways leading to increased ceramide concentrations in inflamed versus non-inflamed adipose tissue were investigated by quantifying expression levels of key enzymes involved in ceramide metabolism. Sphingomyelinases (sphingomyelin phosphodiesterases SMPD1-3) were investigated further using immunohistochemistry to establish their location within adipose tissue, and their mRNA expression levels were determined in subcutaneous and intra-abdominal adipose tissue from both non-obese and obese subject. Results Gene expression levels of sphingomyelinases, enzymes that hydrolyse sphingomyelin to ceramide, rather than enzymes involved in de novo ceramide synthesis, were higher in inflamed compared to non-inflamed adipose tissue of obese women (with high and normal liver fat contents respectively). Sphingomyelinases were localised to both macrophages and adipocytes, but also to blood vessels and to extracellular regions surrounding vessels within adipose tissue. Expression levels of SMPD3 mRNA correlated significantly with concentrations of different ceramides and sphingomyelins. In both non-obese and obese subjects SMPD3 mRNA levels were higher in the more inflamed intra-abdominal compared to the subcutaneous adipose tissue depot. Conclusions Generation of ceramides within adipose tissue as a result of sphingomyelinase action may contribute to inflammation in human adipose tissue. PMID:22974251

Characterization of the endocrine, digestive and morphological adjustments of the intestine in response to food deprivation and torpor in cunner, Tautogolabrus adspersus.

PubMed

Hayes, James; Volkoff, Hélène

2014-04-01

The cunner, Tautogolabrus adspersus, is a marine teleost endemic to the cold waters of the Northwest Atlantic Ocean. The cunner is non-migratory and is known for its remarkable ability to endure the freezing winter months with little to no food by entering a torpid/dormant state. To evaluate the physiological strategies employed by the cunner's intestinal tract to withstand food deprivation, fish were sampled for their gut after a four-week period of acute food deprivation during their summer (active/feeding) state, as well as after 4months of overwinter fasting. Digestive capacity was evaluated by measuring digestive enzyme activity and related mRNA transcript expression for trypsin, alkaline phosphatase, alanine aminopeptidase and lipase. In order to assess how gut hormones affect/are affected by acute fasting and torpor, we examined the intestinal mRNA expression of several putative appetite regulators, i.e. CCK, apelin, orexin and mTOR. Short-term summer fasting induced a reduction in the activity, but not the transcript expression, of all digestive enzymes examined as well as a reduction in gut apelin mRNA. Torpor induced a reduction in the activity of all enzymes with the exception of alanine aminopeptidase, and a decrease in mRNA levels of alanine aminopeptidase, orexin, CCK and mTOR. Our results suggest that both acute fasting and long-term fasting induce a reduction in the intestinal function of cunner, as evidenced by an overall decrease in the activities of digestive enzymes and mRNA expression of several factors involved in feeding and digestion. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression and Functional Activities of Selected Sulfotransferase Isoforms in BeWo Cells and Primary Cytotrophoblast Cells

PubMed Central

Mitra, Pallabi; Audus, Kenneth L.

2009-01-01

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17β-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent Km values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism. PMID:19646966

Endocannabinoids as endometrial inflammatory markers in lactating Holstein cows.

PubMed

Bonsale, R; Seyed Sharifi, R; Dirandeh, E; Hedayat, N; Mojtahedin, A; Ghorbanalinia, M; Abolghasemi, A

2018-06-01

The objective of this study was to consider endocannabinoid system as inflammatory markers in bovine endometrium to better understand the role of this system in regulating many of the functions that are related to inflammatory condition. At day 26 post-partum, fourteen cows were divided into two groups depending on the inflammatory condition: 1- subclinical endometritis (n = 7, with purulent or mucopurulent uterine discharge detectable in the vagina) and 2- healthy (n = 7, No (muco)) purulent discharge. Blood samples were collected at 26 and 30 days relative to calving to determine plasma tumour necrosis factor (TNF) and lipopolysaccharide-binding protein (LBP) concentrations; moreover, uterine biopsy was carried out on day 26 post-partum to measure mRNA abundance of TNF, interleukin-1B (IL1B), interleukin-6 (IL-6), C-X-C motif chemokine ligand 8 (CXCL8), endocannabinoid receptor (CNR2), N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL) by real-time PCR. Results showed mean plasma concentrations of TNF and LBP were lower in healthy cows compared to subclinical endometritis cows (p < .05). Relative mRNA expression for NAAA and FAAH was decreased (p < .05), and relative mRNA expression for CNR2 and NAPEPLD increased in cows with subclinical endometritis compared to healthy cows. In conclusion, relative mRNA expression of TNF, IL1B and CXCL8 and plasma concentration of LBP increased during inflammatory condition along with decreased endocannabinoids hydrolyzing enzyme (NAAA and FAAH), increased enzymes that synthesize endocannabinoids (NAPEPLD) and relative gene expression of the endocannabinoid receptor; together, these contribute to increased endocannabinoids levels during inflammation. Overall, we provide evidence that endocannabinoid system is altered in endometrium tissue during inflammation through increased mRNA expression of CNR2 and synthesis enzyme and decreased mRNA expression of hydrolyzing enzymes interfere with pro-cytokine production and signalling, which may interfere with the onset and progression of inflammation. © 2018 Blackwell Verlag GmbH.

Characterization of the Impact of Life Stage on Xenobiotic Metabolizing Enzyme Expression and Gene -Chemical Interactions in the Liver

EPA Science Inventory

Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). We have carried out a comprehensive analysis of the mRNA expression of XMETs thro...

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae.

PubMed

Kortner, Trond M; Overrein, Ingrid; Oie, Gunvor; Kjørsvik, Elin; Bardal, Tora; Wold, Per-Arvid; Arukwe, Augustine

2011-10-01

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species. Copyright © 2011 Elsevier Inc. All rights reserved.

Regulation of hepatic bile acid transporters Ntcp and Bsep expression.

PubMed

Cheng, Xingguo; Buckley, David; Klaassen, Curtis D

2007-12-03

Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid (CDCA) increased Bsep mRNA expression. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers.

Protein-bound polysaccharide-K augments the anticancer effect of fluoropyrimidine derivatives possibly by lowering dihydropyrimidine dehydrogenase expression in gastrointestinal cancers.

PubMed

Mekata, Eiji; Murata, Satoshi; Sonoda, Hiromichi; Shimizu, Tomoharu; Umeda, Tomoko; Shiomi, Hisanori; Naka, Shigeyuki; Yamamoto, Hiroshi; Abe, Hajime; Edamatsu, Takeo; Fujieda, Ayako; Fujioka, Masaki; Wada, Tsutomu; Tani, Tohru

2013-12-01

Protein-bound polysaccharide-K (PSK) enhances the antitumor effect of anticancer drug when used clinically in combination with such drugs. PSK is known to act by immune-mediated mechanisms; however, the relationship between PSK and metabolic enzymes of anticancer drugs is unknown. We used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) clinically to evaluate the sensitivity of anticancer drugs. In the present study, we modified the CD-DST by adding peripheral blood mononuclear cells (PBMCs) (immuno-CD-DST) and examined the antitumor effect of PSK in combination with anticancer drugs. First, HCT116 human colon cancer cells were cultured with PSK and 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouridine (5'-DFUR) in the presence or absence of PBMCs, and the antiproliferative effects were compared. In the presence of PBMCs, PSK augmented the inhibitory effects of 5-FU and 5'-DFUR on HCT116 cell proliferation. Next, using human gastric cancer and colon cancer cell lines, the effects of PSK on mRNA expression of various metabolic enzymes of fluoropyrimidines: dihydropyrimidine dehydrogenase (DPD), thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase, were examined by real-time PCR. PSK significantly enhanced DPD mRNA expression in all of the cancer cell lines tested, but not those of the other enzymes. Addition of IFN-α and TRAIL, cytokines known to inhibit DPD expression, to the cultures reduced DPD mRNA expression in the cancer cells. When PBMC samples collected from healthy volunteers were cultured with PSK, IFN-α mRNA expression increased in 3 of the 5 PBMC samples, while TRAIL mRNA expression was unchanged. The present results propose the possibility that PSK induces PBMCs to express IFN-α which inhibits DPD expression, and consequently augments the antitumor effect of 5-FU or 5'-DFUR. Immuno-CD-DST is useful for evaluating drugs with immunological mechanisms of action.

MicroRNA-141-3p/200a-3p target and may be involved in post-transcriptional repression of RNA decapping enzyme Dcp2 during renal development.

PubMed

Zhang, Ming-Nan; Tang, Qun-Ye; Li, Rui-Min; Song, Man-Gen

2018-06-18

The RNA decapping enzyme Dcp2 is a crucial enzyme involved in the process of RNA turnover, which can post-transcriptionally regulate gene expression. Dcp2 has been found to be highly expressed in embryonic, but not adult, kidneys. Here we showed that Dcp2 mRNA was expressed, but Dcp2 proteins were absent, in mouse kidneys after postnatal day 10 (P10). In kidneys of adult Dcp2-IRES-EGFP knock-in mice, Dcp2 was undetectable but EGFP was expressed, indicating that Dcp2 mRNA was not completely silenced in adult kidneys. Using luciferase reporter assays, we found that miR-141-3p/200a-3p directly targeted the 3' UTR of Dcp2 mRNA. Overexpression of miR-141-3p and miR-200a-3p downregulated endogenous Dcp2 protein expression. Furthermore, miR-141-3p and miR-200a-3p expression was low in embryonic kidneys but increased dramatically after P10 and was negatively correlated with Dcp2 protein expression during renal development. These results suggest miR-141-3p/200a-3p may be involved in post-transcriptional repression of Dcp2 expression during renal development. IRES: internal ribosome entry site; EGFP: enhanced green fluorescent protein; UTR: untranslated region.

RNA-sequencing quantification of hepatic ontogeny of phase-I enzymes in mice.

PubMed

Peng, Lai; Cui, Julia Y; Yoo, Byunggil; Gunewardena, Sumedha S; Lu, Hong; Klaassen, Curtis D; Zhong, Xiao-Bo

2013-12-01

Phase-I drug metabolizing enzymes catalyze reactions of hydrolysis, reduction, and oxidation of drugs and play a critical role in drug metabolism. However, the functions of most phase-I enzymes are not mature at birth, which markedly affects drug metabolism in newborns. Therefore, characterization of the expression profiles of phase-I enzymes and the underlying regulatory mechanisms during liver maturation is needed for better estimation of using drugs in pediatric patients. The mouse is an animal model widely used for studying the mechanisms in the regulation of developmental expression of phase-I genes. Therefore, we applied RNA sequencing to provide a "true quantification" of the mRNA expression of phase-I genes in the mouse liver during development. Liver samples of male C57BL/6 mice at 12 different ages from prenatal to adulthood were used for defining the ontogenic mRNA profiles of phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldo-keto reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). Two rapidly increasing stages of total phase-I gene expression after birth reflect functional transition of the liver during development. Diverse expression patterns were identified, and some large gene families contained the mRNA of genes that are enriched at different stages of development. Our study reveals the mRNA abundance of phase-I genes in the mouse liver during development and provides a valuable foundation for mechanistic studies in the future.

Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages.

PubMed

Rybicka, Marta; Stachowska, Ewa; Gutowska, Izabela; Parczewski, Miłosz; Baśkiewicz, Magdalena; Machaliński, Bogusław; Boroń-Kaczmarska, Anna; Chlubek, Dariusz

2011-04-27

The aim of this study was to investigate the effect of conjugated linoleic acids (CLAs) on macrophage reactive oxygen species synthesis and the activity and expression of antioxidant enzymes, catalase (Cat), glutathione peroxidase (GPx), and superoxide dismutase (SOD). The macrophages were obtained from the THP-1 monocytic cell line. Cells were incubated with the addition of cis-9,trans-11 CLA or trans-10,cis-12 CLA or linoleic acid. Reactive oxygen species (ROS) formation was estimated by flow cytometry. Enzymes activity was measured spectrophotometrically. The antioxidant enzyme mRNA expression was estimated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was based on nonparametric statistical tests [Friedman analysis of variation (ANOVA) and Wilcoxon signed-rank test]. cis-9,trans-11 CLA significantly increased the activity of Cat, while trans-10,cis-12 CLA notably influenced GPx activity. Both isomers significantly decreased mRNA expression for Cat. Only trans-10,cis-12 significantly influenced mRNA for SOD-2 expression. The CLAs activate processes of the ROS formation in macrophages. Adverse metabolic effects of each isomer action were observed.

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

PubMed Central

Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

2002-01-01

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

Glucose transporters and enzymes related to glucose synthesis in small intestinal mucosa of mid-lactation dairy cows fed 2 levels of starch.

PubMed

Lohrenz, A-K; Duske, K; Schönhusen, U; Losand, B; Seyfert, H M; Metges, C C; Hammon, H M

2011-09-01

Diets containing corn starch may improve glucose supply by providing significant amounts of intestinal starch and increasing intestinal glucose absorption in dairy cows. Glucose absorption in the small intestine requires specific glucose transporters; that is, sodium-dependent glucose co-transporter-1 (SGLT1) and facilitated glucose transporter (GLUT2), which are usually downregulated in the small intestine of functional ruminants but are upregulated when luminal glucose is available. We tested the hypothesis that mRNA and protein expression of intestinal glucose transporters and mRNA expression of enzymes related to gluconeogenesis are affected by variable starch supply. Dairy cows (n=9/group) were fed for 4 wk total mixed rations (TMR) containing either high (HS) or low (LS) starch levels in the diet. Feed intake and milk yield were measured daily. After slaughter, tissue samples of the small intestinal mucosa (mid-duodenum and mid-jejunum) were taken for determination of mRNA concentrations of SGLT1 and GLUT2 as well as pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase by real-time reverse transcription PCR relative to a housekeeping gene. Protein expression of GLUT2 in crude mucosal membranes and of SGLT1 and GLUT2 in brush-border membrane vesicles was quantified by sodium dodecyl sulfate-PAGE and immunoblot. A mixed model was used to examine feeding and time-related changes on feed intake and milk yield and to test feeding and gut site effects on gene or protein expression of glucose transporters and enzymes in the intestinal mucosa. Dry matter intake, but not energy intake, was higher in cows fed HS compared with LS. Abundance of SGLT1 mRNA tended to be higher in duodenal than in jejunal mucosa, and mRNA abundances of pyruvate carboxylase tended to be higher in jejunal than in duodenal mucosa. In brush-border membrane vesicles, SGLT1 and GLUT2 protein expression could be demonstrated. No diet-dependent differences were found concerning mRNA and protein contents of glucose transporter or mRNA level of gluconeogenic enzymes. In conclusion, our investigations on glucose transporters and gluconeogenic enzymes in the small intestinal mucosa of dairy cows did not show significant diet regulation when TMR with different amounts of intestinal starch were fed. Therefore, predicted intestinal glucose absorption after enhanced starch feeding is probably not supported by changes of intestinal glucose transporters in dairy cows. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture

PubMed Central

Meißner, Joachim D; Gros, Gerolf; Scheibe, Renate J; Scholz, Michael; Kubis, Hans-Peter

2001-01-01

The addition of cyclosporin A (500 ng ml−1) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 × 10−7m) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation. PMID:11351029

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland.

PubMed

Falvo, Sara; Chieffi Baccaria, Gabriella; Spaziano, Giuseppe; Rosati, Luigi; Venditti, Massimo; Di Fiore, Maria Maddalena; Santillo, Alessandra

2018-03-01

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology. Copyright © 2018 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain

PubMed Central

Purves-Tyson, T D; Owens, S J; Rothmond, D A; Halliday, G M; Double, K L; Stevens, J; McCrossin, T; Shannon Weickert, C

2017-01-01

The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis. In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action. PMID:28094812

[Effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice].

PubMed

Jin, Xin; Zhang, Hui-xin; Zhang, Yan-fen; Cui, Wen-wen; Bi, Yao; He, Qi-long; Zhou, Sheng-shan

2015-03-01

To study the effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice. Eight male C57BL/6J mice were selected in the normal group (NF), 40 male ApoE -/- mice were fed for 16 weeks, divided into the model group (HF), the rosiglitazone group ( LGLT), the Jinlida low-dose group (JLDL), the Jinlida medium-dose group (JLDM), the Jinlida high-dose group (JLDH) and then orally given drugs for 8 weeks. The organization free fatty acids, BCA protein concentration determination methods were used to determine the skeletal muscle FFA content. The Real-time fluorescent quantitative reverse transcription PCR ( RT-PCR) and Western blot method were adopted to determine mRNA and protein expressions of mice fatty acids transposition enzyme (FAT/CD36), carnitine palm acyltransferase 1 (CPT1), peroxide proliferators-activated receptor α( PPAR α). Jinlida could decrease fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FIns) and raise insulin sensitive index (ISI) in mice to varying degrees. It could also up-regulate mRNA and protein expressions of CPT1 and PPARα, and down-regulate mRNA and protein levels of FAT/CD36. Jinlida can improve fat-induced insulin resistance ApoE -/- in mice by adjusting the changes in expression of skeletal muscle lipid transport enzymes.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

PubMed

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

USDA-ARS?s Scientific Manuscript database

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our objective was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretor...

Relationship Between the DPD and TS mRNA Expression and the Response to S-1-Based Chemotherapy and Prognosis in Patients with Advanced Gastric Cancer.

PubMed

Shen, Xiao-Ming; Zhou, Chong; Lian, Lian; Li, Li-Qun; Li, Wei; Tao, Min

2015-04-01

The aim was to determine changes in dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNAs in the blood of advanced gastric cancer (AGC) patients to see whether these enzymes affected the patients' response to S-1-based chemotherapy and prognosis. For this purpose, pretreatment DPD/TS mRNA expressions were determined in 40 AGC patients using RT-PCR. The patients were then administered with S-1-based regimen (S-1 + cisplatin) and toxicities were recorded. The relationship between the DPD/TS mRNA expressions and the chemotherapy response, drug resistance, and prognosis was analyzed. The data show that DPD mRNA expression correlated significantly with Lauren type while TS mRNA expression correlated with distant metastasis. Patients with higher DPD and/or TS mRNA expression(s) showed poor response, while those with low DPD mRNA expression showed better response to the chemotherapy. Pooled analysis showed that the patients with low DPD/TS mRNA expressions had better therapeutic response. The incidence of bone marrow suppression, diarrhea, and oral mucositis was high in patients with low DPD mRNA expression. Median overall survival (OS) in 40 patients was 13.5 months. It was 17 months for low and 10 months for high DPD (P = 0.044) and TS mRNA expression (P = 0.047). Pooled analysis showed that the patients with both low DPD/TS mRNA expressions had longer OS (P = 0.001). In conclusion, the detection of DPD and/or TS mRNA expression can be used to predict the response to S-1-based chemotherapy, drug resistance, and prognosis in AGC patients as well as to help guide the individualized treatment of gastric cancer.

[Effect of Bushen Huoxue Compound on Retinal Müller Cells in High Glucose or AGEs Conditions].

PubMed

Xie, Xue-jun; Song, Ming-xia; Zhang, Mei; Qin, Wei; Wan, Li; Fang, Yang

2015-06-01

To explore the effect of Bushen Huoxue Compound (BHC) on lactate dehydrogenase (LDH) leakage, expressions of vascular endothelial growth factor (VEGF) and VEGF mRNA in retinal Muller cells under high glucose condition or advanced glycosylation end products (AGEs) condition by using serum pharmacological method. The retinal Müller cells of 5-7 days post-natal Sprague Dawley (SD) rats were cultured with modified enzyme-digestion method. Purified retinal Muller cells were cultured in normal conditions, high glucose condition (50 mmol/L) or AGEs (50 mg/L and 100 mg/L) conditions, and BHC-containing serum was added to culture medium. The LDH leakage and VEGF expressions were measured by enzyme-linked immunosorbent assay (ELISA). In addition, the relative expression of VEGF mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR). Compared with the normal control group, expressions of VEGF and VEGF mRNA were significantly increased in the high glucose group, the low dose AGEs group and the high dose AGEs group (all P < 0.01). The LDH leakage was obviously increased in the high dose AGEs group, when compared with the normal control group and the high glucose group (P < 0.01). The LDH leakage, expressions of VEGF and VEGF mRNA were obviously decreased by BHC-containing serum both in high glucose and AGEs conditions (P < 0.05, P < 0.01). BHC-containing serum had no significant effect on the LDH leakage and expressions of VEGF and VEGF mRNA in normal conditions (P > 0.05). AGEs intervention could obviously lower the stability of Müller cell membrane. Up-regulated expressions of VEGF and VEGF mRNA in cultured Müller cells could be induced by AGEs or high glucose. BHC-containing serum could stabilize the stability of Müller cell membrane, inhibit the transcription of VEGF mRNA and decrease the protein expression of VEGF, which might be one of important mechanisms for preventing and treating diabetic retinopathy.

Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence1

PubMed Central

Wu, Chun-Ta; Leubner-Metzger, Gerhard; Meins, Frederick; Bradford, Kent J.

2001-01-01

β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed. PMID:11457981

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls

PubMed Central

Fassah, Dilla Mareistia; Jeong, Jin Young

2018-01-01

Objective This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Methods Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Results Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Conclusion Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition. PMID:29502393

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls.

PubMed

Fassah, Dilla Mareistia; Jeong, Jin Young; Baik, Myunggi

2018-04-01

This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition.

Fermented wheat aleurone induces enzymes involved in detoxification of carcinogens and in antioxidative defence in human colon cells.

PubMed

Stein, Katrin; Borowicki, Anke; Scharlau, Daniel; Glei, Michael

2010-10-01

Dietary fibre is fermented by the human gut flora resulting mainly in the formation of SCFA, for example, acetate, propionate and butyrate. SCFA, in particular butyrate, may be important for secondary cancer prevention by inducing apoptosis and inhibiting cell growth of cancer cells, thereby inhibiting the promotion and/or progression of cancer. Furthermore, SCFA could also act on primary cancer prevention by activation of detoxifying and antioxidative enzymes. We investigated the effects of fermented wheat aleurone on the expression of genes involved in stress response and toxicity, activity of drug-metabolising enzymes and anti-genotoxic potential. Aleurone was digested and fermented in vitro to obtain samples that reflect the content of the colon. HT29 cells and colon epithelial stripes were incubated with the resulting fermentation supernatant fractions (fs) and effects on mRNA expression of CAT, GSTP1 and SULT2B1 and enzyme activity of glutathione S-transferase (GST) and catalase (CAT) were measured. Fermented aleurone was also used to study the protection against H2O2-induced DNA damage in HT29 cells. The fs of aleurone significantly induced the mRNA expression of CAT, GSTP1 and SULT2B1 (HT29) and GSTP1 (epithelial stripes), respectively. The enzyme activities of GST (HT29) and CAT (HT29, epithelial stripes) were also unambiguously increased (1.4- to 3.7-fold) by the fs of aleurone. DNA damage induced by H2O2 was significantly reduced by the fs of aleurone after 48 h, whereupon no difference was observed compared with the faeces control. In conclusion, fermented aleurone is able to act on primary prevention by inducing mRNA expression and the activity of enzymes involved in detoxification of carcinogens and antioxidative defence.

Divergent expression of 11beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase genes between male morphs in the central nervous system, sonic muscle and testis of a vocal fish.

PubMed

Arterbery, Adam S; Deitcher, David L; Bass, Andrew H

2010-05-15

The vocalizing midshipman fish, Porichthys notatus, has two male morphs that exhibit alternative mating tactics. Only territorial males acoustically court females with long duration (minutes to >1h) calls, whereas sneaker males attempt to steal fertilizations. During the breeding season, morph-specific tactics are paralleled by a divergence in relative testis and vocal muscle size, plasma levels of the androgen 11-ketotestosterone (11KT) and the glucocorticoid cortisol, and mRNA expression levels in the central nervous system (CNS) of the steroid-synthesizing enzyme aromatase (estrogen synthase). Here, we tested the hypothesis that the midshipman's two male morphs would further differ in the CNS, as well as in the testis and vocal muscle, in mRNA abundance for the enzymes 11beta-hydroxylase (11betaH) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) that directly regulate both 11KT and cortisol synthesis. Quantitative real-time PCR demonstrated male morph-specific profiles for both enzymes. Territorial males had higher 11betaH and 11betaHSD mRNA levels in testis and vocal muscle. By contrast, sneaker males had the higher CNS expression, especially for 11betaHSD, in the region containing an expansive vocal pacemaker circuit that directly determines the temporal attributes of natural calls. We propose for territorial males that higher enzyme expression in testis underlies its greater plasma 11KT levels, which in vocal muscle provides both gluconeogenic and androgenic support for its long duration calling. We further propose for sneaker males that higher enzyme expression in the vocal CNS contributes to known cortisol-specific effects on its vocal physiology. Copyright 2010 Elsevier Inc. All rights reserved.

Hormonal control of GTP cyclohydrolase I gene expression and enzyme activity during color pattern development in wings of Precis coenia.

PubMed

Sawada, H; Nakagoshi, M; Reinhardt, R K; Ziegler, I; Koch, P B

2002-06-01

Color patterns of butterfly wings are composed of single color points represented by each scale. In the case of Precis coenia, at the end of pupal development, different types of pigments are synthesized sequentially in the differently colored scales beginning with white (pterins) followed by red (ommatins) and then black (melanin). In order to explain how formation of these different colors is regulated, we examined the expression of an mRNA-encoding guanosine triphosphate-cyclohydrolase I (GTP-CH I; EC 3.5.4.16), the first key enzyme in the biosynthesis of pteridines, during pigment formation in the wings of P. coenia. The strongest positive signal was recognized around pigment formation one day before butterfly emergence. This GTP-CH I gene expression is paralleled by GTP-CH I enzyme activity measured in wing extracts. We also investigated the effect of 20-hydroxyecdysone on the expression of GTP-CH I mRNA and the enzyme activity during color formation. The results strongly suggest that the onset and duration of the expression of a GTP-CH I mRNA is triggered by a declining ecdysteroid hormone titer during late pupal development.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zidong Donna; Klaassen, Curtis D., E-mail: cklaasse@kumc.edu

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors.more » In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs.« less

Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation.

PubMed

Greenfield, R B; Cecava, M J; Donkin, S S

2000-06-01

The objective of this study was to profile phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) mRNA expression in the liver of dairy cattle during the peripartum transition and determine changes in abundance of these mRNA in response to protein fed during the prepartum period. Thirty-eight multiparous Holstein cows were fed diets containing either 12% crude protein (CP) and 26% rumen undegradable protein (RUP), 16% CP and 26% RUP, 16% CP and 33% RUP, or 16% CP and 40% RUP on a dry-matter basis beginning 28 d before expected calving. After calving, all cows were fed a common diet through 56 d in milk (DIM). Northern analysis of RNA from liver biopsy samples obtained on days -28, -14, +1, +28, and +56 relative to calving indicated that PC and PEPCK mRNA expression were responsive to onset of lactation but not to prepartum protein or RUP concentration. Abundance of PEPCK mRNA was similar at -28, -14, and +1 DIM but was elevated by +28 and +56 DIM relative to precalving levels. Liver PC mRNA abundance was elevated on +1 DIM, remained elevated through 28 DIM, and declined to precalving levels by 56 DIM. The activity of PC enzyme was correlated (r2 = 0.89) with PC mRNA abundance. The data demonstrate increased abundance of PC mRNA during the early transition period followed by increased abundance of PEPCK mRNA during the postpartum period and suggest increased potential metabolism of lactate, pyruvate, and amino acids that contribute to the liver pyruvate pool.

Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4

PubMed Central

2010-01-01

Background Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Results Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Conclusions Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms. PMID:20359329

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

PubMed Central

Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

2012-01-01

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

Effects of postprandial starvation on mRNA expression of endocrine-, amino acid and peptide transporter-, and metabolic enzyme-related genes in zebrafish (Danio rerio).

PubMed

Tian, Juan; He, Gen; Mai, Kangsen; Liu, Chengdong

2015-06-01

The goal of this study was to systematically evaluate the molecular activities of endocrine-, amino acid and peptide transporters-, and metabolic enzyme-related genes in 35-day-old mixed-sex zebrafish (Danio rerio) after feeding . Zebrafish with initial body weights ranging from 9 to 11 mg were fasted for 384 h in a controlled indoor environment. Fish were sampled at 0, 3, 6, 12, 24, 48, 96, 192, and 384 h after fed. Overall, the present study results show that the regulatory mechanism that insulin-like growth factor I negative feedback regulated growth hormone is conserved in zebrafish, as it is in mammals, but that regulation of growth hormone receptors is highly intricate. Leptin and cholecystokinin are time-dependent negative feedback signals, and neuropeptide Y may be an important positive neuropeptide for food intake in zebrafish. The amino acid/carnitine transporters B(0,+) (ATB(0,+)) and broad neutral (0) amino acid transporter 1(B(0)AT1) mRNA levels measured in our study suggest that protein may be utilized during 24-96 h of fasting in zebrafish. Glutamine synthetase mRNA levels were downregulated, and glutamate dehydrogenase, alanine aminotransferase, aspartate transaminase, and trypsin mRNA levels were upregulated after longtime fasting in this study. The mRNA expression levels of fatty acid synthetase decreased significantly (P < 0.05), whereas those of lipoprotein lipase rapidly increased after 96 h of fasting. Fasting activated the expression of glucose synthesis genes when fasting for short periods of time; when fasting is prolonged, the mRNA levels of glucose breakdown enzymes and pentose phosphate shunt genes decreased.

Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation and POMC gene expression in β-endorphin-producing POMC neurons of the hypothalamus

PubMed Central

Bekdash, Rola A.; Zhang, Changqing; Sarkar, Dipak K.

2013-01-01

Background Prenatal exposure to ethanol reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, since these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of ethanol on these neurons. Methods Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes, and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunoabsorbent assay. Results Prenatal ethanol treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10) increased the repressive marks (H3K9me2, G9a, Setdb1) and DNA methylating enzyme (Dnmt1) and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP, and elevated corticosterone response to LPS. Gestational choline normalized the ethanol-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1 and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production and the corticosterone response to LPS. Conclusions These data suggest that prenatal ethanol modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction of POMC gene expression. Gestational choline supplementation prevents the adverse effects of ethanol on these neurons. PMID:23413810

Characterization of glucagon-like peptide 2 pathway member expression in bovine gastrointestinal tract.

PubMed

Connor, E E; Baldwin, R L; Capuco, A V; Evock-Clover, C M; Ellis, S E; Sciabica, K S

2010-11-01

Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, has several physiological effects on the intestine of monogastric species, including promotion of growth of intestinal epithelium, reduction of epithelial cell apoptosis, and enhancement of intestinal blood flow, nutrient absorption, and epithelial barrier function. The regulatory functions of GLP-2 in the ruminant gastrointestinal tract (GIT) have not been well studied. The objectives of this investigation were to characterize the mRNA expression of 4 members of the GLP-2 pathway throughout the bovine GIT, including (1) proglucagon (GCG), the parent peptide from which GLP-2 is derived through cleavage by prohormone convertase; (2) prohormone convertase (PCSK1); (3) GLP-2 receptor (GLP2R); and (4) dipeptidyl peptidase IV (DPP4), the enzyme that inactivates GLP-2. Gene expression was evaluated in rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, and rectum collected at slaughter from prepubertal heifers, mature cows in early, mid, and late lactation, and nonlactating cows (n=3 per stage) by a gene expression profiling assay. In addition, mRNA expression of 14 genes involved in nutrient transport, enzyme activity, blood flow, apoptosis, and proliferation were evaluated in the 9 GIT tissues for their association with GCG and GLP2R mRNA expression. Immunohistochemistry was used to localize GLP2R protein in tissues of the lower GIT. Results indicated that mRNA expression of GCG, PCSK1, GLP2R, and DPP4 varies across the 9 GIT tissues, with greatest expression in small and large intestines, and generally nondetectable levels in forestomachs. Expression of DPP4 and GLP2R mRNA varied by developmental stage or lactational state in intestinal tissues. Expression of GCG or GLP2R mRNA was correlated with molecular markers of proliferation, apoptosis, blood flow, enzyme activity, and urea transport, depending on the tissue examined, which suggests a potential for involvement of GLP-2 in these physiological processes in the ruminant GIT. The GLP2R protein was expressed in intestinal crypts of the bovine GIT, which is consistent with the distribution in monogastric species. Our findings support a functional role of the GLP-2 pathway in bovine GIT and the potential for use of GLP-2 as a therapy to improve intestinal function and nutrient absorption in ruminants. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Development of Prostaglandin Endoperoxide Synthase Expression in the Ovine Fetal Central Nervous System and Pituitary

PubMed Central

Gersting, Jason A.; Schaub, Christine E.; Wood, Charles E.

2009-01-01

In this study, we tested the hypothesis that prostaglandin endoperoxide synthase -1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145 days of gestational age (DGA), 1 and 7 days postpartum lambs, and from adult ewes (n=4–5 per group). mRNA and protein were isolated from each region, and expression of Prostaglandin Synthase -1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNA’s were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNA’s were upregulated at 120 DGA. Tissue concentrations of PGE2 correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus. PMID:19706338

Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome.

PubMed

Maissen-Villiger, Carla A; Schweighauser, Ariane; van Dorland, H Anette; Morel, Claudine; Bruckmaier, Rupert M; Zurbriggen, Andreas; Francey, Thierry

2016-01-01

Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. The mRNA abundance of cytokines (IL-1α, IL-1β, IL-8, IL-10, TNF-α, TGF-β) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1β, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.

The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts.

PubMed

Hyland, Paula L; Traynor, Patrick S; Myrillas, Theofilos T; Marley, John J; Linden, Gerard J; Winter, Paul; Leadbetter, Nicola; Cawston, Timothy E; Irwin, Chris R

2003-04-01

The immunosuppressive agent cyclosporin is associated with a number of major side-effects including the development of gingival overgrowth. Although the pathogenesis of cyclosporin-induced gingival overgrowth remains unclear, it has been suggested that the finely regulated balance between extracellular matrix synthesis and degradation may be disturbed, resulting in an accumulation of excess connective tissue components within the gingival tissue. The aim of this study was to investigate the effect of cyclosporin on matrix metalloproteinases (MMP)-1 and tissue inhibitors of MMP (TIMP)-1 expression at the mRNA, protein, and enzyme activity levels. Gingival fibroblasts were grown to confluence and then cultured in serum-free medium supplemented with cyclosporin over the concentration range of 0 to 2000 ng/ml. MMP-1 and TIMP-1 mRNA levels in cultures were determined by reverse transcription polymerase chain reaction (RT-PCR), protein levels in whole conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA), and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. Tissue mRNA levels in normal and overgrown gingiva were also determined by RT-PCR. Results indicated that cyclosporin inhibited MMP-1 expression at both the mRNA and protein level in a dose- and time-dependent fashion. The effects on TIMP-1 expression were less clear, cyclosporin inhibiting mRNA expression, but having no effect on TIMP-1 protein levels at any concentration studied. Addition of the drug resulted in reduced levels of collagenolytic activity in the culture medium. MMP-1 mRNA expression was significantly reduced in overgrown compared to normal tissue. These results add support to the hypothesis that the accumulation of collagen seen in gingival overgrowth can be explained by a cyclosporin-induced inhibition of collagenolytic activity within the gingival tissues.

Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow

NASA Technical Reports Server (NTRS)

Hillsley, M. V.; Frangos, J. A.

1997-01-01

It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteoblasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.

Expression and Enzyme Activity of Catalase in Chilo suppressalis (Lepidoptera: Crambidae) Is Responsive to Environmental Stresses.

PubMed

Lu, Yanhui; Bai, Qi; Zheng, Xusong; Lu, Zhongxian

2017-08-01

Catalase (CAT) is an important antioxidant enzyme that protects organisms against oxidative stresses by eliminating hydrogen peroxide. In this study, we cloned and characterized a full-length cDNA of CAT from Chilo suppressalis (CsCAT) and examined the influence of environmental stresses on CsCAT expression and enzyme activity. The cDNA contains a 1659-bp open reading frame encoding a polypeptide of 553 amino acids most closely related (90.14%) to Papilio polytes catalases. The CsCAT was expressed in all developmental stages with the highest expression in the fat body, and the CsCAT enzyme activity closely mirrored its observed mRNA expression patterns. The CsCAT mRNA was up-regulated when the larvae were exposed to high temperature (≥30 °C), insecticides (abamectin and chlorantraniliprole), chemicals (H2O2, CHP, CdCl2, and CuSO4), and a dead-end trap plant (vetiver grass), and the CsCAT enzyme activity again mirrored the observed CsCAT expression patterns. These results suggest that up-regulation of CsCAT may enhance the defense response of C. suppressalis by weakening the effects of environmental stresses, and provide insight into the role of CsCAT during development of C. suppressalis. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

α-lipoic acid ameliorates n-3 highly-unsaturated fatty acids induced lipid peroxidation via regulating antioxidant defenses in grass carp (Ctenopharyngodon idellus).

PubMed

Shi, Xiao-Chen; Jin, Ai; Sun, Jian; Yang, Zhou; Tian, Jing-Jing; Ji, Hong; Yu, Hai-Bo; Li, Yang; Zhou, Ji-Shu; Du, Zhen-Yu; Chen, Li-Qiao

2017-08-01

This study evaluated the protective effect of α-lipoic acid (LA) on n-3 highly unsaturated fatty acids (HUFAs)-induced lipid peroxidation in grass carp. The result indicated that diets with n-3 HUFAs increased the production of malondialdehyde (MDA) (P < 0.05), thereby inducing lipid peroxidation in liver and muscle of grass carp. Meanwhile, compared with control group, the hepatosomatic index (HSI) and kidney index (KI) of grass carp were markedly increased in n-3 HUFAs-only group. However, diets with LA remarkably inhibited the n-3 HUFAs-induced increase of HSI, KI, and MDA level in serum, liver and muscle (P < 0.05). Interestingly, LA also significantly elevated the ratio of total n-3 HUFAs in fatty acid composition of muscle and liver (P < 0.05). Furthermore, LA significantly promoted the activity of antioxidant enzymes in serum, muscle and liver of grass carp (P < 0.05), including superoxide dismutase (SOD), catalase (CAT), and glutathione s-transferase (GST). The further results showed that LA significantly elevated mRNA expression of antioxidant enzymes with promoting the mRNA expression of NF-E2-related nuclear factor 2 (Nrf2) and decreasing Kelch-like-ECH-associated protein 1 (Keap1) mRNA level. From the above, these results suggested that LA could attenuate n-3 HUFAs-induced lipid peroxidation, remit the toxicity of the lipid peroxidant, and protect n-3 HUFAs against lipid peroxidation to promote its deposition in fish, likely strengthening the activity of antioxidant enzymes through regulating mRNA expressions of antioxidant enzyme genes via mediating Nrf2-Keap1 signaling pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Status of epigenetic chromatin modification enzymes and esophageal squamous cell carcinoma risk in northeast Indian population.

PubMed

Singh, Virendra; Singh, Laishram C; Singh, Avninder P; Sharma, Jagannath; Borthakur, Bibhuti B; Debnath, Arundhati; Rai, Avdhesh K; Phukan, Rup K; Mahanta, Jagadish; Kataki, Amal C; Kapur, Sujala; Saxena, Sunita

2015-01-01

Esophageal cancer incidence is reported in high frequency in northeast India. The etiology is different from other population at India due to wide variations in dietary habits or nutritional factors, tobacco/betel quid chewing and alcohol habits. Since DNA methylation, histone modification and miRNA-mediated epigenetic processes alter the gene expression, the involvement of these processes might be useful to find out epigenetic markers of esophageal cancer risk in northeast Indian population. The present investigation was aimed to carryout differential expression profiling of chromatin modification enzymes in tumor and normal tissue collected from esophageal squamous cell carcinoma (ESCC) patients. Differential mRNA expression profiling and their validation was done by quantitative real time PCR and tissue microarray respectively. Univariate and multiple logistic regression analysis were used to analyze the epidemiological data. mRNA expression data was analyzed by Student t-test. Fisher exact test was used for tissue microarray data analysis. Higher expression of enzymes regulating methylation (DOT1L and PRMT1) and acetylation (KAT7, KAT8, KAT2A and KAT6A) of histone was found associated with ESCC risk. Tissue microarray done in independent cohort of 75 patients revealed higher nuclear protein expression of KAT8 and PRMT1 in tumor similar to mRNA expression. Expression status of PRMT1 and KAT8 was found declined as we move from low grade to high grade tumor. Betel nut chewing, alcohol drinking and dried fish intake were significantly associated with increased risk of esophageal cancer among the study subject. Study suggests the association of PRMT1 and KAT8 with esophageal cancer risk and its involvement in the transition process of low to high grade tumor formation. The study exposes the differential status of chromatin modification enzymes between tumor and normal tissue and points out that relaxed state of chromatin facilitates more transcriptionally active genome in esophageal carcinogenesis.

CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

PubMed Central

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how “human-like” can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs. PMID:19285097

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji

2009-05-15

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carryingmore » humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.« less

Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.

PubMed

Feng, Lin; Gan, Lu; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Tang, Ling; Kuang, Sheng-Yao; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2017-04-01

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill. Copyright © 2017 Elsevier Ltd. All rights reserved.

Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by ToxCast Chemicals

EPA Science Inventory

ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

Skin barrier disruption by sodium lauryl sulfate-exposure alters the expressions of involucrin, transglutaminase 1, profilaggrin, and kallikreins during the repair phase in human skin in vivo.

PubMed

Törmä, Hans; Lindberg, Magnus; Berne, Berit

2008-05-01

Detergents are skin irritants affecting keratinocytes. In this study, healthy volunteers were exposed to water (vehicle) and 1% sodium lauryl sulfate (SLS) under occlusive patch tests for 24 hours. The messenger RNA (mRNA) expression of keratinocyte differentiation markers and of enzymes involved in corneodesmosome degradation was examined in skin biopsies (n=8) during the repair phase (6 hours to 7 days postexposure) using real-time reverse-transcription PCR. It was found that the expression of involucrin was increased at 6 hours, but then rapidly normalized. The expression of transglutaminase 1 exhibited a twofold increase after 24 hours in the SLS-exposed skin. Profilaggrin was decreased after 6 hours. Later (4-7 days), the expression in SLS-exposed areas was >50% above than in control areas. An increased and altered immunofluorescence pattern of involucrin, transglutaminase 1, and filaggrin was also found (n=4). At 6 hours post-SLS exposure, the mRNA expression of kallikrein-7 (KLK-7) and kallikrein-5 (KLK-5) was decreased by 50 and 75%, respectively, as compared with control and water-exposed areas. Thereafter, the expression pattern of KLK-7 and KLK-5 was normalized. Changes in protein expression of KLK-5 were also found. In conclusion, SLS-induced skin barrier defects induce altered mRNA expression of keratinocyte differentiation markers and enzymes degrading corneodesmosomes.

Time- and dose-dependent differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase enzymatic activity and mRNA level by vitamin E in rat blood cells.

PubMed

Hajiani, Maliheh; Razi, Farideh; Golestani, Aboualfazl; Frouzandeh, Mehdi; Owji, Ali Akbar; Khaghani, Shahnaz; Ghannadian, Naghmeh; Shariftabrizi, Ahmad; Pasalar, Parvin

2012-01-01

Vitamin E is the most important lipid-soluble antioxidant. Recently, it has been proposed as a gene regulator, and its gene modulation effects have been observed at different levels of gene expression and cell signaling. This study was performed to investigate the effects of vitamin E on the activity and expression of the most important endogenous antioxidant enzyme, superoxide dismutase (SOD), in rat plasma. Twenty-eight male Sprauge-Dawley rats were divided into four groups: control group and three dosing groups. The control group received the vehicle (liquid paraffin), and the dosing groups received twice-weekly intraperitoneal injections of 10, 30, and 100 mg/kg of vitamin E ((±)-α-Tocopherol) for 6 weeks. Quantitative real-time reverse transcription-polymerase chain reaction and enzyme assays were used to assess the levels of Cu/Zn-SOD and Mn-SOD mRNA and enzyme activity levels in blood cells at 0, 2, 4, and 6 weeks following vitamin E administration. Catalase enzyme activity and total antioxidant capacity were also assessed in plasma at the same time intervals. Mn-SOD activity was significantly increased in the 100 and 30 mg/kg dosing groups after 4 and 6 weeks, with corresponding significant increase in their mRNA levels. Cu/Zn-SOD activity was not significantly changed in response to vitamin E administration at any time points, whereas Cu/Zn-SOD mRNA levels were significantly increased after longer time points with high doses (30 and 100 mg/kg) of vitamin E. Catalase enzyme activity was transiently but significantly increased after 4 weeks of vitamin E treatment in 30 and 100 mg/kg dosing groups. Total antioxidant status was significantly increased after 4 and 6 weeks in the 100 mg/kg dosing group. Only the chronic administration of higher doses of alpha-tocopherol is associated with the increased activity and expression of Mn-SOD in rats. Cu/Zn-SOD activity and expression does not dramatically change in response to vitamin E.

Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles.

PubMed

Lin, Y Q; Xu, Y O; Yue, Y; Jin, S Y; Qu, Y; Dong, F; Li, Y P; Zheng, Y C

2012-08-29

Hypoxia can affect energy metabolism. We examined gene expression and enzyme activity related to mitochondrial energy metabolism, as well as myosin heavy chain (MyHC) types in yaks (Bos grunniens) living at high altitudes. Real-time quantitative PCR assays indicated that the yak has significantly lower levels of carnitine palmitoyltransferase (CPT) mRNA in the biceps femoris and lower levels of uncoupling protein 3 (UCP3) mRNA in both biceps femoris and longissimus dorsi than in Yellow cattle. No significant differences between yak and Yellow cattle were observed in the activities of mitochondrial β-hydroxyacyl-CoA dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase in the same muscles. Semi-quantitative RT-PCR analysis showed that the MyHC 1 mRNA levels in yak biceps femoris was lower than in Yellow cattle. We conclude that the yak has significantly lower mRNA levels of CPT, UCP3, and MyHC 1 in biceps femoris than in Yellow cattle, suggesting that the yak biceps femoris has lower fatty acid oxidation capacity and greater glycolytic metabolic potential.

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

PubMed

Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

2013-07-29

A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

PubMed Central

2013-01-01

Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%. Conclusion We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression. PMID:23895661

Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain.

PubMed

Cain, B M; Connolly, K; Blum, A C; Vishnuvardhan, D; Marchand, J E; Zhu, X; Steiner, D F; Beinfeld, M C

2004-04-01

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Franco, Gilson C.N.; Department of Pharmacology, FOP/UNICAMP, Piracicaba, SP; Kajiya, Mikihito

2011-06-10

Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography andmore » Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.« less

Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

PubMed

Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

2009-12-15

Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC b-Zip transcription factors in estrogen-induced breast carcinogenesis.

PubMed

Chatterjee, Anwesha; Ronghe, Amruta; Singh, Bhupendra; Bhat, Nimee K; Chen, Jie; Bhat, Hari K

2014-12-01

The objective of the present study was to characterize the role of resveratrol (Res) and vitamin C (VC) in prevention of estrogen-induced breast cancer through regulation of cap "n"collar (CNC) b-zip transcription factors. Human breast epithelial cell line MCF-10A was treated with 17β-estradiol (E2) and VC or Res with or without E2. mRNA and protein expression levels of CNC b-zip transcription factors nuclear factor erythroid 2-related factor 1 (Nrf1), nuclear factor erythroid 2 related factor 2 (Nrf2), nuclear factor erythroid 2 related factor 3 (Nrf3), and Nrf2-regulated antioxidant enzymes superoxide dismutase 3 (SOD3) and quinone oxidoreductase 1 (NQO1) were quantified. The treatment with E2 suppressed, whereas VC and Res prevented E2-mediated decrease in the expression levels of SOD3, NQO1, Nrf2 mRNA, and protein in MCF-10A cells. The treatment with E2, Res, or VC significantly increased mRNA and protein expression levels of Nrf1. 17β-Estradiol treatment significantly increased but VC or Res decreased Nrf3 mRNA and protein expression levels. Our studies demonstrate that estrogen-induced breast cancer might be prevented through upregulation of antioxidant enzymes via Nrf-dependent pathways. © 2014 Wiley Periodicals, Inc.

1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

PubMed

Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

2015-12-01

Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

PubMed Central

Sugihara, T; Kobori, A; Imaeda, H; Tsujikawa, T; Amagase, K; Takeuchi, K; Fujiyama, Y; Andoh, A

2010-01-01

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa α-chain linked to a 70-kDa β-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17. PMID:20089077

Prostate Cancer Prevention Through Induction of Phase 2 Enzymes

DTIC Science & Technology

1999-10-01

hypothesis. We have identified sulforaphane , a dietary isothiocyanate found in cucifers, as the most potent phase 2 enzyme inducing agent in human prostate...cancer cell lines compared to over 50 other compounds screened in our laboratory. Sulforaphane readily induced increased expression of quinone...characterizing global changes in mRNA expression for nearly 10,000 genes simultaneously using cDNA microarrays after treatment of prostate cells with sulforaphane

Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

EPA Science Inventory

ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

PubMed

Zeng, X; Wu, J; Wu, Q; Zhang, J

2015-01-01

Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice.

PubMed

Fu, Zidong Donna; Klaassen, Curtis D

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. Copyright © 2013 Elsevier Inc. All rights reserved.

Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L.

PubMed

Qu, Changfeng; He, Yingying; Zheng, Zhou; An, Meiling; Li, Lulu; Wang, Xixi; He, Xiaodong; Wang, Yibin; Liu, Fangming; Miao, Jinlai

2018-01-01

The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.

Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation, and proopiomelanocortin (POMC) gene expression in β-endorphin-producing POMC neurons of the hypothalamus.

PubMed

Bekdash, Rola A; Zhang, Changqing; Sarkar, Dipak K

2013-07-01

Prenatal exposure to ethanol (EtOH) reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, because these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of EtOH on these neurons. Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal- and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunosorbent assay. Prenatal EtOH treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10), and increased the repressive marks (H3K9me2, G9a, Setdb1), DNA-methylating enzyme (Dnmt1), and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP and elevated corticosterone response to LPS. Gestational choline normalized the EtOH-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1, and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production, and the corticosterone response to LPS. These data suggest that prenatal EtOH modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction in POMC gene expression. Gestational choline supplementation prevents the adverse effects of EtOH on these neurons. Copyright © 2013 by the Research Society on Alcoholism.

Metronidazole reduces the expression of cytochrome P450 enzymes in HepaRG cells and cryopreserved human hepatocytes.

PubMed

Kudo, Toshiyuki; Endo, Yumiko; Taguchi, Rina; Yatsu, Masami; Ito, Kiyomi

2015-05-01

1. Blood levels of S-warfarin have been reported to be increased by concomitant administration of metronidazole (MTZ), an antiprotozoal imidazole derivative. 2. To elucidate the mechanism of this interaction and to identify other possible drug-drug interactions, we conducted an in vitro study with the human hepatoma HepaRG cells and cryopreserved human hepatocytes on the ability of MTZ to reduce the expression of cytochrome P450 (CYP) as well as nuclear receptors that regulate the expression of these enzymes. 3. HepaRG cells and cryopreserved human hepatocytes were treated with MTZ (20 to 500 µM) and were then analyzed by real-time RT-PCR to determine mRNA levels of drug-metabolizing enzymes and nuclear receptors. 4. In both cells, the expressions of CYP2C8, CYP2C9, CYP3A4 and constitutive androstane receptor (CAR) were decreased by MTZ treatment. Particularly, in HepaRG cells, their mRNA levels were decreased by MTZ treatment in a concentration-dependent manner. 5. Our findings suggest that the interaction between MTZ and S-warfarin may be due to the MTZ-induced down-regulation of CYP2C9, the primary enzyme responsible for S-warfarin hydroxylation, and CAR, which regulates CYP2C9 expression. We also found that MTZ use may alter the disposition of drugs metabolized by the CYP isozymes investigated.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression

PubMed Central

Fu, Jiaqi; Weise, Amy M.; Falany, Josie L.; Falany, Charles N.; Thibodeau, Bryan J.; Miller, Fred R.; Kocarek, Thomas A.

2013-01-01

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERα and ERβ) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β-hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERα-positive luminal MCF7 breast cancer cells. Low levels of ERα and ERβ mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17βHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17βHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17βHSD1, and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression. PMID:19308726

Selenium-dependent pre- and posttranscriptional mechanisms are responsible for sexual dimorphic expression of selenoproteins in murine tissues.

PubMed

Riese, Cornelia; Michaelis, Marten; Mentrup, Birgit; Götz, Franziska; Köhrle, Josef; Schweizer, Ulrich; Schomburg, Lutz

2006-12-01

Important enzymes for thyroid hormone metabolism, antioxidative defense, and intracellular redox control contain selenocysteine (Sec) in their active centers. Expression of these selenoproteins is tightly controlled, and a sex-specific phenotype is observed on disturbance of selenium (Se) transport in mice. Therefore, we analyzed Se concentrations and expression levels of several selenoproteins including type I iodothyronine deiodinase (Dio1) and glutathione peroxidase (GPx) isozymes in male and female mice. On regular lab chow, serum Se levels were comparable, but serum GPx3 activity was higher in females than males (1.3-fold). Selenoprotein P (SePP) mRNA levels were higher in livers (1.3-fold) and lower in kidneys (to 31%) in female compared with male mice. Orchidectomy alleviated the sex-specific differences in SePP mRNA amounts, indicating modulatory effects of androgens on SePP expression. Female mice expressed higher levels of Dio1 mRNA in kidney (2.6-fold) and liver (1.4-fold) in comparison with male mice. This sexual dimorphic expression of Dio1 mRNA was paralleled by increased Dio1 activity in female kidney (1.8-fold) but not in liver in which males expressed higher Dio1 activity (2.8-fold). Interestingly, Se deficiency decreased Dio1 activity more effectively in males than females, and resulting hepatic enzyme levels were then comparable between the sexes. At the same time, the sex-specific difference of Dio1 activity widened in kidney. Orchidectomy or estradiol treatment of ovariectomized females impacted stronger on renal than hepatic Dio1 expression. Thus, we conclude that Se-dependent posttranscriptional mechanisms are operational that affect either translational efficiency or Dio1 stability in a sex- and tissue-specific manner.

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth1

PubMed Central

Russell, Brenda L.; Rathinasabapathi, Bala; Hanson, Andrew D.

1998-01-01

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family. PMID:9489025

Biological Effects of COMT Haplotypes and Psychosis Risk in 22q11.2 Deletion Syndrome

PubMed Central

Gothelf, Doron; Law, Amanda J.; Frisch, Amos; Chen, Jingshan; Zarchi, Omer; Michaelovsky, Elena; Ren-Patterson, Renee; Lipska, Barbara K.; Carmel, Miri; Kolachana, Bhaskar; Weizman, Abraham; Weinberger, Daniel R.

2013-01-01

Background 22q11.2 deletion syndrome (22q11.2DS) is the most common genetic syndrome associated with schizophrenia. The catechol-o-methyltransferase (COMT) gene is located in the obligatory deletion region, and possible associations between COMT variants and neuropsychiatric manifestations in 22q11.2DS have been reported. The purpose of the current study was to evaluate the effect of COMT hemizygosity and molecular haplotypes on gene expression and enzyme activity and its association with psychotic symptoms in 22q11.2DS. Methods Lymphoblast samples were drawn from 53 individuals with 22q11.2DS and 16 typically developing controls. We measured COMT mRNA and protein expression and enzyme activity using standard procedures. The presence of a psychotic disorder and cognitive deficits were also evaluated using structured testing. Results There was a ~50% reduction in COMT mRNA, protein and enzyme activity levels in 22q11.2DS samples. Haplotype analysis revealed clear phenotypic differences between various Val-containing haplotypes on COMT-3′UTR extended mRNA, S-COMT and MB proteins and enzyme activity. The G variant of rs165599, a 3′UTR SNP, was associated with low levels of COMT expression and with the presence of psychosis and lower performance IQ scores in our 22q11.2DS sample. Finally, we demonstrate that the COMT rs74745580 ‘T’ mutation is associated with absent S-COMT expression and very low COMT activity in two 22q11.2DS individuals. Conclusions Our findings confirm a robust effect of COMT hemizygosity on COMT activity and show complex interactions of variants within the COMT gene that influence COMT biology and confound conclusions based on associations with the Val158Met genotype alone. PMID:23992923

Ascorbic acid transported by sodium-dependent vitamin C transporter 2 stimulates steroidogenesis in human choriocarcinoma cells.

PubMed

Wu, Ximei; Iguchi, Takuma; Itoh, Norio; Okamoto, Kousuke; Takagi, Tatsuya; Tanaka, Keiichi; Nakanishi, Tsuyoshi

2008-01-01

Reduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48-h dose dependently increased progesterone and estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the maximal velocity of AA uptake by approximately 50%, but apparent affinity values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and estradiol production. In addition, inhibition of MAPK kinase-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MAPK kinase-ERK signaling is not involved in AA-induced steroidogenesis.

Why is golden rice golden (yellow) instead of red?

PubMed

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-05-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.

Why Is Golden Rice Golden (Yellow) Instead of Red?1[w

PubMed Central

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-01-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, ζ-carotene desaturase, carotene cis-trans-isomerase, β-lycopene cyclase, and β-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that β-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, α/β-lycopene cyclase, β-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or ζ-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the β-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed. PMID:15821145

Inhibition and Avoidance of mRNA Degradation by RNA Viruses

PubMed Central

Moon, Stephanie L.; Barnhart, Michael D.; Wilusz, Jeffrey

2012-01-01

The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. Thus, to maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described below. PMID:22626865

The In Vitro Effect of Ivermectin on the Activity of Trehalose Synthesis Pathway Enzymes and Their mRNA Expression in the Muscle of Adult Female Ascaris suum (Nematoda)

PubMed Central

Łopieńska-Biernat, Elżbieta; Zaobidna, Ewa Anna

2014-01-01

The in vitro effect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, and tpp genes) in the muscle of adult female Ascaris suum was investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression of tps1 and tps2, and unchanged tpp expression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression of tps2 gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case of tpp gene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway. PMID:25405239

The in vitro effect of ivermectin on the activity of trehalose synthesis pathway enzymes and their mRNA expression in the muscle of adult female Ascaris suum (Nematoda).

PubMed

Dmitryjuk, Małgorzata; Łopieńska-Biernat, Elżbieta; Zaobidna, Ewa Anna

2014-01-01

The in vitro effect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, and tpp genes) in the muscle of adult female Ascaris suum was investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression of tps1 and tps2, and unchanged tpp expression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression of tps2 gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case of tpp gene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway.

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

PubMed

Wojtczak, Blazej A; Sikorski, Pawel J; Fac-Dabrowska, Kaja; Nowicka, Anna; Warminski, Marcin; Kubacka, Dorota; Nowak, Elzbieta; Nowotny, Marcin; Kowalska, Joanna; Jemielity, Jacek

2018-05-09

The 5' cap consists of 7-methylguanosine (m 7 G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m 7 GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients

PubMed Central

Wedler, Alice; Rot, Swetlana; Keßler, Jacqueline; Kehlen, Astrid; Holzhausen, Hans-Jürgen; Bache, Matthias; Würl, Peter; Kappler, Matthias

2017-01-01

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (rs = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients’ disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression. PMID:29215551

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients.

PubMed

Greither, Thomas; Wedler, Alice; Rot, Swetlana; Keßler, Jacqueline; Kehlen, Astrid; Holzhausen, Hans-Jürgen; Bache, Matthias; Würl, Peter; Taubert, Helge; Kappler, Matthias

2017-12-07

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (r s = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients' disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression.

Sex differences in spatiotemporal expression of AR, ERα, and ERβ mRNA in the perinatal mouse brain.

PubMed

Mogi, Kazutaka; Takanashi, Haruka; Nagasawa, Miho; Kikusui, Takefumi

2015-01-01

It has been shown that every masculinized function might be organized by a particular contribution of androgens vs. estrogens in a critical time window. Here, we aimed to investigate the sex differences in brain testosterone levels and in the spatiotemporal dynamics of steroid receptor mRNA expression in perinatal mice, by using enzyme immunoassay and real-time PCR, respectively. We found that testosterone levels in the forebrain transiently increased around birth in male mice. During the perinatal period, levels of androgen receptor mRNA in the hypothalamus (hypo) and prefrontal cortex (PFC) were higher in male mice than in female mice. Estrogen receptor α (ERα) mRNA levels in the hypo and hippocampus were higher in male mice than in female mice before birth. In contrast, ERβ mRNA expression in the PFC was higher in female mice immediately after birth. These spatiotemporal sex differences in steroid receptor expression might contribute to organizing sex differences of not only reproductive function, but also anxiety, stress responses, and cognition in mice. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Technical variations in low-input RNA-seq methodologies.

PubMed

Bhargava, Vipul; Head, Steven R; Ordoukhanian, Phillip; Mercola, Mark; Subramaniam, Shankar

2014-01-14

Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.

Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

PubMed

Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

2010-10-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

PubMed

Löffler, T; Al-Robaiy, S; Bigl, M; Eschrich, K; Schliebs, R

2001-06-01

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Concerted elevation of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) activity through independent stimulation of mRNA expression of DGAT1 and DGAT2 by carbohydrate and insulin.

PubMed

Meegalla, Rupalie L; Billheimer, Jeffrey T; Cheng, Dong

2002-11-01

Glucose and insulin are anabolic signals which upregulate the transcriptions of a series of lipogenic enzymes to convert excess carbohydrate into triglycerides for efficient energy storage. These enzymes include ATP-citrate lyase (ACL), acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (G3PA). Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is important to synthesize fatty acids into triglycerides. Two DGATs from different gene families have recently been identified. In the current study, we report that glucose preferentially enhances DGAT1 mRNA expression, whereas insulin specifically increases the level of DGAT2 mRNA. Treatment of adipocytes with glucose and insulin together results in higher DGAT activity in the membrane than cells treated with either of the agents alone, indicating that glucose and insulin have additive effect on DGAT activation. In mice treated with fast/refeeding protocol, DGAT2 mRNA decreased upon fasting and was replenished upon refeeding in adipose tissue and liver. This pattern of change was not observed for DGAT1. Inasmuch as DGAT1 mRNA is less abundant in liver, we suggest that DGAT1 is more involved in fat absorption in the intestine and in basal level triglyceride synthesis in adipose tissue where it is more highly expressed. In contrast, DGAT2 is more likely to play important roles in assembly of de novo synthesized fatty acids into VLDL particles in the liver.

A Cellular High-Throughput Screening Approach for Therapeutic trans-Cleaving Ribozymes and RNAi against Arbitrary mRNA Disease Targets

PubMed Central

Yau, Edwin H.; Butler, Mark C.; Sullivan, Jack M.

2016-01-01

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization. PMID:27233447

Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components.

PubMed

Gilliam-Davis, Shea; Gallagher, Patricia E; Payne, Valerie S; Kasper, Sherry O; Tommasi, Ellen N; Westwood, Brian M; Robbins, Michael E; Chappell, Mark C; Diz, Debra I

2011-07-14

In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT(1)) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT(1) antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT(1) receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT(1b), AT(2), and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT(1) receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1-7) and prevent age-related declines in the leptin receptor and its signaling pathway.

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Hepatic Xenobiotic Metabolizing Enzyme Gene Expression ...

EPA Pesticide Factsheets

BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species. RESULTS: Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD) 19), neonatal (postnatal day (PND) 7), prepubescent (PND32), middle age (12 months), and old age (18 and 24 months) in the C57BL/6J (C57) mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I), conjugation (Phase II) and excretion (Phase III). In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs w

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level

PubMed Central

Meißner, Joachim D; Kubis, Hans-Peter; Scheibe, Renate J; Gros, Gerolf

2000-01-01

The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression. PMID:10673542

mRNA stability in mammalian cells.

PubMed Central

Ross, J

1995-01-01

This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

Mechanical Loading of Articular Cartilage Reduces IL-1-Induced Enzyme Expression

PubMed Central

Torzilli, P. A.; Bhargava, M.; Chen, C. T.

2011-01-01

Objective: Exposure of articular cartilage to interleukin-1 (IL-1) results in increased synthesis of matrix degrading enzymes. Previously mechanical load applied together with IL-1 stimulation was found to reduce aggrecan cleavage by ADAMTS-4 and 5 and MMP-1, -3, -9, and -13 and reduce proteoglycan loss from the extracellular matrix. To further delineate the inhibition mechanism the gene expression of ADAMTS-4 and 5; MMP-1, -3, -9, and -13; and TIMP-1, -2, and -3 were measured. Design: Mature bovine articular cartilage was stimulated with a 0.5 MPa compressive stress and 10 ng/ml of IL-1α for 3 days and then allowed to recover without stimulation for 1 additional day. The media was assayed for proteoglycan content on a daily basis, while chondrocyte gene expression (mRNA) was measured during stimulation and 1 day of recovery. Results: Mechanical load alone did not change the gene expression for ADAMTS, MMP, or TIMP. IL-1 caused an increase in gene expression for all enzymes after 1 day of stimulation while not affecting the TIMP levels. Load applied together with IL-1 decreased the expression levels of ADAMTS-4 and -5 and MMP-1 and -3 and increased TIMP-3 expression. Conclusions: A mechanical load appears to modify cartilage degradation by IL-1 at the cellular level by reducing mRNA. PMID:22039566

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir

2014-03-01

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependentmore » increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of antioxidant proteins is regulated via MAP kinases, Nrf2 and caveolae. • 4-HNE is an effective signaling molecule in keratinocytes.« less

Fructose intake during gestation and lactation differentially affects the expression of hippocampal neurosteroidogenic enzymes in rat offspring.

PubMed

Mizuno, Genki; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Murase, Yuri; Kondo, Kanako; Ishikawa, Hiroaki; Teradaira, Ryoji; Suzuki, Koji; Ohashi, Koji

2017-02-01

Neurosteroids, steroidal hormones synthesized de novo from cholesterol within the brain, stimulate hippocampal functions such as neuron protection and synapse formation. Previously, we examined the effect of maternal fructose on the transcriptional regulation of neurosteroidogenic enzymes. We found that the mRNA expression level of the steroidogenic acute regulatory protein (StAR), peripheral benzodiazepine receptor (PBR), cytochrome P450(11β), 11β-hydroxysteroid dehydrogenase (HSD), and 17β-HSD was altered. However, we could not determine whether maternal fructose intake played a role in the gestation or lactation period because the dam rats were fed fructose solution during both periods. Thus, in this study, we analyzed the hippocampi of the offspring of dams fed fructose during the gestation or lactation period. Maternal fructose consumption during either the gestation or lactation period did not affect the mRNA levels of StAR, P450(17α), 11β-HSD-2, and 17β-HSD-1. PBR expression was down-regulated, even when rats consumed fructose during the lactation period only, while fructose consumption during gestation tended to activate the expression of P450(11β)-2. We found that maternal fructose intake during gestation and lactation differentially affected the expression of hippocampal neurosteroidogenic enzymes in the offspring.

Semicomprehensive analysis of the postnatal age-related changes in the mRNA expression of sex steroidogenic enzymes and sex steroid receptors in the male rat hippocampus.

PubMed

Kimoto, Tetsuya; Ishii, Hirotaka; Higo, Shimpei; Hojo, Yasushi; Kawato, Suguru

2010-12-01

Although sex steroids play a crucial role in the postnatal brain development, the age-related changes in the hippocampal steroidogenesis remain largely unknown. We performed comprehensive investigations for the mRNA expressions of 26 sex steroidogenic enzymes/proteins and three sex steroid receptors in the male rat hippocampus, at the ages of postnatal day (PD) 1, PD4, PD7, PD10, PD14, 4 wk, and 12 wk (adult), by RT-PCR/Southern blotting analysis. The relative expression levels of these enzymes/receptors at PD1 were Srd5a1 > Star > Ar ∼ Hsd17b4 ∼ Hsd17b1 ∼ Hsd17b7 ∼ Esr1 ∼ Srd5a2 > Hsd17b3 > Esr2 > Cyp11a1 > Cyp17a1 > Cyp19a1 ∼ Hsd17b2 > 3β-hydroxysteroid dehydrogenase I. The mRNA levels of essential enzymes for progesterone/testosterone/estradiol metabolisms (Cyp17a1, Hsd17b7, and Cyp19a1) were approximately constant between PD1 and PD14 and then declined toward the adult levels. Cyp11a1 increased during PD4-PD14 and then considerably decreased toward the adult level (∼8% of PD1). Hsd17b1, Hsd17b2, and 3β-hydroxysteroid dehydrogenase I mRNA decreased approximately monotonously. Hsd17b3 increased to approximately 200% of PD1 during PD4-PD14 and was maintained at this high level. The 5α-reductase mRNA was maintained constant (Srd5a1) or decreased monotonically (Srd5a2) toward the adult level. The Esr1 level peaked at PD4 and decreased toward the adult level, whereas Ar greatly increased during PD1-PD14 and was maintained at this high level. The Star and Hsd17b4 levels were maintained constant from neonate to adult. These results suggest that the hippocampal sex steroidogenic properties are substantially altered during the postnatal development processes, which might contribute to brain sexual maturation.

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

PubMed Central

Kreth, Simone; Thon, Niklas; Eigenbrod, Sabina; Lutz, Juergen; Ledderose, Carola; Egensperger, Rupert; Tonn, Joerg C.; Kretzschmar, Hans A.; Hinske, Ludwig C.; Kreth, Friedrich W.

2011-01-01

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined. PMID:21365007

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

PubMed

el Meanawy, M A; Aji, T; Phillips, N F; Davis, R E; Salata, R A; Malhotra, I; McClain, D; Aikawa, M; Davis, A H

1990-07-01

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Proteomic analysis of soybean hypocotyl during recovery after flooding stress.

PubMed

Khan, Mudassar Nawaz; Sakata, Katsumi; Komatsu, Setsuko

2015-05-21

Soybean is a nutritionally important crop, but exhibits reduced growth and yields under flooding stress. To investigate soybean responses during post-flooding recovery, a gel-free proteomic technique was used to examine the protein profile in the hypocotyl. Two-day-old soybeans were flooded for 2 days and hypocotyl was collected under flooding and during the post-flooding recovery period. A total of 498 and 70 proteins were significantly changed in control and post-flooding recovering soybeans, respectively. Based on proteomic and clustering analyses, three proteins were selected for mRNA expression and enzyme activity assays. Pyruvate kinase was increased under flooding, but gradually decreased during post-flooding recovery period at protein abundance, mRNA, and enzyme activity levels. Nucleotidylyl transferase was decreased under flooding and increased during post-flooding recovery at both mRNA expression and enzyme activity levels. Beta-ketoacyl reductase 1 was increased under flooding and decreased during recovery at protein abundance and mRNA expression levels, but its enzyme activity gradually increased during the post-flooding recovery period. These results suggest that pyruvate kinase, nucleotidylyl transferase, and beta-ketoacyl reductase play key roles in post-flooding recovery in soybean hypocotyl by promoting glycolysis for the generation of ATP and regulation of secondary metabolic pathways. This study analyzed post-flooding recovery response mechanisms in soybean hypocotyl, which is a model organ for studying secondary growth, using a gel-free proteomic technique. Mass spectrometry analysis of proteins extracted from soybean hypocotyls identified 20 common proteins between control and flooding-stressed soybeans that changed significantly in abundance over time. The hypocotyl proteins that changed during post-flooding recovery were assigned to protein, development, secondary metabolism, and glycolysis categories. The analysis revealed that three proteins, pyruvate kinase, nucleotidylyl transferase, and beta-ketoacyl reductase, were increased in hypocotyl under flooding conditions and during post-flooding recovery. The proteins are involved in glycolysis, nucleotide synthesis and amino acid activation, and complex fatty acid biosynthesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Infrasound-induced changes on sexual behavior in male rats and some underlying mechanisms.

PubMed

Zhuang, Zhiqiang; Pei, Zhaohui; Chen, Jingzao

2007-01-01

To investigate some bioeffects of infrasound on copulation as well as underlying mechanisms, we inspected the changes of sexual behavior, serum testosterone concentration and mRNA expression levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in testes of rats exposed to infrasound of 8Hz at 90 or 130dB for 1, 7, 14 and 21 days (2h/day), respectively. Rats exposed to 90dB exhibited significant decrement in sexual behavior, serum testosterone levels and mRNA expression levels of StAR and P450scc at the time point of 1 day but not at the rest time points, and no significantly change of SF-1 mRNA expression was observed over the period of 21 days in spite of mild fluctuation. Rats exposed to 130dB exhibited significant decrement in all aspects above, which became more profound with prolonged exposure. Our conclusion is that adverse bioeffects of infrasound on reproduction depend on some exposure parameters, the mechanism of which could involve in the decreased expression of some key enzymes or regulator for testosterone biosynthesis. Copyright © 2006. Published by Elsevier B.V.

Current Status of Messenger RNA Delivery Systems.

PubMed

Stanton, Matthew G

2018-06-01

Messenger RNA is emerging as a highly versatile biological construct for creation of impactful medicines. mRNA vaccines directed toward infectious disease and cancer are in clinical development with encouraging early reads on tolerability and efficacy. The use of mRNA to direct intense but transient expression of paracrine factors is finding utility in reprogramming progenitor cells for wound healing and cardiac regeneration and for stimulation of antitumor immune responses, at least preclinically as we await clinical results. The use of mRNA for prolonged and repeated expression of proteins and enzymes to treat rare, typically monogenic disease is nearing clinical entry. These uses of mRNA require delivery solutions, and the application of and improvement to existing nanoparticle nucleic acid delivery systems have jump started the pace of development and reenergized the field of particle based nucleic acid delivery. The current status of mRNA delivery is reviewed in this article with an eye toward clinical tractability.

[Effect of soy isoflavone on gene expression of leptin and insulin sensibility in insulin-resistant rats].

PubMed

Chen, Shi-wei; Zhang, Li-shi; Zhang, Hong-min; Feng, Xiao-fan; Peng, Xiao-li

2006-04-18

To explore the effects of soy isoflavone (SIF) on gene expression of leptin and insulin sensibility in insulin-resistant (IR) rats induced by high-fat, and to reveal the mechanisms of SIF in ameliorating insulin sensibility. IR rats were randomly divided into four groups based on their insulin-resistant indexes (IRI): one model control group and three SIF groups that were gavaged with water solutions with SIF at doses of 0 mg/kg, 50 mg/kg, 150 mg/kg, and 450 mg/kg, respectively. After one month, fasting glucose, fasting insulin, leptin in serum, and leptin mRNA in the perirenal adipocyte were detected by enzymic method, radioimmunoassay, enzyme linked immunosorbent assay, and real time quantitative RT-PCR, respectively. The model control group was used to compare against the other groups: (1) Insulin and IRI were lower in the 150 mg/kg and 450 mg/kg groups; (2) In the 450 mg/kg group, body weight and leptin mRNA expression were lower, serum leptin content was higher. These results indicate that soy isoflavone might decrease body weight of rats and leptin mRNA, increase serum leptin level, and ameliorate leptin and insulin sensitivities.

Garlic Influences Gene Expression In Vivo and In Vitro.

PubMed

Charron, Craig S; Dawson, Harry D; Novotny, Janet A

2016-02-01

There is a large body of preclinical research aimed at understanding the roles of garlic and garlic-derived preparations in the promotion of human health. Most of this research has targeted the possible functions of garlic in maintaining cardiovascular health and in preventing and treating cancer. A wide range of outcome variables has been used to investigate the bioactivity of garlic, ranging from direct measures of health status such as cholesterol concentrations, blood pressure, and changes in tumor size and number, to molecular and biochemical measures such as mRNA gene expression, protein concentration, enzyme activity, and histone acetylation status. Determination of how garlic influences mRNA gene expression has proven to be a valuable approach to elucidating the mechanisms of garlic bioactivity. Preclinical studies investigating the health benefits of garlic far outnumber human studies and have made frequent use of mRNA gene expression measurement. There is an immediate need to understand mRNA gene expression in humans as well. Although safety and ethical constraints limit the types of available human tissue, peripheral whole blood is readily accessible, and measuring mRNA gene expression in whole blood may provide a unique window to understanding how garlic intake affects human health. © 2016 American Society for Nutrition.

Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

PubMed Central

Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

2014-01-01

Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

PubMed Central

2014-01-01

Background Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission. PMID:24898526

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

PubMed

Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

2014-06-04

Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

The effect of androgen excess on maternal metabolism, placental function and fetal growth in obese dams.

PubMed

Fornes, Romina; Maliqueo, Manuel; Hu, Min; Hadi, Laila; Jimenez-Andrade, Juan M; Ebefors, Kerstin; Nyström, Jenny; Labrie, Fernand; Jansson, Thomas; Benrick, Anna; Stener-Victorin, Elisabet

2017-08-14

Pregnant women with polycystic ovary syndrome (PCOS) are often overweight or obese. To study the effects of maternal androgen excess in obese dams on metabolism, placental function and fetal growth, female C57Bl6J mice were fed a control (CD) or a high fat/high sucrose (HF/HS) diet for 4-10 weeks, and then mated. On gestational day (GD) 15.5-17.5, dams were injected with dihydrotestosterone (CD-DHT, HF/HS-DHT) or a vehicle (CD-Veh, HF/HS-Veh). HF/HS dams had higher fat content, both before mating and on GD18.5, with no difference in glucose homeostasis, whereas the insulin sensitivity was higher in DHT-exposed dams. Compared to the CD groups, the livers from HF/HS dams weighed more on GD18.5, the triglyceride content was higher, and there was a dysregulation of liver enzymes related to lipogenesis and higher mRNA expression of Fitm1. Fetuses from HF/HS-Veh dams had lower liver triglyceride content and mRNA expression of Srebf1c. Maternal DHT exposure, regardless of diet, decreased fetal liver Pparg mRNA expression and increased placental androgen receptor protein expression. Maternal diet-induced obesity, together with androgen excess, affects maternal and fetal liver function as demonstrated by increased triglyceride content and dysfunctional expression of enzymes and transcription factors involved in de novo lipogenesis and fat storage.

Phenobarbital reduces blood glucose and gluconeogenesis through down-regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression in rats.

PubMed

Oda, Hiroaki; Okuda, Yuji; Yoshida, Yukiko; Kimura, Noriko; Kakinuma, Atsushi

2015-10-23

The regulatory mechanism of phosphoenolpyruvate carboykinase (GTP) (EC 4.1.1.32) (PEPCK) gene expression and gluconeogenesis by phenobarbital (PB), which is known to induce drug-metabolizing enzymes, was investigated. Higher level of PEPCK mRNA was observed in spherical rat primary hepatocytes on EHS-gel than monolayer hepatocytes on TIC (type I collagen). We found that PB directly suppressed PEPCK gene expression in spherical hepatocytes on EHS-gel, but not in those on TIC. PB strongly suppressed cAMP-dependent induction of PEPCK gene expression. Tyrosine aminotransferase (TAT), another gluconeogenic enzyme, was induced by cAMP, but not suppressed by PB. Chronic administration of PB reduced hepatic PEPCK mRNA in streptozotocin-induced diabetic and nondiabetic rats, and PB reduced blood glucose level in diabetic rats. Increased TAT mRNA in diabetic rats was not suppressed by PB. These results indicated that PB-dependent reduction is specific to PEPCK. From pyrvate challenge test, PB suppressed the increased gluconeogenesis in diabetic rats. PEPCK gene promoter activity was suppressed by PB in HepG2 cells. In conclusion, we found that spherical hepatocytes cultured on EHS-gel are capable to respond to PB to suppress PEPCK gene expression. Moreover, our results indicate that hypoglycemic action of PB result from transcriptional repression of PEPCK gene and subsequent suppression of gluconeogenesis. Copyright © 2015. Published by Elsevier Inc.

Phenolic compounds increase the transcription of mouse intestinal maltase-glucoamylase and sucrase-isomaltase.

PubMed

Simsek, Meric; Quezada-Calvillo, Roberto; Nichols, Buford L; Hamaker, Bruce R

2017-05-24

Diverse natural phenolic compounds show inhibition activity of intestinal α-glucosidases, which may constitute the molecular basis for their ability to control systemic glycemia. Additionally, phenolics can modify mRNA expression for proteins involved in nutritional, metabolic or immune processes. To explore the possibility that phenolics can regulate the mRNA expression, enzymatic activity, and protein synthesis/processing of intestinal Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), small intestinal explants from Balb/c mice were cultured for 24 h in the presence or absence of gallic acid, caffeic acid, and (+)-catechin at 0.1, 0.5, and 1 mM. We measured the levels of MGAM and SI mRNA expression by qRT-PCR, maltase and sucrase activities by a standard colorimetric method and the molecular size distribution of MGAM and SI proteins by western blotting. mRNA expression for MGAM was induced by the three phenolic compounds at 0.1 mM. mRNA expression for SI was induced by caffeic and gallic acids, but not by (+)-catechin. Caffeic acid was the most effective inducer of mRNA expression of these enzymes. Total maltase and sucrase activities were not affected by treatment with phenolics. The proportion of high molecular size forms of MGAM was significantly increased by two of the three phenolic compounds, but little effect was observed on SI proteins. Thus, changes in the protein synthesis/processing, affecting the proportions of the different molecular forms of MGAM, may account for the lack of correlation between mRNA expression and enzymatic activity.

Effects of hypothermia and cerebral ischemia on cold-inducible RNA-binding protein mRNA expression in rat brain.

PubMed

Liu, Aijun; Zhang, Zhiwen; Li, Anmin; Xue, Jinghui

2010-08-06

CIRP (cold-inducible RNA-binding protein) mRNA is highly expressed in hypothermic conditions in mammalian cells, and the relationship between CIRP and neuroprotection for cerebral ischemia under hypothermia has been focused upon. At present, however, the expression characteristics of CIRP under hypothermia and cerebral ischemia in vivo are not clearly elucidated. In this study, CIRP mRNA expression in various regions of rat brain was examined by reverse transcriptase polymerase chain reaction (RT-PCR). CIRP expression levels were found to be similar in the hippocampus and cortex. Real-time quantitative PCR analysis revealed increasing CIRP mRNA expression in the cortex during the 24-h observation period following treatment with hypothermia or cerebral ischemia, with a greater increase in the hypothermia group. When cerebral ischemia was induced following hypothermia, CIRP mRNA expression in the cortex again showed a significant increasing tendency, but ischemia delayed the appearance of this increase. To reveal the relationship between CIRP and energy metabolism in the rat brain, lactate and pyruvate concentrations in the cortex of the rats treated with hypothermia, ischemia and ischemia after hypothermia were determined by spectrophotometric assay, and levels of phosphofructokinas-1 (PFK-1), the major regulatory enzyme of the glycolytic pathway, in the rat cortex in the three groups was also analyzed by Western blot. Using linear correlation, lactate and pyruvate concentrations, and PFK-1 levels, were each analyzed in the three groups in association with CIRP mRNA expression levels. The analysis did not reveal any correlation between the three metabolic parameters and CIRP mRNA expression induced by hypothermia, suggesting that while playing a role in neuroprotection under hypothermia, CIRP does not affect cerebral energy metabolism. Copyright 2010. Published by Elsevier B.V.

Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

PubMed

Wojciechowska, A; Mlynarczuk, J; Kotwica, J

2017-01-15

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3β-hydroxysteroid dehydrogenase (3βHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits 1

PubMed Central

DellaPenna, Dean; Kates, David S.; Bennett, Alan B.

1987-01-01

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:16665727

Transcriptome analysis of the Tan sheep testes: Differential expression of antioxidant enzyme-related genes and proteins in response to dietary vitamin E supplementation.

PubMed

Xu, Chenchen; Zuo, Zhaoyun; Liu, Kun; Jia, Huina; Zhang, Yuwei; Luo, Hailing

2016-03-15

Gene-chip technology was employed to study the effect of dietary vitamin E on gene expression in sheep testes based on our previous research. Thirty-five male Tan sheep (20-30 days after weaning) with similar body weight were randomly allocated into five groups and supplemented 0, 20, 100, 200 and 2,000 IU sheep(-1)day(-1) vitamin E (treatments denoted as E0, E20, E100, E200, and E2000, respectively) for 120 days. At the end of the study the sheep were slaughtered and the testis samples were immediately collected and stored in liquid nitrogen. Differences in gene expression between different treated groups were identified. Based on GO enrichment analysis and the KEGG database to evaluate the gene expression data we found that vitamin E might affect genes in the testes by modulating the oxidation level, by affecting the expression of various receptors and transcription factors in biological pathways, and by regulating the expression of metabolism-associated genes. The effect of vitamin E supplementation on the expression of oxidative enzyme-related genes was detected by quantitative real-time PCR (qRT-PCR) and Western blot. The results show that dietary vitamin E, at various doses, can significantly increase (P<0.05) the mRNA and protein expression of Glutathione peroxidase 3 and Glutathione S-transferase alpha 1. In addition, the results of qRT-PCR of the antioxidant enzyme genes were consistent with those obtained using the gene chip microarray analysis. In summary, the dietary vitamin E treatment altered the expression of a number of genes in sheep testes. The increase in the mRNA and protein levels of antioxidant enzyme genes, coupled with the elevation in the activity of the antioxidant enzymes were primarily responsible for the improved reproductive performance promoted by dietary vitamin E. Copyright © 2015 Elsevier B.V. All rights reserved.

Collagenase IV plays an important role in regulating hair cycle by inducing VEGF, IGF-1, and TGF-β expression

PubMed Central

Hou, Chun; Miao, Yong; Wang, Jin; Wang, Xue; Chen, Chao-Yue; Hu, Zhi-Qi

2015-01-01

Background It has been reported that collagenases (matrix metalloproteinase 2 [MMP-2] and matrix metalloproteinase 9 [MMP-9]) are associated with hair cycle, whereas the mechanism of the association is largely unknown. Methods The mice were randomly allocated into four groups: saline, and 5, 10, and 15 nM SB-3CT. Immunohistochemical analysis was employed to examine MMP-2 and MMP-9 protein. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay were performed to determine mRNA and protein levels of VEGF, IGF-1, TGF-β, and GAPDH. Growing hair follicles from anagen phase III–IV were scored based on hematoxylin and eosin staining. Hair regrowth was also evaluated. Results Results showed that mRNA expressions of enzymes changed with a peak at late anagen and a trough at telogen after depilation. Immunostaining showed that the highest expression of MMP-2 was more than that of MMP-9, and the highest expression of enzymes changed during anagen. The localizations of MMP-2 changed from dermal papilla, keratinocyte strand, out of root sheath, and basal plate at early anagen, to hair bulb, inner root sheath, and outer root sheath at late anagen. The localization of MMP-9 changed from partial keratinocyte to dermal papilla at early anagen and to outer root sheath at late anagen. VEGF, IGF-1, and TGF-β have been shown to regulate hair growth. We found mRNA and protein expressions of VEGF and IGF-1 fluctuated with a peak at anagen and a decrease at catagen to telogen. In contrast, mRNA and protein expressions of TGF-β changed with highest and lowest levels at anagen and telogen, respectively. With selective inhibitor of collagenase IV, SB-3CT, mice showed significant suppressed hair growth and decreased expression of VEGF, IGF-1, and TGF-β. The MMPs agonist also significantly increased expression of VEGF, IGF-1, and TGF-β. Meanwhile, SB-3CT treatment significantly suppressed hair growth. Conclusion All these data suggest that the type IV collagenases, MMP-2 and MMP-9, play important roles in hair cycle, and this could be mediated by induced expression of VEGF, IGF-1, and TGF-β. PMID:26451090

Characterization of the impact of life stage on gene -chemical interactions in the liver

EPA Science Inventory

Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). We have carried out a comprehensive analysis of the mRNA expression of XMETs thro...

Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos.

PubMed

Cheng, Shih-Lin; Lai, Yi-Ling; Lee, Ming-Che; Shen, Perng-Chih; Liu, Shyh-Shyan; Liu, Bing-Tsan

2014-07-03

The deer velvet or its extracts has been widely used in clinic. It has been used in promoting reproductive performances and treating of oxidation and aging process. The aim of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer; Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro. Mouse 4-cells embryos were divided into 16 groups for 72 h in vitro incubation. The embryonic development stages and morphology were evaluated every 12h in experimental period. The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the blastocysts. The 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant stress challenged. The blastocyst developmental rate (90.0-90.4%, P>0.05) and normal morphological rate (84.4-85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group (90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25 μM) and subsequently incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue development; the blastocyst developmental rate of these groups were similar to that in the control group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did not significantly differ among the designed DV treatment groups (P>0.05). The FSD velvet extract in adequate concentration could promote anti-oxidative enzymes mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under oxidative stress, and maintain the blastocyst developmental ability in vitro. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

PubMed

Tomankova, Veronika; Liskova, Barbora; Skalova, Lenka; Bartikova, Hana; Bousova, Iva; Jourova, Lenka; Anzenbacher, Pavel; Ulrichova, Jitka; Anzenbacherova, Eva

2015-07-15

Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane). Copyright © 2015 Elsevier Inc. All rights reserved.

Augmented Rac1 Expression and Activity are Associated with Oxidative Stress and Decline of β Cell Function in Obesity.

PubMed

Zhou, Shutong; Yu, Dongni; Ning, Shangyong; Zhang, Heli; Jiang, Lei; He, Lei; Li, Miao; Sun, Mingxiao

2015-01-01

The aim of this study was to clarify the relationship among Rac1 expression and activation, oxidative stress and β cell dysfunction in obesity. In vivo, serum levels of glucose, insulin, oxidative stress markers and Rac1 expression were compared between ob/ob mice and C57BL/6J controls. Then, these variables were rechecked after the administration of the specific Rac1 inhibitor-NSC23766 in ob/ob mice. In vitro, NIT-1 β cells were cultured in a hyperglycemic and/or hyperlipidemic state with or without NSC23766, and the differences of Rac1 expression and translocation, NADPH oxidase(Nox) enzyme activity, reactive oxygen species (ROS) and insulin mRNA were observed. ob/ob mice displayed abnormal glycometabolism, oxidative stress and excessive expression of Rac1 in the pancreas. NSC23766 injection inhibited the expression of Rac1 in the pancreas, along with amelioration of oxidative stress and glycometabolism in obese mice. Under hyperglycemic and/or hyperlipidemic conditions, Rac1 translocated to the cellular membrane, induced activation of the NADPH oxidase enzyme and oxidative stress, and simultaneously reduced the insulin mRNA expression in NIT-1 β cells. Inhibiting Rac1 activity could alleviate oxidative stress and meliorate the decline of insulin mRNA in β cells. Rac1 might contribute to oxidative stress systemically and locally in the pancreas in obesity. The excessive activation and expression of Rac1 in obesity were associated with β cell dysfunction through ROS production. © 2015 S. Karger AG, Basel.

Altered Expression of Urea Cycle Enzymes in Amyloid-β Protein Precursor Overexpressing PC12 Cells and in Sporadic Alzheimer's Disease Brain.

PubMed

Jęśko, Henryk; Lukiw, Walter J; Wilkaniec, Anna; Cieślik, Magdalena; Gąssowska-Dobrowolska, Magdalena; Murawska, Emilia; Hilgier, Wojciech; Adamczyk, Agata

2018-01-01

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

AMPKα2 regulates expression of estrogen-related receptor alpha, a metabolic transcription factor related to heart failure development

PubMed Central

Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Fassett, John; Zhang, Ying; Xin, Yi; Hall, Jennifer L.; Viollet, Benoit; Bache, Robert J.; Huang, Yimin; Chen, Yingjie

2011-01-01

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial Estrogen-Related Receptor alpha (ERRα), but not PPAR gamma coactivator-1 alpha (PGC1α), in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of LV dysfunction, pulmonary congestion and decreases of a group of myocardial energy metabolism related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 KO decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial PGC1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, over-expression of ERRα in AMPKα2 KO neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes. PMID:21825219

BAG3 directly stabilizes Hexokinase 2 mRNA and promotes aerobic glycolysis in pancreatic cancer cells.

PubMed

An, Ming-Xin; Li, Si; Yao, Han-Bing; Li, Chao; Wang, Jia-Mei; Sun, Jia; Li, Xin-Yu; Meng, Xiao-Na; Wang, Hua-Qin

2017-12-04

Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents. © 2017 An et al.

Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

PubMed

Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

2009-05-01

The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

Effect of Caloric Restriction and AMPK Activation on Hepatic Nuclear Receptor, Biotransformation Enzyme, and Transporter Expression in Lean and Obese Mice

PubMed Central

Kulkarni, Supriya R.; Xu, Jialin; Donepudi, Ajay C.; Wei, Wei

2014-01-01

Purpose Fatty liver alters liver transporter expression. Caloric restriction (CR), the recommended therapy to reverse fatty liver, increases Sirtuin1 deacetylase activity in liver. This study evaluated whether CR and CR mimetics reversed obesity-induced transporter expression in liver and hepatocytes. Methods mRNA and protein expression was determined in adult lean (lean) and leptin-deficient obese (OB) mice fed ad libitum or placed on 40% (kCal) reduced diet. Hepatocytes were isolated from lean and OB mice, treated with AMP Kinase activators, and gene expression was determined. Results CR decreased Oatp1a1, Oatp1b2, and Abcb11 mRNA expression in lean, but not OB mice. CR increased Abcc2 mRNA OB livers, whereas protein expression increased in both genotypes. CR increased Abcc3 protein expression increased in OB livers. CR did not alter Abcc1, 4 and 5 mRNA expression in lean mice but decreased expression in livers of OB mice. CR increased Abcc4 protein in lean, but not OB mice. Conclusions CR restriction reversed the expression of some, but not all transporters in livers of OB mice. Overall, these data indicate a potential for CR to restore some hepatic transporter changes in OB mice, but suggest a functional leptin axis is needed for reversal of expression for some transporters. PMID:23949303

High-Fat Diet Induces Oxidative Stress and MPK2 and HSP83 Gene Expression in Drosophila melanogaster.

PubMed

Trindade de Paula, Mariane; Poetini Silva, Márcia Rósula; Machado Araujo, Stífani; Cardoso Bortolotto, Vandreza; Barreto Meichtry, Luana; Zemolin, Ana Paula Pegoraro; Wallau, Gabriel L; Jesse, Cristiano Ricardo; Franco, Jeferson Luís; Posser, Thaís; Prigol, Marina

2016-01-01

The consumption of a high-fat diet (HFD) causes alteration in normal metabolism affecting lifespan of flies; however molecular mechanism associated with this damage in flies is not well known. This study evaluates the effects of ingestion of a diet supplemented with 10% and 20% of coconut oil, which is rich in saturated fatty acids, on oxidative stress and cells stress signaling pathways. After exposure to the diet for seven days, cellular and mitochondrial viability, lipid peroxidation and antioxidant enzymes SOD and CAT activity, and mRNA expression of antioxidant enzymes HSP83 and MPK2 were analyzed. To confirm the damage effect of diet on flies, survival and lifespan were investigated. The results revealed that the HFD augmented the rate of lipid peroxidation and SOD and CAT activity and induced a higher expression of HSP83 and MPK2 mRNA. In parallel, levels of enzymes involved in lipid metabolism (ACSL1 and ACeCS1) were increased. Our data demonstrate that association among metabolic changes, oxidative stress, and protein signalization might be involved in shortening the lifespan of flies fed with a HFD.

Characterization, molecular modelling and developmental expression of zebrafish manganese superoxide dismutase.

PubMed

Lin, Chi-Tsai; Tseng, Wen-Chung; Hsiao, Nai-Wan; Chang, Hsiao-Huang; Ken, Chuian-Fu

2009-08-01

A 977 bp cDNA containing an open reading frame encoding 224 amino acid residues of manganese superoxide dismutase was cloned from zebrafish (zMn-SOD). The deduced amino acid sequence showed high identity with the sequences of Mn-SODs from human (85.1%) to nematode (61.6%). The 3-D structure model was superimposed on the relative domains of human Mn-SOD with the root mean square (rms) deviation of 0.0919 A. The recombinant mature zMn-SOD with enzyme activity was purified using His-tag technique. The half-life of the enzyme is approximately 48 min and its thermal inactivation rate constant k(d) is 0.0154 min(-1)at 70 degrees C. The enzyme was active under a broad pH (2.2-11.2) and in the presence of up to 4% SDS. Real-time RT-PCR assay was used to detect the zMn-SOD mRNA expression during the developmental stages following a challenge with paraquat. A high level expression of Mn-SOD mRNA was detected at the cleavage stage, but decreased significantly under paraquat treatment. The results indicated that Mn-SOD plays an important role during embryonic development.

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

PubMed

Trejo, Sebastián A; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L; Canals, Francesc; Avilés, Francesc X

2009-07-01

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

Enhanced UGT1A1 Gene and Protein Expression in Endometriotic Lesions.

PubMed

Piccinato, Carla A; Neme, Rosa M; Torres, Natália; da Silva Victor, Elivane; Brudniewski, Heloísa F; Rosa E Silva, Júlio C; Ferriani, Rui A

2018-01-01

The cellular function in endometriosis lesions depends on a highly estrogenic milieu. Lately, it is becoming evident that, besides the circulating levels of estrogens, the balance of synthesis versus inactivation (metabolism) of estrogens by intralesion steroid-metabolizing enzymes also determines the local net estrogen availability. In order to extend the knowledge of the role of estrogen-metabolizing enzymes in endometriosis, we investigated the gene and protein expression of a key uridine diphospho-glucuronosyltransferase (UGT) for estrogen glucuronidation, UGT1A1, in eutopic endometrial samples obtained from nonaffected and endometriosis-affected women and also from endometriotic lesions. Although UGT1A1 messenger RNA (mRNA) expression was detected at similar frequencies in endometriotic lesions and in eutopic endometrial samples, the levels of mRNA expression were greater in deep-infiltrating endometriotic lesions and in non-deep-infiltrating lesions when compared with either control endometrium or eutopic endometrium from women with endometriosis. Overall, we observed that protein expression of UGT1A1 was significantly more frequent in samples from endometriotic lesions in comparison with endometria. In addition, expression of UGT1A1 protein was greater in deep-infiltrating than in non-deep-infiltrating endometriotic lesions. We suggest that the finding of increased expression of UGT1A1 in lesions versus endometria might be related to impairment of regulatory mechanisms, in response to a highly estrogenic milieu, and that this enzyme may be a new target for therapy.

Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary

PubMed Central

Ortega, Israel; Sokalska, Anna; Villanueva, Jesus A.; Cress, Amanda B.; Wong, Donna H.; Stener-Victorin, Elisabet; Stanley, Scott D.; Duleba, Antoni J.

2012-01-01

Objective To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model. Design In vivo and in vitro studies. Setting Research laboratory. Animal(s) Immature Sprague-Dawley female rats. Intervention(s) In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 µg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. Main Outcome Measure(s) Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. Result(s) In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. Conclusion(s) These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary. PMID:23200686

Developmental changes in drug-metabolizing enzyme expression during metamorphosis of Xenopus tropicalis.

PubMed

Mori, Junpei; Sanoh, Seigo; Kashiwagi, Keiko; Hanada, Hideki; Shigeta, Mitsuki; Suzuki, Ken-Ichi T; Yamamoto, Takashi; Kotake, Yaichiro; Sugihara, Kazumi; Kitamura, Shigeyuki; Kashiwagi, Akihiko; Ohta, Shigeru

2017-01-01

A large number of chemicals are routinely detected in aquatic environments, and these chemicals may adversely affect aquatic organisms. Accurate risk assessment requires understanding drug-metabolizing systems in aquatic organisms because metabolism of these chemicals is a critical determinant of chemical bioaccumulation and related toxicity. In this study, we evaluated mRNA expression levels of nuclear receptors and drug-metabolizing enzymes as well as cytochrome P450 (CYP) activities in pro-metamorphic tadpoles, froglets, and adult frogs to determine how drug-metabolizing systems are altered at different life stages. We found that drug-metabolizing systems in tadpoles were entirely immature, and therefore, tadpoles appeared to be more susceptible to chemicals compared with metamorphosed frogs. On the other hand, cyp1a mRNA expression and CYP1A-like activity were higher in tadpoles. We found that thyroid hormone (TH), which increases during metamorphosis, induced CYP1A-like activity. Because endogenous TH concentration is significantly increased during metamorphosis, endogenous TH would induce CYP1A-like activity in tadpoles.

Expression of lysozyme in the life history of the house fly (Musca domestica l.).

PubMed

Nayduch, Dana; Joyner, Chester

2013-07-01

From egg to adult, all life history stages of house flies associate with septic environments teeming with bacteria. House fly lysozyme was first identified in the larval midgut, where it is used for digestion of microbe-rich meals because of its broad-spectrum activity against gram-positive and gram-negative bacteria as well as fungi. This study aimed to determine the temporal expression of lysozyme in the life history of house flies (from egg through adults) on both the mRNA and protein level, and to determine the tissue-specific expression of lysozyme in adult flies induced by feeding Staphylococcus aureus. From 30-min postoviposition through adulthood, all life history stages of the house fly express lysozyme on the mRNA level. In adult flies, lysozyme is expressed both locally in the alimentary canal and systemically in the fat body. Interestingly, we found that during the normal life history of flies, lysozyme protein was only detected in larval stages and older adults, likely because of ingestion of immune-stimulating levels of bacteria, not experienced during egg, pupa, and teneral adult stages. Constitutive expression on the mRNA level implies that this effector is a primary defense molecule in all stages of the house fly life history, and that a mechanism for posttranscriptional control of mature lysozyme enzyme expression may be present. Lysozyme active enzyme primarily serves both a digestive and defensive function in larval and adult flies, and may be a key player in the ability of Musca domestica L. to thrive in microbe-rich environments.

Redox regulation of antioxidant enzymes: post-translational modulation of catalase and glutathione peroxidase activity by resveratrol in diabetic rat liver.

PubMed

Sadi, Gökhan; Bozan, Davut; Yildiz, Huseyin Bekir

2014-08-01

Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.

Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

PubMed Central

Kayampilly, Pradeep P.; Wanamaker, Brett L.; Stewart, James A.; Wagner, Carrie L.; Menon, K. M. J.

2010-01-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation. PMID:20810561

Feeding glycerol-enriched yeast culture improves lactation performance, energy status, and hepatic gluconeogenic enzyme expression of dairy cows during the transition period.

PubMed

Ye, G; Liu, J; Liu, Y; Chen, X; Liao, S F; Huang, D; Huang, K

2016-06-01

This study aimed to evaluate the effects of feeding glycerol-enriched yeast culture (GY) on feed intake, lactation performance, blood metabolites, and expression of some key hepatic gluconeogenic enzymes in dairy cows during the transition period. Forty-four multiparous transition Holstein cows were blocked by parity, previous 305-d mature equivalent milk yield, and expected calving date and randomly allocated to 4 dietary treatments: Control (no additive), 2 L/d of GY (75.8 g/L glycerol and 15.3 g/L yeast), 150 g/d of glycerol (G; 0.998 g/g glycerol), and 1 L/d of yeast culture (Y; 31.1 g/L yeast). All additives were top-dressed and hand mixed into the upper one-third of the total mixed ration in the morning from -14 to +28 d relative to calving. Results indicated that the DMI, NE intake, change of BCS, and milk yields were not affected by the treatments ( > 0.05). Supplementation of GY or Y increased milk fat percentages, milk protein percentages, and milk protein yields relative to the Control or G group ( < 0.05). Cows fed GY or G had higher glucose levels and lower β-hydroxybutyric acid (BHBA) and NEFA levels in plasma than cows fed the Control ( < 0.05) and had lower NEFA levels than cows fed Y ( < 0.05). On 14 d postpartum, cows fed GY or G had higher enzyme activities, mRNA, and protein expression of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; < 0.05); higher enzyme activities ( < 0.05) and a tendency toward higher mRNA expression ( < 0.10) of glycerol kinase (GK); and a tendency toward higher enzyme activities of pyruvate carboxylase (PC) in the liver ( < 0.10) when compared with cows fed Control or Y. The enzyme activities, mRNA, and protein expression of PEPCK-C, PC, and GK did not differ between cows fed GY and G ( > 0.10). In conclusion, dietary GY or Y supplementation increased the milk fat and protein content of the cows in early lactation and GY or G supplementation improved the energy status as indicated by greater plasma glucose and lower plasma BHBA and NEFA concentrations and upregulated the hepatic gluconeogenic enzymes of dairy cows during the transition period. Feeding cows with a GY mixture in the peripartum period combined the effects of yeast on lactation performance and the effects of glycerol on energy status in dairy cows.

mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

PubMed

De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

2010-07-01

Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Effects of triiodothyronine on turnover rate and metabolizing enzymes for thyroxine in thyroidectomized rats.

PubMed

Nagao, Hidenori; Sasaki, Makoto; Imazu, Tetsuya; Takahashi, Kenjo; Aoki, Hironori; Minato, Kouichi

2014-10-29

Previous studies in rats have indicated that surgical thyroidectomy represses turnover of serum thyroxine (T4). However, the mechanism of this process has not been identified. To clarify the mechanism, we studied adaptive variation of metabolic enzymes involved in T4 turnover. We compared serum T4 turnover rates in thyroidectomized (Tx) rats with or without infusion of active thyroid hormone, triiodothyronine (T3). Furthermore, the levels of mRNA expression and activity of the metabolizing enzymes, deiodinase type 1 (D1), type 2 (D2), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase were also compared in several tissues with or without T3 infusion. After the T3 infusion, the turnover rate of serum T4 in Tx rats returned to normal. Although mRNA expression and activity of D1 decreased significantly in both liver and kidneys without T3 infusion, D2 expression and activity increased markedly in the brain, brown adipose tissue, and skeletal muscle. Surprisingly, hepatic UGT mRNA expression and activity in Tx rats increased significantly in comparison with normal rats, and returned to normal after T3 infusion. This study suggests that repression of the disappearance of serum T4 in rats after Tx is a homeostatic response to decreased serum T3 concentrations. Additionally, T4 glucuronide is a storage form of T4, but may also have biological significance. These results suggest strongly that repression of deiodination of T4 by D1 in the liver and kidneys plays a major role in thyroid hormone homeostasis in Tx rats, and that hepatic UGT also plays a key role in this mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components

PubMed Central

Gilliam-Davis, Shea; Gallagher, Patricia E.; Payne, Valerie S.; Kasper, Sherry O.; Tommasi, Ellen N.; Westwood, Brian M.; Robbins, Michael E.; Chappell, Mark C.

2011-01-01

In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT1) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT1 antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT1 receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT1b, AT2, and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT1 receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1–7) and prevent age-related declines in the leptin receptor and its signaling pathway. PMID:21540301

Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I.

PubMed Central

Teruel, T; Valverde, A M; Alvarez, A; Benito, M; Lorenzo, M

1995-01-01

Rat brown adipocytes at day 22 of foetal development showed greater size, higher mitochondria content and larger amounts of lipids, as determined by flow cytometry, than 20-day foetal cells. Simultaneously, an inhibition on the percentage of brown adipocytes into S+G2/M phases of the cell cycle was observed between days 20 and 22 of foetal development. The expression of several adipogenesis-related genes, such as fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase and insulin-regulated glucose transporter, increased at the end of foetal life in brown adipose tissue. In addition, the lipogenic enzyme activities and the lipogenic flux increased during late foetal development, resulting in mature brown adipocytes showing a multilocular fat droplet phenotype. Concurrently, brown adipocytes induced the expression of the uncoupling protein (UP) mRNA and UP protein, as visualized by immunofluorescence. The three isoforms of CCAAT enhancer-binding proteins (C/EBPs) were expressed at the mRNA level in brown adipose tissue at day 20. C/EBP alpha decreased and C/EBP beta and delta increased their expression between days 20 and 22 of foetal development, respectively. Brown adipose tissue constitutively expressed insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNAs. Moreover, IGF-IR mRNA content increased between days 20 and 22 in parallel with the occurrence of tissue differentiation. Images Figure 2 Figure 3 Figure 4 PMID:7575409

Paraoxonase 1 and oxidative stress in paediatric non-alcoholic steatohepatitis.

PubMed

Desai, Sonal; Baker, Susan S; Liu, Wensheng; Moya, Diana A; Browne, Richard W; Mastrandrea, Lucy; Baker, Robert D; Zhu, Lixin

2014-01-01

Non-alcoholic steatohepatitis (NASH) in children is a significant public health concern. Oxidative stress is an important component in the pathophysiology of NASH. Several enzymatic antioxidant mechanisms protect the liver from oxidative injury. Examination of the expression of these enzymes in NASH livers may provide insight on the roles for these antioxidant mechanisms in the pathophysiology of NASH. The mRNA expression of catalase, glutathione peroxidase 1 (GPX1), glutathione reductase (GSR), paraoxonase 1 (PON1) and other reactive oxygen species-related genes was evaluated by microarray and quantitative real-time PCR analyses. The PON1 protein levels were evaluated in liver and serum by Western blot analyses. Serum enzymatic activities of GPX, GSR and PON1 (paraoxonase and arylesterase activities) were examined. NASH livers exhibited elevated mRNA expression of catalase and PON1, but not GPX1 or GSR. No difference in serum GPX or GSR activity was detected between NASH patients and controls. Elevated expression of PON1 mRNA and protein was detected in NASH livers, but serum PON1 protein and activities were not elevated. Elevated expression of catalase and PON1 suggests protective roles for these antioxidants in NASH livers. Given the importance of oxidative stress in the pathophysiology of NASH, future studies focusing on these enzymes could identify important targets for therapeutic or preventive interventions for NASH patients. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Abnormal regulation for progesterone production in placenta with prenatal cocaine exposure in rats.

PubMed

Wu, L; Yan, J; Qu, S C; Feng, Y Q; Jiang, X L

2012-12-01

Cocaine abuse in pregnant women is currently a significant public hygiene problem and is tightly associated with elevated risk for preterm delivery. Placental steroidogenesis especially progesterone production was essential for success and maintenance of pregnancy in humans and rodents. In the present study, we determined the impact of prenatal cocaine exposure on pathways of placental progesterone synthesis in rats. Pregnant rats were treated cocaine twice daily (15 mg/kg/day) during the third trimester, and the maternal and fetal plasma progesterone and pregnenolone concentrations were detected. We also examined both the protein and mRNA expression of some key enzymes and regulators for progesterone production in placenta. Results showed that, after maternal cocaine use during pregnancy, progesterone and pregnenolone concentrations in both maternal and fetal rats were significantly decreased. Although prenatal cocaine exposure had no effects on placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) expression, protein and mRNA expression of the cholesterol side-chain cleavage enzyme (P450scc/CYP11a) in placenta was significantly inhibited. Moreover, protein and mRNA expressions of MLN64 that regulating cholesterol transport and activating protein 2γ (AP2γ/Tfap2c) that controlling P450scc/CYP11a gene expression in placenta were both decreased following maternal cocaine use in pregnancy. Collectively, this study suggested that prenatal cocaine exposure could insult the placental progesterone production in rats possibly associated with the high risk for preterm delivery. Copyright © 2012 Elsevier Ltd. All rights reserved.

Naproxen sodium decreases prostaglandins secretion from cultured human endometrial stromal cells modulating metabolizing enzymes mRNA expression.

PubMed

Carrarelli, Patrizia; Funghi, Lucia; Bruni, Simone; Luisi, Stefano; Arcuri, Felice; Petraglia, Felice

2016-01-01

Dysmenorrhea, defined as painful cramps occurring immediately before or during the menstrual period, is a common symptom of different gynecological diseases. An acute uterine inflammatory response driven by prostaglandins (PGs) is responsible for painful symptoms. Progesterone withdrawal is responsible for activation of cyclooxygenase (COX-2) enzyme and decrease of hydroxyprostaglandin dehydrogenase (HPDG) with consequent increased secretion of PGs secretion, inducing uterine contractility and pain. The most widely used drugs for the treatment of pelvic pain associated with menstrual cycle are non steroidal anti-inflammatory drugs (NSAIDs). The uterine site of action of these drugs is still not defined and the present study evaluated the effect of naproxen sodium in cultured human endometrial stromal cells (HESC) collected from healthy women. PGE2 release was measured by ELISA; COX-2 and HPDG mRNA expression were assessed by qRT-PCR. Naproxen sodium did not affect HESC vitality. Naproxen sodium significantly decreased PGE2 secretion (p < 0.01) and COX-2 mRNA expression (p < 0.01). TNF-α induced PGE2 release was reduced in presence of naproxen sodium (p < 0.05), in association with decreased COX-2 and increased HPDG mRNAs expression. Naproxen sodium decreases endometrial PGE2 release induced by inflammatory stimulus acting on endometrial COX-2 and HPDG expression, suggesting endometrial synthesis of prostaglandins as a possible target for reduction of uterine inflammatory mechanism in dysmenorrhea.

Associations of ACE Gene Insertion/Deletion Polymorphism, ACE Activity, and ACE mRNA Expression with Hypertension in a Chinese Population

PubMed Central

He, Qingfang; Fan, Chunhong; Yu, Min; Wallar, Gina; Zhang, Zuo-Feng; Wang, Lixin; Zhang, Xinwei; Hu, Ruying

2013-01-01

Background The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D, rs4646994) polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored. Methods The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension. Results The results of the multivariate logistic regression analysis showed that the additive model (ID, DD versus II) of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls. Conclusion ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. PMID:24098401

Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation.

PubMed

Hood, D A; Simoneau, J A; Kelly, A M; Pette, D

1992-10-01

The effect of thyroid status on the expression of cytochrome c oxidase (CYTOX) and the activities of citrate synthase (CS) and phosphofructokinase (PFK) were examined in chronically stimulated (10 Hz; 35 days) and contralateral, nonstimulated rat tibialis anterior muscle of hypothyroid, hyperthyroid, and euthyroid animals. Stimulation increased CYTOX activity by 2.7-, 3.2-, and 4.9-fold in hyperthyroid, euthyroid, and hypothyroid animals, respectively, to similar absolute values. CS displayed similar increases. Stimulation reduced PFK activity in hypothyroid and euthyroid animals to 45% and 60% of control values. This effect was abolished with hyperthyroidism. Thus stimulation and thyroid hormone act antagonistically on PFK activity. Stimulation increased CYTOX subunit III (mitochondrially encoded) mRNA by 2.5- and 2.9-fold in hyperthyroid and euthyroid animals. Similar increases were observed in the nuclear-encoded mRNAs of CYTOX subunit VIc in euthyroid muscle. In hyperthyroid and euthyroid conditions, the mRNA changes paralleled the increases in enzyme activity. In hypothyroid muscle, the increase in mRNA was less for subunit VIc than III, suggesting that hypothyroidism upsets the coordinate expression of nuclear and mitochondrial genes. Further, the increases in CYTOX activity exceeded that of both subunit mRNAs in hypothyroid muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.).

PubMed

Lin, L S; Varner, J E

1991-05-01

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening."

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

PubMed Central

Hosaka, K; Murakami, T; Kodaki, T; Nikawa, J; Yamashita, S

1990-01-01

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance. Images FIG. 3 FIG. 4 PMID:2156807

Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

PubMed

Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2015-12-01

Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Copyright © 2015 Elsevier Inc. All rights reserved.

Overexpression of peptide deformylase in breast, colon, and lung cancers.

PubMed

Randhawa, Harsharan; Chikara, Shireen; Gehring, Drew; Yildirim, Tuba; Menon, Jyotsana; Reindl, Katie M

2013-07-01

Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.

Overexpression of peptide deformylase in breast, colon, and lung cancers

PubMed Central

2013-01-01

Background Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. Methods The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. Results PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. Conclusions This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation. PMID:23815882

Skeletal muscle deiodinase type 2 regulation during illness in mice.

PubMed

Kwakkel, J; van Beeren, H C; Ackermans, M T; Platvoet-Ter Schiphorst, M C; Fliers, E; Wiersinga, W M; Boelen, A

2009-11-01

We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is upregulated in an animal model of acute illness. However, human studies on the expression of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of skeletal muscle D2 and D2-regulating factors in two mouse models of illness that differ in timing and severity of illness: 1) turpentine-induced inflammation, and 2) Streptococcus pneumoniae infection. During turpentine-induced inflammation, D2 mRNA and activity increased compared to pair-fed controls, most prominently at day 1 and 2, whereas after S. pneumoniae infection D2 mRNA decreased. We evaluated the association of D2 expression with serum thyroid hormones, (de-)ubiquitinating enzymes ubiquitin-specific peptidase 33 and WD repeat and SOCS box-containing 1 (Wsb1), cytokine expression and activation of inflammatory pathways and cAMP pathway. During chronic inflammation the increased muscle D2 expression is associated with the activation of the cAMP pathway. The normalization of D2 5 days after turpentine injection coincides with increased Wsb1 and tumor necrosis factor alpha expression. Muscle interleukin-1beta (Il1b) expression correlated with decreased D2 mRNA expression after S. pneumoniae infection. In conclusion, muscle D2 expression is differentially regulated during illness, probably related to differences in the inflammatory response and type of pathology. D2 mRNA and activity increases in skeletal muscle during the acute phase of chronic inflammation compared to pair-fed controls probably due to activation of the cAMP pathway. In contrast, muscle D2 mRNA decreases 48 h after a severe bacterial infection, which is associated with local Il1b mRNA expression and might also be due to diminished food-intake.

[Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

PubMed

Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

2015-10-01

To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 mRNA workers exposed to manganese increased and protect nerve cells from related to Mn stimulation induced lipid peroxidation damag.

Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential.

PubMed

Arakawa, Hiroshi; Kamioka, Hiroki; Jomura, Tomoko; Koyama, Satoshi; Idota, Yoko; Yano, Kentaro; Kojima, Hajime; Ogihara, Takuo

2017-01-01

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events.

Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

PubMed

Yu, Hong; Bao, En-Dong; Zhao, Ru-Qian; Lv, Qiong-Xia

2007-11-01

To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. 24 pigs (mean weight, 20 +/- 1 kg). Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

PubMed

Dicken, Matthew S; Hughes, Alexander R; Hentges, Shane T

2015-11-01

The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus

PubMed Central

Dicken, Matthew S.; Hughes, Alexander R.; Hentges, Shane T.

2016-01-01

The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. PMID:26370162

L-DOPA decarboxylase mRNA expression is associated with tumor stage and size in head and neck squamous cell carcinoma: a retrospective cohort study

PubMed Central

2012-01-01

Background Head and neck squamous cell carcinoma (HNSCC) represents one of the most commonly diagnosed malignancies worldwide. The DDC gene encodes L-DOPA decarboxylase, an enzyme catalyzing the decarboxylation of L-DOPA to dopamine. We have recently shown that DDC mRNA is a significant predictor of patients’ prognosis in colorectal adenocarcinoma and prostate cancer. The aim of the current study was to analyze the DDC mRNA expression in HNSCC patients. Methods 53 malignant tumors were resected from the larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissues of HNSCC patients, and were homogenized. Total RNA was isolated and converted into first-strand cDNA. An ultrasensitive real-time PCR method based on the SYBR Green chemistry was used for DDC mRNA quantification in head and neck tissue specimens. Relative quantification was performed using the comparative Ct (2-ddCt) method. Results DDC mRNA levels were lower in squamous cell carcinomas (SCCs) of the larynx and tongue than in adjacent non-cancerous tissue specimens. Furthermore, low DDC mRNA expression was noticed in laryngeal and tongue tumors of advanced TNM stage or bigger size, compared to early-stage or smaller tumors, respectively. No statistically significant differences were observed between SCCs resected from pharynx, buccal mucosa, or nasal cavity, and their normal counterparts. Conclusion This is the first study examining the DDC mRNA expression in HNSCC. According to our results, DDC mRNA expression may constitute a potential prognostic biomarker in tongue and/or larynx SCCs, which principally represent the overwhelming majority of HNSCC cases. PMID:23083099

L-DOPA decarboxylase mRNA expression is associated with tumor stage and size in head and neck squamous cell carcinoma: a retrospective cohort study.

PubMed

Geomela, Panagiota-Aikaterini; Kontos, Christos K; Yiotakis, Ioannis; Fragoulis, Emmanuel G; Scorilas, Andreas

2012-10-20

Head and neck squamous cell carcinoma (HNSCC) represents one of the most commonly diagnosed malignancies worldwide. The DDC gene encodes L-DOPA decarboxylase, an enzyme catalyzing the decarboxylation of L-DOPA to dopamine. We have recently shown that DDC mRNA is a significant predictor of patients' prognosis in colorectal adenocarcinoma and prostate cancer. The aim of the current study was to analyze the DDC mRNA expression in HNSCC patients. 53 malignant tumors were resected from the larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissues of HNSCC patients, and were homogenized. Total RNA was isolated and converted into first-strand cDNA. An ultrasensitive real-time PCR method based on the SYBR Green chemistry was used for DDC mRNA quantification in head and neck tissue specimens. Relative quantification was performed using the comparative Ct (2-ddCt) method. DDC mRNA levels were lower in squamous cell carcinomas (SCCs) of the larynx and tongue than in adjacent non-cancerous tissue specimens. Furthermore, low DDC mRNA expression was noticed in laryngeal and tongue tumors of advanced TNM stage or bigger size, compared to early-stage or smaller tumors, respectively. No statistically significant differences were observed between SCCs resected from pharynx, buccal mucosa, or nasal cavity, and their normal counterparts. This is the first study examining the DDC mRNA expression in HNSCC. According to our results, DDC mRNA expression may constitute a potential prognostic biomarker in tongue and/or larynx SCCs, which principally represent the overwhelming majority of HNSCC cases.

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

PubMed

Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

Expression of Leptin and Visfatin in Gingival Tissues of Chronic Periodontitis With and Without Type 2 Diabetes Mellitus: A Study Using Enzyme-Linked Immunosorbent Assay and Real-Time Polymerase Chain Reaction.

PubMed

Ghallab, Noha A; Amr, Eman M; Shaker, Olfat G

2015-07-01

The aim of this study is to investigate the protein and gene expression of leptin and visfatin in gingival tissue from patients with chronic periodontitis (CP), patients with CP and type 2 diabetes mellitus (T2DM), and healthy individuals. The study includes 50 individuals: 10 healthy individuals, 20 patients with CP, and 20 patients with CP and T2DM. Plaque index, gingival index, probing depth, and clinical attachment loss were measured, and gingival biopsies were obtained. Leptin and visfatin protein expression in gingival tissues was determined using enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was measured via real-time polymerase chain reaction. The highest leptin mRNA and protein expression was observed in the control group and was significantly (P ≤0.05) different from the CP and CP+T2DM groups. Gingival tissues from patients with CP and T2DM had a significant increase in visfatin and a decrease in leptin gene and protein expression (P <0.05) compared with both controls and patients with CP. Expression of leptin and visfatin in the gingival tissues suggests a possible role for these adipokines in the pathogenesis of CP and T2DM.

Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus.

PubMed

Zheng, Jinbin; Mao, Yong; Su, Yongquan; Wang, Jun

2016-11-01

Nitrite accumulation in aquaculture systems is a potential risk factor that may trigger stress responses in aquatic organisms. However, the mechanisms regulating the responses of shrimp to nitrite stress remain unclear. In this study, full-length cDNA sequences of two apoptosis-related genes, caspase-3 and defender against apoptotic death (DAD-1), were cloned from Marsupenaeus japonicus for the first time, and their expression levels and tissue distribution were analyzed by quantitative real-time PCR (qRT-PCR). The full lengths of Mjcaspase-3 and MjDAD-1 were 1203 bp and 640 bp respectively, with deduced amino acid (AA) sequences of 321 and 114 AA. Mjcaspase-3 was predominantly expressed in haemocytes and weakly expressed in the seven other tissues tested. MjDAD-1 was mainly expressed in the defense and digestive tissues, especially in the hepatopancreas and hemocytes. To explore the influence of nitrite stress on the genetic response of antioxidant enzymes, immune-related genes and apoptosis-related proteins, the mRNA expression profiles of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 in response to nitrite stress were analyzed by qRT-PCR. The mRNA levels of MjCAT, MjMnSOD, Mj-ilys, Mj-sty, Mjcaspase-3 and MjDAD-1 show both time- and dose-dependent changes in response to nitrite stress. The mRNA expression levels of MjCAT and MjSOD peaked at 6 h for all nitrite concentrations tested (p < 0.05) and the up-regulated of MjCAT and MjSOD exhibited a positive correlation with the nitrite concentration. The mRNA expression levels of Mj-ilys and Mj-sty gradually decreased during the experiment period. Mjcaspase-3 mRNA level reached a maximum at 6 h (p < 0.05), and MjDAD-1 reached its peak at 12 h and 48 h in 10 mg/L and 20 mg/L nitrite, respectively. In addition, CAT and SOD activity showed changes in response to nitrite stress that mirrored the induced expression of MjCAT and MjMnSOD, and prolonged nitrite exposure reduced the activity of CAT. This study provided basic data for further elucidating the responses of shrimp to nitrite stress at the molecular level. Copyright © 2016. Published by Elsevier Ltd.

Chylomicron remnants and nonesterified fatty acids differ in their ability to inhibit genes involved in lipogenesis in rats.

PubMed

Kohan, Alison B; Qing, Yang; Cyphert, Holly A; Tso, Patrick; Salati, Lisa M

2011-02-01

Primary hepatocytes treated with nonesterified PUFA have been used as a model for analyzing the inhibitory effects of dietary polyunsaturated fats on lipogenic gene expression. Although nonesterified fatty acids play an important signaling role in starvation, they do not completely recapitulate the mechanism of dietary fat presentation to the liver, which is delivered via chylomicron remnants. To test the effect of remnant TG on lipogenic enzyme expression, chylomicron remnants were generated from the lymph of rats intubated with either safflower oil or lard. The remnants were added to the medium of primary rat hepatocytes in culture and the accumulation of mRNA for genes involved in carbohydrate and lipid metabolism was measured. Both PUFA-enriched remnants and nonesterified PUFA inhibited the expression and maturation of sterol response element binding protein-1c (SREBP-1c) and the expression of lipogenic genes regulated by this transcription factor. These remnants also inhibited the expression of glucose-6-phosphate dehydrogenase (G6PD), a gene regulated at post-transcriptional steps. In contrast, PUFA-enriched remnants did not inhibit the accumulation of mRNA for malic enzyme, glucokinase, and L-pyruvate kinase, whereas nonesterified fatty acids caused a decrease in these mRNA. These genes are regulated independently of SREBP-1c. SFA-enriched remnants did not inhibit lipogenic gene expression, which is consistent with a lack of inhibition of lipogenesis by dietary saturated fats. Thus, the inhibitory action of dietary polyunsaturated fats on lipogenesis involves a direct action of chylomicron remnants on the liver.

Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection

PubMed Central

Mehdy, Mona C.; Lamb, Christopher J.

1987-01-01

The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene encoding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A λgt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 105 recombinants with an antiserum to purified bean CHI. The identity of the cloned sequences was confirmed by hybrid-select translation and the production of antigenic polypeptides from transcripts synthesized in vitro. Addition of elicitor to cell cultures resulted in the rapid accumulation of CHI mRNA, with maximum levels achieved 3–4 h after elicitation. CHI mRNA also accumulated during the natural infection of hypocotyls with the fungal pathogen Colletotrichum lindemuthianum, and in mechanically wounded hypocotyls. The kinetics of accumulation of CHI mRNA in response to these environmental signals were strikingly similar to those of mRNAs encoding two other phenylpropanoid pathway enzymes, phenylalanine ammonialyase and chalcone synthase. In contrast to the multi-gene families encoding these two enzymes, chalcone isomerase is encoded by a single gene which is regulated by several environmental stimuli. ImagesFig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 9. PMID:16453768

Squalene epoxidase, located on chromosome 8q24.1, is upregulated in 8q+ breast cancer and indicates poor clinical outcome in stage I and II disease.

PubMed

Helms, M W; Kemming, D; Pospisil, H; Vogt, U; Buerger, H; Korsching, E; Liedtke, C; Schlotter, C M; Wang, A; Chan, S Y; Brandt, B H

2008-09-02

Gains of chromosomes 7p and 8q are associated with poor prognosis among oestrogen receptor-positive (ER+) stage I/II breast cancer. To identify transcriptional changes associated with this breast cancer subtype, we applied suppression subtractive hybridisation method to analyse differentially expressed genes among six breast tumours with and without chromosomal 7p and 8q gains. Identified mRNAs were validated by real-time RT-PCR in tissue samples obtained from 186 patients with stage I/II breast cancer. Advanced statistical methods were applied to identify associations of mRNA expression with distant metastasis-free survival (DMFS). mRNA expression of the key enzyme of cholesterol biosynthesis, squalene epoxidase (SQLE, chromosomal location 8q24.1), was associated with ER+ 7p+/8q+ breast cancer. Distant metastasis-free survival in stage I/II breast cancer cases was significantly inversely related to SQLE mRNA in multivariate Cox analysis (P<0.001) in two independent patient cohorts of 160 patients each. The clinically favourable group associated with a low SQLE mRNA expression could be further divided by mRNA expression levels of the oestrogen-regulated zinc transporter LIV-1. The data strongly support that SQLE mRNA expression might indicate high-risk ER+ stage I/II breast cancers. Further studies on tumour tissue from standardised treated patients, for example with tamoxifen, may validate the role of SQLE as a novel diagnostic parameter for ER+ early stage breast cancers.

Effect of lipoic acid on paraoxonase-1 and paraoxonase-3 protein levels, mRNA expression and arylesterase activity in liver hepatoma cells.

PubMed

Ozgun, Eray; Sayilan Ozgun, Gulben; Tabakcioglu, Kiymet; Suer Gokmen, Selma; Sut, Necdet; Eskiocak, Sevgi

2017-10-01

Paraoxonase-1 (PON1) and PON3 (PON3) are anti-atherosclerotic enzymes, synthesized primarily in liver and bound to HDL in circulation. The aim of the present study was to investigate the effects of therapeutic doses of lipoic acid on PON1 and PON3 protein levels, mRNA expression and arylesterase activity in liver. We treated HepG2 cells with 10, 40 and 200 μM lipoic acid for 72 h. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. PON1 and PON3 protein levels were measured by Western blotting, their mRNA expression was measured by quantitative PCR and arylesterase activity was measured spectrophotometrically. 200 µM lipoic acid caused a significant increase on PON1 and PON3 protein levels and arylesterase activity as compared with control, 10 µM and 40 µM lipoic acid-treated cells. 200 µM lipoic acid also caused a significant decrease on PON1 mRNA expression whereas on a significant increase PON3 mRNA expression as compared with control, 10 µM and 40 µM lipoic acid-treated cells. Our study showed that although lipoic acid up-regulates PON3 but down-regulates PON1 mRNA expression, it increases both PON1 and PON3 protein levels and arylesterase activity in HepG2 cells. We can report that lipoic acid may be useful for preventing atherosclerosis at therapeutic doses.

Prostaglandin G/H synthase (PGHS)-2 expression in bovine myometrium: influence of steroid hormones and PGHS inhibitors.

PubMed

Doualla-Bell, F; Guay, J M; Bourgoin, S; Fortier, M A

1998-12-01

Prostaglandins (PGs) are important mediators regulating uterine functions during the reproductive process. The objective of this study was to examine, in myocytes from the circular and longitudinal layers of bovine myometrium, the relative levels of mRNA and proteins corresponding to the gene expression of key enzymes (phospholipase A2; prostaglandin G/H synthase-1 [PGHS-1]; prostaglandin G/H synthase-2 [PGHS-2]; prostaglandin I2 synthase) involved in PG biosynthesis. We examined the influence of estradiol-17beta and progesterone on the expression and activity of these enzymes. Treatment of myocytes with progesterone (P4: 10 nM, 24 h) in the absence or presence of estradiol-17beta (E2: 1 nM, 72 h) suppressed PG biosynthesis by approximately 60% in both myometrial layers. No significant effect was observed after E2 treatment. The combined effect of E2 and P4 on PG accumulation was correlated with the modulation of PGHS-2 protein and mRNA levels in the two myometrial layers without affecting other enzymes of the PG cascade. Selective or nonselective inhibition of PGHS activity with CGP 28238 (PGHS-2-specific; a product from Ciba-Geigy: 6-[2, 4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone) or indomethacin (PGHS-1 and -2) reduced prostacyclin accumulation (measured as 6-keto-PGF1alpha in the culture medium) in a dose-dependent manner in the two myometrial layers. A significant inhibitory effect was obtained at a low concentration of indomethacin (1 nM, p < 0.05) compared to CGP 28238 (10 nM, p < 0. 05). In both myometrial layers, the maximal effect of indomethacin and/or CGP 28238 on PG accumulation was observed at 100 nM and represented 85% and 65% inhibition, respectively. In the presence of phorbol 12-myristate (100 nM), CGP 28238 (10 nM) significantly suppressed PGHS-2 mRNA level by 44.80 +/- 7.67% (p < 0.01) and 27.83 +/- 7.62% (p < 0.05) in the longitudinal and circular layer, respectively. In contrast, indomethacin did not have any significant effect. These data constitute the first quantitative analysis of key enzymes involved in PG biosynthesis in separated myometrial layers. Furthermore, the results provide interesting information on the CGP 28238 drug modulating both enzymatic activity and mRNA expression of PGHS-2.

Cocaine-Induced Behavioral Sensitization Is Associated With Changes in the Expression of Endocannabinoid and Glutamatergic Signaling Systems in the Mouse Prefrontal Cortex

PubMed Central

Blanco, Eduardo; Pavón, Francisco J.; Palomino, Ana; Luque-Rojas, María Jesús; Serrano, Antonia; Rivera, Patricia; Bilbao, Ainhoa; Alen, Francisco; Vida, Margarita; Suárez, Juan

2015-01-01

Background: Endocannabinoids modulate the glutamatergic excitatory transmission by acting as retrograde messengers. A growing body of studies has reported that both signaling systems in the mesocorticolimbic neural circuitry are involved in the neurobiological mechanisms underlying drug addiction. Methods: We investigated whether the expression of both endocannabinoid and glutamatergic systems in the prefrontal cortex (PFC) were altered by an acute and/or repeated cocaine administration schedule that resulted in behavioral sensitization. We measured the protein and mRNA expression of the main endocannabinoid metabolic enzymes and the cannabinoid receptor type 1 (CB1). We also analyzed the mRNA expression of relevant components of the glutamate-signaling system, including glutamate-synthesizing enzymes, metabotropic receptors, and ionotropic receptors. Results: Although acute cocaine (10mg/kg) produced no significant changes in the endocannabinoid-related proteins, repeated cocaine administration (20mg/kg daily) induced a pronounced increase in the CB1 receptor expression. In addition, acute cocaine administration (10mg/kg) in cocaine-sensitized mice (referred to as cocaine priming) induced a selective increase in the endocannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). These protein changes were accompanied by an overall decrease in the ratios of endocannabinoid synthesis/degradation, especially the N-acyl phosphatidylethanolamine phospholipase D/FAAH and diacylglycerol lipase alpha/MAGL ratios. Regarding mRNA expression, while acute cocaine administration produced a decrease in CB1 receptors and N-acyl phosphatidylethanolamine phospholipase D, repeated cocaine treatment enhanced CB1 receptor expression. Cocaine-sensitized mice that were administered priming injections of cocaine mainly displayed an increased FAAH expression. These endocannabinoid changes were associated with modifications in glutamatergic transmission-related genes. An overall decrease was observed in the mRNA expression of the glutamate-synthesizing gene kidney-type glutaminase (KGA), the metabotropic glutamate receptors (mGluR3 and GluR), and subunits of NMDA ionotropic receptors (NR1, NR2A, NR2B and NR2C) after acute cocaine administration, while mice repeatedly exposed to cocaine only displayed an increase in NR2C. However, in cocaine-sensitized mice primed with cocaine, this inhibition was reversed and a strong increase was detected in the mGluR5, NR2 subunits, and both GluR1 and GluR3. Conclusions: These findings indicate that cocaine sensitization is associated with an endocannabinoid downregulation and a hyperglutamatergic state in the PFC that, overall, contribute to an enhanced glutamatergic input into PFC-projecting areas. PMID:25539508

The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2

PubMed Central

Smid, Kees; de Klerk, Daniël; van Kuilenburg, André B. P.; Meinsma, Rutger; Lee, Young B.; Kim, Deog J.; Peters, Godefridus J.

2016-01-01

Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117. PMID:27612203

Short- and long-term salinity challenge, Osmoregulatory ability, and (Na+, K+)-ATPase KINETICS AND α-SUBUNIT mRNA expression in the gills of the thinstripe hermit CRAB Clibanarius symmetricus.

PubMed

Faleiros, Rogério O; Garçon, Daniela P; Lucena, Malson N; McNamara, John C; Leone, Francisco A

2018-06-19

The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6 h) or long-term acclimation (10 days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na + , K + )-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15‰S (Salinity, g L -1 ) but weakly hyper-regulates up to ≈27‰S. After 6 h exposure to 35‰S and 45‰S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10 days acclimation to these salinities. (Na + , K + )-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na + , K + )-ATPase α-subunit mRNA expression increases only after 10 days acclimation to 5‰S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na + , K + )-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes. Copyright © 2018. Published by Elsevier Inc.

Interactive effects of hypoxia and PCB co-exposure on expression of CYP1A and its potential regulators in Atlantic croaker liver.

PubMed

Rahman, Md Saydur; Thomas, Peter

2018-04-01

Although marine and coastal environments which are contaminated with xenobiotic organic compounds often become hypoxic during the summer, the interactive effects of hypoxia and xenobiotic exposure on marine species such as teleost fishes remain poorly understood. The expression and activity of monooxygenase enzyme cytochrome P450-1A (CYP1A) in fishes are upregulated by exposure to polychlorinated biphenyls (PCBs), whereas they are down-regulated during hypoxia exposure. We investigated the interactive effects of hypoxia and PCB co-exposure on hepatic CYP1A expression in Atlantic croaker and on potential regulators of CYP1A. Croaker were exposed to hypoxia (1.7 mg/L dissolved oxygen), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, dose: 2 and 8 µg/g body weight), and Aroclor 1254 (a common PCB mixture, dose: 0.5 and 1 µg/g body weight), alone and in combination for 4 weeks. PCB 77 exposure markedly increased hepatic CYP1A mRNA and protein expression, and ethoxyresorufin-O-deethylase (EROD, an indicator of CYP1A enzyme) activity and increased endothelial nitric oxide synthase (eNOS) protein expression. PCB 77 treatment also increased interleukin-1β (IL-1β, a cytokine) mRNA levels and protein carbonyl (PC, an indicator of reactive oxygen species, ROS) contents. These marked PCB 77- and Aroclor 1254-induced increases in CYP1A mRNA levels and EROD activity were significantly attenuated by co-exposure to hypoxia, whereas the increases in hepatic eNOS protein and IL-1β mRNA expression, and PC contents were augmented by hypoxia co-exposure. The results suggest that biotransformation of organic xenobiotics by CYP1A is reduced in fish during co-exposure to hypoxia and is accompanied by alterations in eNOS, ROS, and IL-1β levels. © 2018 Wiley Periodicals, Inc.

Modulation of xenobiotic biotransformation system and hormonal responses in Atlantic salmon (Salmo salar) after exposure to tributyltin (TBT).

PubMed

Mortensen, Anne Skjetne; Arukwe, Augustine

2007-04-01

Multiple biological effects of tributyltin (TBT) on juvenile salmon have been investigated. Fish were exposed for 7 days to waterborne TBT at nominal concentrations of 50 and 250 microg/L dissolved in dimethyl sulfoxide (DMSO). Hepatic samples were analyzed for gene expression patterns in the hormonal and xenobiotic biotransformation pathways using validated real-time PCR method. Immunochemical and several cytochrome P450 (CYP)-mediated enzyme activity (ethoxyresorufin: EROD, benzyloxyresorufin: BROD, methoxyresorufin: MROD and pentoxyresorufin: PROD) assays were analyzed. Our data show that TBT produced concentration-specific decrease of estrogen receptor-alpha (ERalpha), vitellogenin (Vtg), zona radiata protein (Zr-protein) and increase of estrogen receptor-beta (ERbeta) and androgen receptor-beta (ARbeta) in the hormonal pathway. In the xenobiotic biotransformation pathway, TBT produced apparent increase and decrease at respective low and high concentration, on aryl hydrocarbon receptor-alpha (AhRalpha), AhR nuclear translocator (ARNT) and AhR repressor (AhRR) mRNA. The expression of CYP1A1 and GST showed a TBT concentration-dependent decrease. The AhRbeta, CYP3A and uridine diphosphoglucuronosyl transferase (UGT) mRNA expressions were significantly induced after exposure to TBT. Immunochemical analysis of CYP3A and CYP1A1 protein levels confirmed the TBT effects observed at the transcriptional levels. The effect of TBT on the biotransformation enzyme gene expressions partially co-related but did not directly parallel enzyme activity levels for EROD, BROD, MROD and PROD. In general, these findings confirm previous reports on the endocrine effects of TBT, in addition to effects on hepatic CYP1A isoenzyme at the transcriptional level that transcends to protein and enzymatic levels. The induced expression patterns of CYP3A and UGT mRNA after TBT exposure, suggest the involvement of CYP3A and UGT in TBT metabolism in fish. The effect of TBT on CYP3A is proposed to represent another hormonal effect of TBT not previously reported in any fish or lower vertebrate. The proposed androgenic effect is supported by the observation that TBT also induced ARbeta mRNA expression in a concentration-specific manner. To our knowledge, this is the first study that has simultaneously studied multiple responses after exposure to TBT in fish.

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation.

PubMed

Rivière, Guillaume; Lienhard, Daniel; Andrieu, Thomas; Vieau, Didier; Frey, Brigitte M; Frey, Felix J

2011-04-01

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

PubMed

Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

2013-01-01

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

Function of glutathione peroxidases in legume root nodules.

PubMed

Matamoros, Manuel A; Saiz, Ana; Peñuelas, Maria; Bustos-Sanmamed, Pilar; Mulet, Jose M; Barja, Maria V; Rouhier, Nicolas; Moore, Marten; James, Euan K; Dietz, Karl-Josef; Becana, Manuel

2015-05-01

Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Osthole, a coumadin analog from Cnidium monnieri (L.) Cusson, stimulates corticosterone secretion by increasing steroidogenic enzyme expression in mouse Y1 adrenocortical tumor cells.

PubMed

Pan, Zhiqiang; Fang, Zhaoqin; Lu, Wenli; Liu, Xiaomei; Zhang, Yuanyuan

2015-12-04

Osthole is an O-methylated coumadin, which was isolated and purified from the seeds of Cnidium monnieri (L.) Cusson. Osthole is a commonly used traditional Chinese medicine to treat patients with Kidney-Yang deficiency patients, who exhibit clinical signs similar to those of glucocorticoid withdrawal. However, the mechanism of action of osthole is not fully understood. This study was designed to reveal the effects of osthole on corticosterone production in mouse Y1 cell. Mouse Y1 adrenocortical cells were used to evaluate corticosterone production, which was quantified by enzyme-linked immunosorbent assay (ELISA) kits. Cell viability was tested using the MTT assay, and the mRNA and protein expression of genes encoding steroidogenic enzymes and transcription factors was monitored by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Osthole stimulated corticosterone secretion from mouse Y1 cells in a dose- and time-dependent manner, and osthole enhanced the effect of dibutyryl-cAMP (Bu2cAMP) on corticosterone production. Further, osthole also increased StAR and CYP11B1 mRNA expression in a dose-dependent manner and enhanced the expression of transcription factors such as HSD3B1, FDX1, POR and RXRα as well as immediate early genes such as NR4A1. Moreover, osthole significantly increased SCARB1(SRB1) mRNA and StAR protein expression in the presence or absence of Bu2cAMP; these proteins are an important for the transport of the corticosteroid precursor cholesterol transport into mitochondria. Our results show that the promotion of corticosterone biosynthesis and secretion is a novel effect of osthole, suggesting that this agent can be utilized for the prevention and treatment of Kidney-Yang deficiency syndrome. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Effect of L-arginine supplementation on the hepatic phosphatidylinositol 3-kinase signaling pathway and gluconeogenic enzymes in early intrauterine growth-restricted rats

PubMed Central

Luo, Kaiju; Chen, Pingyang; Li, Suping; Li, Wen; He, Mingfeng; Wang, Tao; Chen, Juncao

2017-01-01

The present study aimed to investigate the response of the phosphatidylinositol 3-kinase (PI3K) signaling pathway and gluconeogenic enzymes in intrauterine growth-restricted rats to dietary L-arginine (L-Arg) supplementation during the lactation period early in life. Pregnant Sprague-Dawley rats were randomly divided into a control group (CON), an intrauterine growth restriction group (IUGR) and an L-Arg group (LA). The pregnant rats in the CON group were fed a 21% protein diet, and those in the IUGR and LA groups were fed a 10% low protein diet, and all rats were fed a 21% protein diet after delivery. Water was available ad libitum to the pregnant rats during the 21-day lactation period, and the water provided to the LA group included 200 mg/kg/day L-Arg. Blood glucose, serum insulin, homeostasis model of assessment for insulin resistance (HOMA-IR), PI3K and protein kinase B (PKB) protein expression, and phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) mRNA expression in the offspring rats were measured postnatally at 1, 3 and 8 weeks. No significant difference in blood glucose, serum insulin and HOMA-IR were identified at any time point among the three groups. PI3K and PKB expression was lower in the IUGR group offspring compared with that in the CON group offspring, but both were increased by dietary L-Arg supplementation. PEPCK mRNA and G-6-Pase mRNA expression levels in the offspring of the IUGR group were higher compared with those in the CON group but were downregulated following L-Arg supplementation. These results suggest that dietary L-Arg supplementation during the early lactation period promoted catch-up growth and reversed abnormalities in hepatic insulin signaling and gene expression of gluconeogenic enzymes in IUGR offspring rats. PMID:28962167

TEMPORAL PATTERNS OF LIPOPEROXIDATION AND ANTIOXIDANT ENZYMES ARE MODIFIED IN THE HIPPOCAMPUS OF VITAMIN A-DEFICIENT RATS

PubMed Central

Navigatore Fonzo, Lorena S.; Golini, Rebeca S.; Delgado, Silvia M.; Ponce, Ivana T.; Bonomi, Myrta R.; Rezza, Irma G.; Gimenez, María S.; Anzulovich, Ana C.

2011-01-01

Animals can adapt their behavior to predictable temporal fluctuations in the environment through both, memory-and-learning processes and an endogenous time-keeping mechanism. Hippocampus plays a key role in memory and learning and is especially susceptible to oxidative stress. In compensation, antioxidant enzymes activity, such as Catalase (CAT) and Glutathione peroxidase (GPx), has been detected in this brain region. Daily rhythms of antioxidant enzymes activitiy, as well as of glutathione and lipid peroxides levels, have been described in brain. Here, we investigate day/night variations in lipoperoxidation, CAT and GPx expression and activity, as well as the temporal fluctuations of two key components of the endogenous clock, BMAL1 and PER1, in the rat hippocampus and evaluate to which extent vitamin A deficiency may affect their amplitude or phase. Holtzman male rats from control, vitamin A-deficient and vitamin A-refed groups were sacrificed throughout a 24-h period. Daily levels of clock proteins, lipoperoxidation, CAT and GPx mRNA, protein, and activity, were determined in the rat hippocampus obtained every 4 or 5 h. Gene expression of RARα and RXRβ was also quantified in the hippocampus of the three groups of rats. Our results show significant daily variations of BMAL1 and PER1 protein expression. Rhythmic lipoperoxidation, CAT, and GPx, expression and activity, were also observed in the rat hippocampus. Vitamin A deficiency reduced RXRβ mRNA level, as well as the amplitude of BMAL1 and PER1 daily oscillation, phase-shifted the daily peak of lipoperoxidation, and had a differential effect on the oscillating CAT and GPx mRNA, protein, and activity. Learning how vitamin A deficiency affects the circadian gene expression in the hippocampus may have an impact on the neurobiology, nutritional and chronobiology fields, emphasizing for the first time the importance of nutritional factors, such as dietary micronutrients, in the regulation of circadian parameters in this brain memory-and-learning-related region. PMID:19308957

NADPH Oxidase Isoform 2 (NOX2) Is Involved in Drug Addiction Vulnerability in Progeny Developmentally Exposed to Ethanol.

PubMed

Contreras, Marcela L; de la Fuente-Ortega, Erwin; Vargas-Roberts, Sofía; Muñoz, Daniela C; Goic, Carolina A; Haeger, Paola A

2017-01-01

Ethanol exposure increases oxidative stress in developing organs, including the brain. Antioxidant treatment during maternal ethanol ingestion improves behavioral deficits in rodent models of fetal alcohol spectrum disorder (FASD). However, the impact of general antioxidant treatment in their adult offspring and the Specific Reactive Species (ROS)-dependent mechanism, are not fully understood. We hypothesized that pre and early postnatal ethanol exposure (PEE) modifies redox homeostasis, in particular NOX2 function during reward signaling in the mesocorticolimbic pathway, which reinforces the effects of alcohol. We developed a FASD rat model which was evaluated during adolescence (P21) and adulthood (P70). We first studied whether redox homeostasis is affected in PEE animals, by analyzing mRNA expression of SOD1, CAT, and Gpx1. We found that PEE reduced the mRNA levels of these three anti-oxidant enzymes in PFC and HIPP at P21 and in the VTA at P70. We also analyzed basal mRNA and protein expression of NOX2 subunits such as gp91phox, p22 phox, and p47 phox, in mesocorticolimbic brain areas of PEE rat brains. At P21, gp91 phox, and p47 phox levels in the VTA were decreased. At P70, gp91 phox mRNA levels was decreased in HIPP and both mRNA and protein levels were decreased in PFC. Since NOX2 is regulated by the N-methyl-D-aspartate Receptor (NMDAR), we analyzed NMDAR mRNA expression and found differential expression of NMDAR subunits (NR1 and NR2B) in the PFC that was age dependent, with levels decreased at P21 and increased at P70. The analysis also revealed decreased NR2B mRNA expression in HIPP and VTA at P70. Offspring from maternal ethanol users consumed 25% more ethanol in a free choice alcohol consumption test than control rats, and showed place preference for an alcohol-paired compartment. In vivo inhibition of NOX2 using apocynin in drinking water, or infusion of blocked peptide gp91 phox ds in the VTA normalized alcohol place preference, suggesting that NOX2 plays an important role in addictive like behavior. Taken together, PEE significantly affects the expression of antioxidant enzymes, NOX2, NMDAR in an age, and brain region dependent manner. Moreover, we demonstrate that NOX2 regulates alcohol seeking behavior.

Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice.

PubMed

Jourová, L; Anzenbacher, P; Lišková, B; Matušková, Z; Hermanová, P; Hudcovic, T; Kozáková, H; Hrnčířová, L; Anzenbacherová, E

2017-11-01

Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host's health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum NIZO2877 ; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.

Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

PubMed

Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

2008-12-01

The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

Isoquercetin ameliorates hyperglycemia and regulates key enzymes of glucose metabolism via insulin signaling pathway in streptozotocin-induced diabetic rats.

PubMed

Jayachandran, Muthukumaran; Zhang, Tongze; Ganesan, Kumar; Xu, Baojun; Chung, Stephen Sum Man

2018-06-15

Among the foremost common flavonoids within the human diet, quercetin glycosides possess neuroprotective, cardioprotective, anti-oxidative, chemopreventive, and anti-allergic properties. Isoquercetin is one such promising candidate with anti-diabetic potential. However, complete studies of its molecular action on insulin signaling pathway and carbohydrate metabolizing enzymes remain unclear. Hence, we have designed this study to accumulate the experimental evidence in support of anti-diabetic effects of isoquercetin. Male albino Wistar rats were divided into seven groups. Rats (Groups 3-7) were administered a single intraperitoneal injection of streptozotocin (STZ; 40 mg/kg b.w) to induce diabetes mellitus. As an extension, STZ rats received isoquercetin at three different doses (20, 40 and 80 mg/kg b.w), and Group 7 rats received glibenclamide (standard drug) (600 μg/kg b.w). The results showed that STZ exaggerated blood sugar, decreased insulin, altered metabolizing enzymes, and impaired the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. Supplementation with isoquercetin significantly normalized blood sugar levels, insulin and regulated the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. The results achieved with isoquercetin are similar to that of standard drug glibenclamide. The findings suggest isoquercetin could be a possible therapeutic agent for treating diabetes mellitus in the near future. Copyright © 2018 Elsevier B.V. All rights reserved.

Deficient Differentiation of Mast Cells in the Skin of mi/mi Mice

PubMed Central

Kasugai, Tsutomu; Oguri, Kayoko; Jippo-Kanemoto, Tomoko; Morimoto, Masahiro; Yamatodani, Atsushi; Yoshida, Keiichi; Ebi, Yoshitaka; Isozaki, Koji; Tei, Hideki; Tsujimura, Tohru; Nomura, Shintaro; Okayama, Minoru; Kitamura, Yukihiko

1993-01-01

The staining property of skin mast cells changed from Alcian blue+/berberine sulfate- to Alcian blue +/berberine sulfate+ in the skin of normal (+/+) and Wv/Wv mice. In contrast, this change did not occur in the skin of mi/mi mice. Heparin content and histamine content per a mi/mi skin mast cell were estimated to be 34% and 18% those of a +/+ skin mast cell, respectively. The low heparin content of mi/mi skin mast cells seemed to be consistent with the Alcian blue+/berberine sulfate- staining property. Expression of genes encoding mast cell-specific proteolytic enzymes was examined by Northern blotting and in situ hybridization. Messenger RNA of mast cell carboxypeptidase A was expressed most of all by +/+, WV Wv/W+ and mi/mi skin mast cells, but mRNA of mouse mast cell protease (MMCP)-6 was expressed by approximately a half of +/+ and Wv/Wv skin mast cells and by only 3% of mi/mi skin mast cells. A significant amount of MMCP-2 mRNA was not expressed in the skin of all +/+, Wv/Wv and mi/mi mice. This shows the presence of at least three phenotypes in skin mast cells of mice: berberine sulfate+/MMCP-6+, berberine sulfate+/MMCP-6-, and berberine sulfate-/ MMCP-6-. The in situ hybridization of mRNA of mast cell-specific proteolytic enzymes seemed to be useful to describe abnormalities of mast cell differentiation in the skin of mi/mi mice. ImagesFigure 4Figure 5 PMID:8238251

Transcript Levels of Androgen Receptor Variant 7 and Ubiquitin-Conjugating Enzyme 2C in Hormone Sensitive Prostate Cancer and Castration-Resistant Prostate Cancer.

PubMed

Lee, Chan Ho; Ku, Ja Yoon; Ha, Jung Min; Bae, Sun Sik; Lee, Jeong Zoo; Kim, Choung-Soo; Ha, Hong Koo

2017-01-01

This study is designed to identify the androgen receptor variant 7 (AR-V7) status, clinical significance of AR-V7 in hormone sensitive prostate cancer (HSPC). Then, we evaluated AR-V7 and changes of its target gene, ubiquitin-conjugating enzyme E2C (UBE2C) which is an anaphase-promoting complex/cyclosome (APC/C)-specific ubiquitin-conjugating enzyme, in castration-resistant prostate cancer (CRPC) in serial tumor biopsies from patients receiving androgen deprivation therapy. We used RT-PCR and Q-PCR assay to evaluate AR-V7, androgen receptor full length (AR-FL), and UBE2C in tumor biopsies from patients with HSPC and CRPC. We examined associations between mRNA expression of AR-V7 and clinicopathologic factors. Furthermore, to identify other potential genes involved in the development of CRPC, RNA sequencing was conducted, using paired prostate cancer (PCa) tissues obtained immediately prior to treatment and at the time of therapeutic resistance. A total of 13 HSPC patients and three CRPC patients were enrolled. Neither a high Gleason score (score of 8 and 9) nor a high risk of PCa (a high risk of locally advanced PCa according to NCCN guidelines) was correlated with mRNA expression of AR-V7 in HSPC (P = 0.153 and P = 0.215). The mRNA expression of AR-FL, but not AR-V7, was significantly associated with the mRNA expression of UBE2C level in HSPC (P = 0.007). However, increased expression of AR-V7, not AR-FL, paralleled increased expression of UBE2C in the CRPC specimens (P = 0.03). AR-V7 expression status before ADT was likely related to shorter CRPC development in patients treating ADT. The result of the RNA-sequencing analysis using serial samples from the same patient before and after castration demonstrated an increased level of the PI3K regulatory subunit 1 (P = 0.018). Our study revealed the role of UBE2C as a marker of the androgen signaling pathway in PCa. Differential gene expression analysis using serial samples from the same patient before and after castration revealed potential genes and pathways involved in development of CRPC. Further studies are needed to determine whether these genes and pathways are potential therapeutic target for CRPC. Prostate 77:60-71, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Determination of Dietary Iron Requirements by Full Expression of Iron-Containing Enzymes in Various Tissues of Broilers.

PubMed

Ma, Xinyan; Liao, Xiudong; Lu, Lin; Li, Sufen; Zhang, Liyang; Luo, Xugang

2016-11-01

The current dietary iron requirement (80 mg/kg) of broilers is mainly based on growth, hemoglobin concentration, or hematocrit data obtained in a few early studies; however, expressions of iron-containing enzymes might be more sensitive novel criteria to evaluate dietary iron requirements. The objective of this study was to determine dietary iron requirements of broilers for the full expression of succinate dehydrogenase (SDH), catalase, and cytochrome c oxidase (COX) in various tissues. A total of 336 1-d-old Arbor Acres male chicks were randomly assigned to 1 of 7 treatments with 6 replicates and fed a basal corn and soybean-meal diet (control, containing 67 mg Fe/kg) and the basal diet supplemented with 20, 40, 60, 80, 100, or 120 mg Fe/kg from FeSO 4 ⋅ 7H 2 O for 21 d. Regression analysis was performed to estimate the optimal dietary iron concentration with the use of broken-line or quadratic models. SDH activity in the liver and heart, COX and catalase activity in the liver, Sdh mRNA levels in the liver, and Cox mRNA levels in the liver and heart of broilers were affected (P < 0.027) by supplemental iron concentration, and increased quadratically (P < 0.004) as dietary iron concentration increased. Dietary iron requirements estimated on the basis of fitted broken-line or quadratic-curve models (P < 0.005) of the above indexes were 97-136 mg/kg. SDH activity in the liver and heart, COX and catalase activity in the liver, Sdh mRNA levels in the liver, and Cox mRNA levels in the liver and heart are, to our knowledge, new and sensitive criteria to evaluate the dietary iron requirements of broilers, and the dietary iron requirements would be 97-136 mg/kg to support the full expression of the above iron-containing enzymes in various tissues of broiler chicks from 1 to 21 d of age, which are higher than the current NRC iron requirement. © 2016 American Society for Nutrition.

Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count.

PubMed

Loureiro, Bárbara; Ereno, Ronaldo L; Favoreto, Mauricio G; Barros, Ciro M

2016-07-15

Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus animals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concentration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concentration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV.

PubMed Central

Adamski, J; Normand, T; Leenders, F; Monté, D; Begue, A; Stéhelin, D; Jungblut, P W; de Launoit, Y

1995-01-01

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7487879

Effects of an Antimutagenic 1,4-Dihydropyridine AV-153 on Expression of Nitric Oxide Synthases and DNA Repair-related Enzymes and Genes in Kidneys of Rats with a Streptozotocin Model of Diabetes Mellitus.

PubMed

Ošiņa, Kristīne; Rostoka, Evita; Isajevs, Sergejs; Sokolovska, Jelizaveta; Sjakste, Tatjana; Sjakste, Nikolajs

2016-11-01

Development of complications of diabetes mellitus (DM), including diabetic nephropathy, is a complex multi-stage process, dependent on many factors including the modification of nitric oxide (NO) production and an impaired DNA repair. The goal of this work was to study in vivo effects of 1,4-dihydropyridine AV-153, known as antimutagen and DNA binder, on the expression of several genes and proteins involved in NO metabolism and DNA repair in the kidneys of rats with a streptozotocin (STZ)-induced model of DM. Transcription intensity was monitored by means of real-time RT-PCR and the expression of proteins by immunohistochemistry. Development of DM significantly induced PARP1 protein expression, while AV-153 (0.5 mg/kg) administration decreased it. AV-153 increased the expression of Parp1 gene in the kidneys of both intact and diabetic animals. Expression of H2afx mRNA and γH2AX histone protein, a marker of DNA breakage, was not changed in diabetic animals, but AV-153 up-regulated the expression of the gene without any impact on the protein expression. Development of DM was followed by a significant increase in iNOS enzyme expression, while AV-153 down-regulated the enzyme expression up to normal levels. iNos gene expression was also found to be increased in diabetic animals, but unlike the protein, the expression of mRNA was found to be enhanced by AV-153 administration. Expression of both eNOS protein and eNos gene in the kidneys was down-regulated, and the administration of AV-153 normalized the expression level. The effects of the compound in the kidneys of diabetic animals appear to be beneficial, as a trend for the normalization of expression of NO synthases is observed. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Expression of genes of the cardiac and renal renin-angiotensin systems in preterm piglets: is this system a suitable target for therapeutic intervention?

PubMed

Kim, Eleanor; Eiby, Yvonne; Lumbers, Eugenie; Boyce, Amanda; Gibson, Karen; Lingwood, Barbara

2015-10-01

The newborn circulating, cardiac and renal renin-angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets. Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term. Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs. © The Author(s), 2015.

Aberrant expression of erythropoietin in uterine leiomyoma: implications in tumor growth.

PubMed

Asano, Ryoko; Asai-Sato, Mikiko; Miyagi, Yohei; Mizushima, Taichi; Koyama-Sato, Makiko; Nagashima, Yoji; Taguri, Masataka; Sakakibara, Hideya; Hirahara, Fumiki; Miyagi, Etsuko

2015-08-01

Myomatous erythrocytosis syndrome is a rare complication of uterine leiomyoma caused by erythropoietin (EPO) that is produced by tumor cells. We assessed the EPO expression in leiomyomas and investigated the effects of EPO on the tumor growth. Tissue samples were collected from 114 patients with uterine leiomyomas who underwent myomectomy or hysterectomy in Yokohama City University Hospital. From 17 patients, the corresponding normal myometrium was also collected. All samples were analyzed for EPO messenger RNA (mRNA) expression by real-time reverse transcription-polymerase chain reaction. EPO protein expression was determined by an enzyme-linked immunosorbent assay. The relationships between EPO expression and clinicopathological features were retrospectively analyzed using the patients' charts. Blood vessel density and maturity were assessed using hematoxylin-eosin staining and CD34 immunohistochemistry. EPO mRNA expression was detected in 108 of 114, or 95%, of the leiomyomas. The mean EPO mRNA expression in the leiomyoma was higher than the corresponding normal myometrium (3836 ± 4122 vs 1455 ± 2141; P = .025 by Wilcoxon rank test). The EPO mRNA expression in the leiomyomas varied extensively among samples, ranging from undetectable levels to 18-fold above the mean EPO mRNA of normal myometrium. EPO protein production was observed concomitant with mRNA expression. A positive correlation of leiomyoma size and EPO mRNA expression was shown by Spearman rank correlation coefficient (ρ = 0.294; P = .001), suggesting the involvement of EPO in leiomyoma growth. The blood vessel maturity was also significantly increased in EPO-producing leiomyomas (high vessel maturity in high vs low EPO group: 67% vs 20%; P = .013 by Fisher exact test). This report demonstrates that EPO is produced in most of conventional leiomyomas and supports a model in which EPO accelerates tumor growth, possibly by inducing vessel maturity. Our study suggests one possible mechanism by which some uterine leiomyomas reach a large size, and the understanding of EPO expression patterns in these tumors may be useful for management of the patients with leiomyomas. Copyright © 2015 Elsevier Inc. All rights reserved.

Type 2 iodothyronine deiodinase in skeletal muscle: effects of hypothyroidism and fasting.

PubMed

Heemstra, Karen A; Soeters, Maarten R; Fliers, Eric; Serlie, Mireille J; Burggraaf, Jacobus; van Doorn, Martijn B; van der Klaauw, Agatha A; Romijn, Johannes A; Smit, Johannes W; Corssmit, Eleonora P; Visser, Theo J

2009-06-01

The iodothyronine deiodinases D1, D2, and D3 enable tissue-specific adaptation of thyroid hormone levels in response to various conditions, such as hypothyroidism or fasting. The possible expression of D2 mRNA in skeletal muscle is intriguing because this enzyme could play a role in systemic as well as local T3 production. We determined D2 activity and D2 mRNA expression in human skeletal muscle biopsies under control conditions and during hypothyroidism, fasting, and hyperinsulinemia. This was a prospective study. The study was conducted at a university hospital. We studied 11 thyroidectomized patients with differentiated thyroid carcinoma (DTC) on and after 4 wk off T4( replacement and six healthy lean subjects in the fasting state and during hyperinsulinemia after both 14 and 62 h of fasting. D2 activity and D2 mRNA levels were measured in skeletal muscle samples. No differences were observed in muscle D2 mRNA levels in DTC patients on and off T4 replacement therapy. In healthy subjects, muscle D2 mRNA levels were lower after 62 h compared to 14 h of fasting. Insulin increased mRNA expression after 62 h, but not after 14 h of fasting. Skeletal muscle D2 activities were very low and not influenced by hypothyroidism and fasting. Human skeletal muscle D2 mRNA expression is modulated by fasting and insulin, but not by hypothyroidism. The lack of a clear effect of D2 mRNA modulation on the observed low D2 activities questions the physiological relevance of D2 activity in human skeletal muscle.

Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

PubMed

Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

2014-09-01

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

Effect of resveratrol analogue, DMU-212, on antioxidant status and apoptosis-related genes in rat model of hepatocarcinogenesis.

PubMed

Piotrowska, H; Kujawska, M; Nowicki, M; Petzke, E; Ignatowicz, E; Krajka-Kuźniak, V; Zawierucha, P; Wierzchowski, M; Murias, M; Jodynis-Liebert, J

2017-02-01

The aim of the study was to examine whether antioxidant properties of 3,4,4',5-tetramethoxystilbene (DMU-212) contribute to its anticarcinogenic activity and whether DMU-212 affects the expression of apoptosis-related genes. Two-stage model of hepatocarcinogenesis was used; male Wistar rats were challenged with N-nitrosodiethylamine (NDEA), 200 mg/kg body weight (b.w.), intraperitoneal, then phenobarbital (PB) in drinking water (0.05%) was administered. Simultaneously, DMU-212 was given per os at a dose 20 or 50 mg/kg b.w. two times a week for 16 weeks. DMU-212 caused a moderate decrease in hepatic thiobarbituric acid reactive substances and protein carbonyls concentration elevated in rats treated with NDEA/PB. The activity of antioxidant enzymes examined reduced by NDEA/PB treatment was not restored in rats coadministered with DMU-212. Effects of DMU-212 on messenger RNA (mRNA) expression of antioxidant enzymes in rats challenged with NDEA/PB were diversified; no changes in their protein expression were noted in any of the groups. The expression of 17,000 genes was analyzed by Affymetrix® Rat Gene 1.1 ST Array; 15 apoptosis-related genes were selected and validated by RT-q PCR. The combined treatment with NDEA/PB and DMU-212 increased the mRNA level of some genes driving mitochondria-mediated apoptosis, whereas the mRNA expression of some anti-apoptotic genes triggering receptor-mediated apoptosis was reduced. The expression of genes encoding caspases-4, -8, -9, and -12 was also increased in rats treated with DMU-212. Although antioxidant effect of DMU-212 in rats challenged with NDEA/PB was moderate, its potential anticarcinogenic properties were demonstrated as evidenced by modulation of apoptosis-related genes.

ACTH-independent macronodular adrenocortical hyperplasia reveals prevalent aberrant in vivo and in vitro responses to hormonal stimuli and coupling of arginine-vasopressin type 1a receptor to 11β-hydroxylase

PubMed Central

2013-01-01

Background Adrenal Cushing’s syndrome caused by ACTH-independent macronodular adrenocortical hyperplasia (AIMAH) can be accompanied by aberrant responses to hormonal stimuli. We investigated the prevalence of adrenocortical reactions to these stimuli in a large cohort of AIMAH patients, both in vivo and in vitro. Methods In vivo cortisol responses to hormonal stimuli were studied in 35 patients with ACTH-independent bilateral adrenal enlargement and (sub-)clinical hypercortisolism. In vitro, the effects of these stimuli on cortisol secretion and steroidogenic enzyme mRNA expression were evaluated in cultured AIMAH and other adrenocortical cells. Arginine-vasopressin (AVP) receptor mRNA levels were determined in the adrenal tissues. Results Positive serum cortisol responses to stimuli were detected in 27/35 AIMAH patients tested, with multiple responses within individual patients occurring for up to four stimuli. AVP and metoclopramide were the most prevalent hormonal stimuli triggering positive responses in vivo. Catecholamines induced short-term cortisol production more often in AIMAH cultures compared to other adrenal cells. Short- and long-term incubation with AVP increased cortisol secretion in cultures of AIMAH cells. AVP also increased steroidogenic enzyme mRNA expression, among which an aberrant induction of CYP11B1. AVP type 1a receptor was the only AVPR expressed and levels were high in the AIMAH tissues. AVPR1A expression was related to the AVP-induced stimulation of CYP11B1. Conclusions Multiple hormonal signals can simultaneously induce hypercortisolism in AIMAH. AVP is the most prevalent eutopic signal and expression of its type 1a receptor was aberrantly linked to CYP11B1 expression. PMID:24034279

Time Course of mRNA Induction Elicited by Salt Stress in the Common Ice Plant (Mesembryanthemum crystallinum) 1

PubMed Central

Michalowski, Christine B.; Olson, Steven W.; Piepenbrock, Mechtild; Schmitt, Jürgen M.; Bohnert, Hans J.

1989-01-01

In the facultative halophyte Mesembryanthemum crystallinum (common ice plant), irrigation with solutions containing NaCl induces an alternate mode of carbon dioxide fixation, Crassulacean acid metabolism (CAM). The salt stress protocol which we have established facilitates the study of CAM induction and the correlation of changes in metabolism and gene expression. We have studied the time course of mRNA induction for phosphoenolpyruvate carboxylase (PEPCase) (gene: ppc) and several other enzymes of carbon metabolism during stress. While CAM is not fully established for at least 10 days after the start of stress, mRNA amounts for PEPCase and for other CAM enzymes, such as Pyruvate orthophosphate dikinase, increase between day 2 and 3 after stress induction. Increases continue for at least 5 days. Concomitant with the increase of CAM transcripts, fluctuations in the mRNA amounts for genes rbcS and cab were observed. Transcript levels for these proteins decreased several-fold during a 3 to 4 day period. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16666626

HSP90 gene expression induced by aspirin is associated with damage remission in a chicken myocardial cell culture exposed to heat stress.

PubMed

Zhang, X; Qian, Z; Zhu, H; Tang, S; Wu, D; Zhang, M; Kemper, N; Hartung, J; Bao, E

2016-08-01

To understand the potential protection of heat shock protein 90 (HSP90) induced by aspirin against heat stress damage in chicken myocardial cells, enzyme activities related to stress damage, cytopathological changes, the expression and distribution of HSP90, and HSP90 mRNA levels in the myocardial cells exposed to heat stress (42°C) for different durations with or without aspirin administration (1 mg/ml, 2 h prior) in vitro were investigated. Significant increase of enzyme levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterised by acute degeneration, karyopyknosis and karyorrhexis were observed, compared to non-treated cells. However, the lesions of cells treated with aspirin were milder, characterised by earlier recovery of enzyme levels to the control levels and no obvious heat stress-related cellular necrosis. Stronger positive signals in the cytoplasm and longer retention of HSP90 signal in nuclei were observed in aspirin-treated myocardial cells than those of only heat-stressed cells. HSP90 level in the aspirin-treated myocardial cells was 11.1-fold higher than that in non-treated cells, and remained at a high level at the early stage of heat stress, whereas it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to a low level. Overexpression of HSP90 mRNA in aspirin-treated cells was observed throughout the experiment, whereas HSP90 mRNA decreased significantly only in heat-stressed cells. The early higher HSP90 expression induced by aspirin during heat stress was accompanied by decreased heat stress damage, suggesting that aspirin might play an important role in preventing myocardial cells from heat stress damage in vitro.

Expression analysis and clinical utility of L-Dopa decarboxylase (DDC) in prostate cancer.

PubMed

Avgeris, Margaritis; Koutalellis, Georgios; Fragoulis, Emmanuel G; Scorilas, Andreas

2008-10-01

L-Dopa decarboxylase (DDC) is a pyridoxal 5'-phosphate-dependent enzyme that was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of DDC in prostate tissues and to evaluate its clinical utility in prostate cancer (CaP). Total RNA was isolated from 118 tissue specimens from benign prostate hyperplasia (BPH) and CaP patients and a highly sensitive quantitative real-time RT-PCR (qRT-PCR) method for DDC mRNA quantification has been developed using the SYBR Green chemistry. LNCaP prostate cancer cell line was used as a calibrator and GAPDH as a housekeeping gene. DDC was found to be overexpressed, at the mRNA level, in the specimens from prostate cancer patients, in comparison to those from benign prostate hyperplasia patients (p<0.001). Logistic regression and ROC analysis have demonstrated that the DDC expression has significant discriminatory value between CaP and BPH (p<0.001). DDC expression status was compared with other established prognostic factors, in prostate cancer. High expression levels of DDC were found more frequently in high Gleason's score tumors (p=0.022) as well as in advanced stage patients (p=0.032). Our data reveal the potential of DDC expression, at the mRNA level, as a novel biomarker in prostate cancer.

Silver nanoparticles administered to chicken affect VEGFA and FGF2 gene expression in breast muscle and heart

NASA Astrophysics Data System (ADS)

Hotowy, Anna; Sawosz, Ewa; Pineda, Lane; Sawosz, Filip; Grodzik, Marta; Chwalibog, André

2012-07-01

Nanoparticles of colloidal silver (AgNano) can influence gene expression. Concerning trials of AgNano application in poultry nutrition, it is useful to reveal whether they affect the expression of genes crucial for bird development. AgNano were administered to broiler chickens as a water solution in two concentrations (10 and 20 ppm). After dissection of the birds, breast muscles and hearts were collected. Gene expression of FGF2 and VEGFA on the mRNA and protein levels were evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay methods. The results for gene expression in the breast muscle revealed changes on the mRNA level ( FGF2 was up-regulated, P < 0.05) but not on the protein level. In the heart, 20 ppm of silver nanoparticles in drinking water increased the expression of VEGFA ( P < 0.05), at the same time decreasing FGF2 expression both on the transcriptional and translational levels. Changes in the expression of these genes may lead to histological changes, but this needs to be proven using histological and immunohistochemical examination of tissues. In general, we showed that AgNano application in poultry feeding influences the expression of FGF2 and VEGFA genes on the mRNA and protein levels in growing chicken.

Recurrent hypoinsulinemic hyperglycemia in neonatal rats increases PARP-1 and NF-κB expression and leads to microglial activation in the cerebral cortex.

PubMed

Gisslen, Tate; Ennis, Kathleen; Bhandari, Vineet; Rao, Raghavendra

2015-11-01

Hyperglycemia is a common metabolic problem in extremely low-birth-weight preterm infants. Neonatal hyperglycemia is associated with increased mortality and brain injury. Glucose-mediated oxidative injury may be responsible. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair and cell survival. However, PARP-1 overactivation leads to cell death. NF-κB is coactivated with PARP-1 and regulates microglial activation. The effects of recurrent hyperglycemia on PARP-1/NF-κB expression and microglial activation are not well understood. Rat pups were subjected to recurrent hypoinsulinemic hyperglycemia of 2 h duration twice daily from postnatal (P) day 3-P12 and killed on P13. mRNA and protein expression of PARP-1/NF-κB and their downstream effectors were determined in the cerebral cortex. Microgliosis was determined using CD11 immunohistochemistry. Recurrent hyperglycemia increased PARP-1 expression confined to the nucleus and without causing PARP-1 overactivation and cell death. NF-κB mRNA expression was increased, while IκB mRNA expression was decreased. inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) mRNA expressions were decreased. Hyperglycemia significantly increased the number of microglia. Recurrent hyperglycemia in neonatal rats is associated with upregulation of PARP-1 and NF-κB expression and subsequent microgliosis but not neuronal cell death in the cerebral cortex.

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

PubMed Central

Gunawardana, Dilantha; Cheng, Heung-Chin; Gayler, Kenwyn R.

2008-01-01

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. PMID:18025047

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

PubMed

Lim, Ratana; Barker, Gillian; Menon, Ramkumar; Lappas, Martha

2016-11-01

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF 2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway. © 2016 by the Society for the Study of Reproduction, Inc.

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: a prospective multi-institutional study in Japan.

PubMed

Eguchi, Keisuke; Oyama, Takahiko; Tajima, Atsushi; Abiko, Tomohiro; Sawafuji, Makoto; Horio, Hirotoshi; Hashizume, Toshinori; Matsutani, Noriyuki; Kato, Ryoichi; Nakayama, Mitsuo; Kawamura, Masafumi; Kobayashi, Koichi

2015-01-01

This investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. 236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2 cm or greater), IB, and II tumors were given a dose of 250 mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Among 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS. The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Expression of cyclooxygenase-2 in the canine lower urinary tract with regard to the effects of gonadal status and gender.

PubMed

Ponglowhapan, S; Church, D B; Khalid, M

2009-05-01

As pituitary gonadotrophins can induce prostaglandin (PG) synthesis and receptors for LH and FSH are present in the canine lower urinary tract (LUT), the objectives of this study were to (i) investigate the expression of COX-2, a key rate-limiting enzyme in PG production, in the canine LUT and (ii) determine if COX-2 expression differs between gender, gonadal status (intact and gonadectomised) and LUT regions. Four regions (body and neck of the bladder as well as proximal and distal urethra) of the LUT were obtained from 20 clinically healthy dogs (5 intact males, 5 intact anoestrous females, 4 castrated males, 6 spayed females). In situ hybridization and immunohistochemistry were performed to determine the presence of COX-2 mRNA and protein, respectively. The mRNA and protein expression was semi-quantitatively assessed. The scoring system combined both the distribution and intensity of positive staining and was carried out separately on the three tissue layers (epithelium, sub-epithelial stroma and muscle) for each of four regions of the LUT. In comparison to intact dogs, lower expression (P<0.001) of COX-2 and its mRNA in gonadectomised males and females was observed in all tissue layers of each region of the LUT except in the distal urethra where there was no difference in mRNA expression between gonadal statuses. Regardless of region and tissue layer, intact females expressed more (P<0.05) COX-2 and its mRNA than intact males. However, in gonadectomised dogs, mRNA expression of COX-2 did not differ between genders; males had higher (P<0.001) protein level of COX-2 compared to females. In conclusion, both COX-2 and its mRNA were expressed in the canine LUT and COX-2-regulated PG synthesis in the canine LUT may differ between gonadal statuses and genders. The lower expression of COX-2 in gonadectomised dogs may impair normal function of the LUT and probably implicated in the development of neutering-induced urinary incontinence in the dog.

Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer.

PubMed

Wojtczyk-Miaskowska, Anita; Presler, Malgorzata; Michajlowski, Jerzy; Matuszewski, Marcin; Schlichtholz, Beata

2017-01-01

This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006). This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive prognostic markers in this pathology. © 2017 The Author(s). Published by S. Karger AG, Basel.

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

Targeting FASN for Breast Cancer Treatment by Repositioning PPIs

DTIC Science & Technology

2017-01-01

the sole cytosolic enzyme responsible for de novo synthesis of palmitate. Recently, it was found that FASN up-regulation contributes to drug and...increased whereas Flag-p65 (NF-κB) over-expression significantly reduced PARP-1 protein and mRNA levels. Knocking down p65 using shRNA significantly...increased PARP-1 protein and mRNA levels (Fig. 2C), whereas activation of p65 with TNF-α had contrary effect (Fig. 2D). Taken together, these results

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex.

PubMed

Łopieńska-Biernat, E; Zaobidna, E A; Dmitryjuk, M

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen-trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)-in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

PubMed Central

Łopieńska-Biernat, E.; Zaobidna, E. A.; Dmitryjuk, M.

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes. PMID:26783451

Expression and regulation of Icer mRNA in the Syrian hamster pineal gland.

PubMed

Diaz, Elena; Garidou, Marie-Laure; Dardente, Hugues; Salingre, Anthony; Pévet, Paul; Simonneaux, Valérie

2003-04-10

Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

Hepatic and extrahepatic distribution of ornithine urea cycle enzymes in holocephalan elephant fish (Callorhinchus milii).

PubMed

Takagi, Wataru; Kajimura, Makiko; Bell, Justin D; Toop, Tes; Donald, John A; Hyodo, Susumu

2012-04-01

Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Posttranscriptional regulation of adrenal TH gene expression contributes to the maladaptive responses triggered by insulin-induced recurrent hypoglycemia.

PubMed

Kudrick, Necla; Chan, Owen; La Gamma, Edmund F; Kim, Juhye Lena; Tank, Arnold William; Sterling, Carol; Nankova, Bistra B

2015-02-01

Acute metabolic stress such as insulin-induced hypoglycemia triggers a counterregulatory response during which the release of catecholamines (epinephrine), the activation of tyrosine hydroxylase (TH) enzyme and subsequent compensatory catecholamine biosynthesis occur in the adrenal medulla. However, recurrent exposure to hypoglycemia (RH), a consequence of tight glycemic control in individuals with type 1 and type 2 diabetes compromises this physiological response. The molecular mechanisms underlying the maladaptive response to repeated glucose deprivation are incompletely understood. We hypothesize that impaired epinephrine release following RH reflects altered regulation of adrenal catecholamine biosynthesis. To test this hypothesis, we compared the effect of single daily (RH) and twice-daily episodes of insulin-induced hypoglycemia (2RH) on adrenal epinephrine release and production in normal rats. Control animals received saline injections under similar conditions (RS and 2RS, respectively). Following 3 days of treatment, we assessed the counterregulatory hormonal responses during a hypoglycemic clamp. Changes in adrenal TH gene expression were also analyzed. The counterregulatory responses, relative TH transcription and TH mRNA levels and Ser40-TH phosphorylation (marker for enzyme activation) were induced to a similar extent in RS, 2RS, and RH groups. In contrast, epinephrine and glucagon responses were attenuated in the 2RH group and this was associated with a limited elevation of adrenal TH mRNA, rapid inactivation of TH enzyme and no significant changes in TH protein. Our results suggest that novel posttranscriptional mechanisms controlling TH mRNA and activated TH enzyme turnover contribute to the impaired epinephrine responses and may provide new therapeutic targets to prevent HAAF. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Posttranscriptional regulation of adrenal TH gene expression contributes to the maladaptive responses triggered by insulin-induced recurrent hypoglycemia

PubMed Central

Kudrick, Necla; Chan, Owen; La Gamma, Edmund F; Kim, Juhye Lena; Tank, Arnold William; Sterling, Carol; Nankova, Bistra B

2015-01-01

Acute metabolic stress such as insulin-induced hypoglycemia triggers a counterregulatory response during which the release of catecholamines (epinephrine), the activation of tyrosine hydroxylase (TH) enzyme and subsequent compensatory catecholamine biosynthesis occur in the adrenal medulla. However, recurrent exposure to hypoglycemia (RH), a consequence of tight glycemic control in individuals with type 1 and type 2 diabetes compromises this physiological response. The molecular mechanisms underlying the maladaptive response to repeated glucose deprivation are incompletely understood. We hypothesize that impaired epinephrine release following RH reflects altered regulation of adrenal catecholamine biosynthesis. To test this hypothesis, we compared the effect of single daily (RH) and twice-daily episodes of insulin-induced hypoglycemia (2RH) on adrenal epinephrine release and production in normal rats. Control animals received saline injections under similar conditions (RS and 2RS, respectively). Following 3 days of treatment, we assessed the counterregulatory hormonal responses during a hypoglycemic clamp. Changes in adrenal TH gene expression were also analyzed. The counterregulatory responses, relative TH transcription and TH mRNA levels and Ser40-TH phosphorylation (marker for enzyme activation) were induced to a similar extent in RS, 2RS, and RH groups. In contrast, epinephrine and glucagon responses were attenuated in the 2RH group and this was associated with a limited elevation of adrenal TH mRNA, rapid inactivation of TH enzyme and no significant changes in TH protein. Our results suggest that novel posttranscriptional mechanisms controlling TH mRNA and activated TH enzyme turnover contribute to the impaired epinephrine responses and may provide new therapeutic targets to prevent HAAF. PMID:25713330

Modulation of steroidogenesis by vitamin D3 in granulosa cells of the mouse model of polycystic ovarian syndrome.

PubMed

Bakhshalizadeh, Shabnam; Amidi, Fardin; Alleyassin, Ashraf; Soleimani, Masoud; Shirazi, Reza; Shabani Nashtaei, Maryam

2017-06-01

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women of reproductive age characterized by polycystic ovarian morphology, anovulation or oligomenorrhea, and hyperandrogenism. It is shown that disruption in the steroidogenesis pathway caused by excess androgen in PCOS is a critical element of abnormal folliculogenesis and failure in dominant follicle selection. Vitamin D plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells. In the present study, we investigated the effects of vitamin D3 on steroidogenic enzyme expression and activities in granulosa cell using a PCOS mouse model. In our study, the PCOS mouse model was developed by the injection of dehydroepiandrosterone (DHEA) for 20 days. The mRNA and protein expression levels of genes involved in steroidogenesis in granulosa cells were compared between polycystic and normal ovaries using real-time PCR and Western blotting assays. Granulosa cells of DHEA-induced PCOS mice were then cultured with and without vitamin D3 and mRNA and protein expression levels of steroidogenic enzymes and serum 17beta-estradiol and progesterone levels were investigated using qRT-PCR, western blot, and radioimmunoassay, respectively. Steroidogenic enzymes including Cyp11a1, StAR, Cyp19a1, and 3β-HSD were upregulated in granulosa cells of PCOS mice when compared to normal mice. Treatment with vitamin D3 decreased mRNA and protein expression levels of steroidogenic enzymes in cultured granulosa cells. Vitamin D3 also decreased aromatase and 3β-HSD activity that leads to decreased 17beta-estradiol and progesterone release. This study suggests that vitamin D3 could modulate the steroidogenesis pathway in granulosa cells of PCOS mice that may lead to improving follicular development and maturation. This is a step towards a possible conceivable treatment for PCOS. AMHR-II: anti-müllerian hormone receptor-II; 3β-HSD: 3β-hydroxysteroid dehydrogenase; Cyp11a1: Cytochrome P450 Family 11 Subfamily A Member 1; Cyp19a1: cytochrome P450 aromatase; DHEA: dehydroepiandrosterone; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; IVF: in vitro fertilization; 25OHD: 25-hydroxy vitamin D; OHSS: ovarian hyperstimulation syndrome; PCOS: polycystic ovarian syndrome; P450scc: P450 side-chain cleavage enzyme; StAR: steroidogenic acute regulatory protein; VDRs: vitamin D receptors.

Activation of pregnane X receptor and induction of MDR1 by dietary phytochemicals.

PubMed

Satsu, Hideo; Hiura, Yuto; Mochizuki, Keiichi; Hamada, Mika; Shimizu, Makoto

2008-07-09

The pregnane X receptor (PXR) is understood to be the key regulator for gene expression of such drug-metabolizing enzymes and transporters as multidrug-resistant protein 1 (MDR1) and the cytochrome P450 (CYP) family. We examined the effect of dietary phytochemicals on the PXR-dependent transcriptional activity in human intestinal LS180 cells by using a reporter assay. Among approximately 40 kinds of phytochemicals, tangeretin and ginkgolides A and B markedly induced the PXR-dependent transcriptional activity and also the activity of the human MDR1 promoter. The expression levels of MDR1 mRNA as well as of CYP3A4 mRNA, another gene regulated by PXR, were significantly increased by these phytochemicals. Furthermore, an increase was observed of the MDR1 protein and its functional activity by tangeretin and by ginkgolides A and B. These findings strongly suggest that tangeretin and ginkgolides A and B activated PXR, thereby regulating detoxification enzymes and transporters in the intestines.

Proteinases during Early Development of the Pacific Whiteleg Shrimp Penaeus vannamei.

PubMed

Hernandez-Cortes, Patricia; Rivera-Pérez, Crisalejandra; García-Carreño, Fernando; Martínez-Alarcón, Diana

2017-02-01

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation.

PubMed

Guillot, Flora; Garcia, Alexandra; Salou, Marion; Brouard, Sophie; Laplaud, David A; Nicot, Arnaud B

2015-07-04

Astrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35-55 immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes. The spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes. We described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.

Hepatic expression of transcription factors affecting developmental regulation of UGT1A1 in the Han Chinese population.

PubMed

Nie, Ya-Li; He, Hang; Li, Jiang-Feng; Meng, Xiang-Guang; Yan, Liang; Wang, Pei; Wang, Shu-Jie; Bi, Hong-Zheng; Zhang, Li-Rong; Kan, Quan-Cheng

2017-01-01

Complete or partial inactivity of UGT1A1, the unique enzyme responsible for bilirubin glucuronidation, is commonly associated with hyperbilirubinemia. We investigated the dynamic expression of UGT1A1, and that of the transcription factors (TFs) involved in its developmental regulation, during human hepatic growth in Han Chinese individuals. Eighty-eight prenatal, pediatric, and adult liver samples were obtained from Han Chinese individuals. Quantitative real-time polymerase chain reaction was used to evaluate mRNA expression of UGT1A1 and TFs including PXR, CAR, HNF1A, HNF4A, PPARA, etc. UGT1A1 protein levels and metabolic activity were determined by western blotting and high-performance liquid chromatography. Direct sequencing was employed to genotype UGT1A1*6 (211G˃A) and UGT1A1*28 (TA6˃TA7) polymorphisms. UGT1A1 expression was minimal in prenatal samples, but significantly elevated during pediatric and adult stages. mRNA and protein levels and metabolic activity were prominently increased (120-, 20-, and 10-fold, respectively) in pediatric and adult livers compared to prenatal samples. Furthermore, expression did not differ appreciably between pediatric and adult periods. Dynamic expression of TFs, including PXR, CAR, HNF1A, HNF4A, and PPARA, was consistent with UGT1A1 levels at each developmental stage. A pronounced correlation between expression of these TFs and that of UGT1A1 (P < 0.001) was observed. Moreover, UGT1A1*6 and UGT1A1*28 polymorphisms reduced levels of UGT1A1 by up to 40-60 %. Hepatic expression of transcription factors is associated with developmental regulation of UGT1A1 in the Han Chinese population. Moreover, UGT1A1 polymorphisms are associated with reduced expression of UGT1A1 mRNA and protein, as well as enzyme activity.

The effects of emodin on cell viability, respiratory burst and gene expression of Nrf2-Keap1 signaling molecules in the peripheral blood leukocytes of blunt snout bream (Megalobrama amblycephala).

PubMed

Zhao, Zhenxin; Xie, Jun; Liu, Bo; Ge, Xianping; Song, Changyou; Ren, Mingchun; Zhou, Qunlan; Miao, Linghong; Zhang, Huimin; Shan, Fan; Yang, Zhenfei

2017-03-01

We determined the effects of emodin on the cell viability, respiratory burst activity, mRNA levels of antioxidative enzymes (Cu-Zn SOD, CAT and NOX2), and gene expressions of the Nrf2-Keap1 signaling molecules in the peripheral blood leukocytes of blunt snout bream. Triplicate groups of cultured cells were treated with different concentrations of emodin (0.04-25 μg/ml) for 24 h. Results showed that the emodin caused a dramatic loss in cell viability, and occurred in a dose-dependent manner. Emodin exposure (1-25 μg/ml) were significantly induced the ROS generation compared to the control. The respiratory burst and NADPH oxidase activities were significantly induced at a concentration of 0.20 μg/ml, and inhibited at 25 μg/ml. Besides, mRNA levels of antioxidant enzyme genes were dramatically regulated by emodin exposure for 24 h. During low concentrations of exposure, mRNA levels of Cu-Zn SOD in the cells treated with 0.04, 0.20 μg/ml, CAT, NOX2 and Nrf2 in the cells treated with 1 μg/ml were sharply increased, respectively. Whereas, high concentrations were dramatically down-regulated the gene expressions of CAT in the cells treated with 5, 25 μg/ml and NOX2 in the cells treated with 25 μg/ml. Furthermore, sharp increase in Keap1and Bach1 expression levels were observed a dose-dependent manner. In conclusion, this study demonstrated that emodin could induce antioxidant defenses which were involved in cytotoxic activities, respiratory burst and the transcriptional regulation levels of antioxidant enzymes and Nrf2-Keap1 signaling molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

PubMed

Goppelt-Struebe, M; Schaefer, D; Habenicht, A J

1997-10-01

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

PubMed Central

Hahnvajanawong, Chariya; Chaiyagool, Jariya; Seubwai, Wunchana; Bhudhisawasdi, Vajarabhongsa; Namwat, Nisana; Khuntikeo, Narong; Sripa, Banchob; Pugkhem, Ake; Tassaneeyakul, Wichittra

2012-01-01

AIM: To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). METHODS: The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. RESULTS: Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). CONCLUSION: OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients. PMID:22912546

Captopril reduces cardiac inflammatory markers in spontaneously hypertensive rats by inactivation of NF-kB

PubMed Central

2010-01-01

Background Captopril is an angiotensin-converting enzyme (ACE) inhibitor widely used in the treatment of arterial hypertension and cardiovascular diseases. Our objective was to study whether captopril is able to attenuate the cardiac inflammatory process associated with arterial hypertension. Methods Left ventricle mRNA expression and plasma levels of pro-inflammatory (interleukin-1β (IL-1β) and IL-6) and anti-inflammatory (IL-10) cytokines, were measured in spontaneously hypertensive rats (SHR) and their control normotensive, Wistar-Kyoto (WKY) rats, with or without a 12-week treatment with captopril (80 mg/Kg/day; n = six animals per group). To understand the mechanisms involved in the effect of captopril, mRNA expression of ACE, angiotensin II type I receptor (AT1R) and p22phox (a subunit of NADPH oxidase), as well as NF-κB activation and expression, were measured in the left ventricle of these animals. Results In SHR, the observed increases in blood pressures, heart rate, left ventricle relative weight, plasma levels and cardiac mRNA expression of IL-1β and IL-6, as well as the reductions in the plasma levels and in the cardiac mRNA expression of IL-10, were reversed after the treatment with captopril. Moreover, the mRNA expressions of ACE, AT1R and p22phox, which were enhanced in the left ventricle of SHR, were reduced to normal values after captopril treatment. Finally, SHR presented an elevated cardiac mRNA expression and activation of the transcription nuclear factor, NF-κB, accompanied by a reduced expression of its inhibitor, IκB; captopril administration corrected the observed changes in all these parameters. Conclusion These findings show that captopril decreases the inflammation process in the left ventricle of hypertensive rats and suggest that NF-κB-driven inflammatory reactivity might be responsible for this effect through an inactivation of NF-κB-dependent pro-inflammatory factors. PMID:20462420

ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

PubMed Central

Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

2015-01-01

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

Serum sex steroids and steroidogenesis-related enzyme expression in skeletal muscle during experimental weight gain in men.

PubMed

Sato, K; Samocha-Bonet, D; Handelsman, D J; Fujita, S; Wittert, G A; Heilbronn, L K

2014-12-01

Low-circulating testosterone is associated with development of type 2 diabetes in obese men. In this study, we examined the effects of experimental overfeeding and weight gain on serum levels of sex hormones and skeletal muscle expression of steroidogenic enzymes in healthy men with (FH+) and without (FH-) a family history of type 2 diabetes. Following a 3-day lead in energy balanced diet, FH+ (n = 9) and FH- men (n = 11) were overfed by 5200 kJ/day (45% fat) for 28 days. Body weight, fasting glucose, insulin, sex steroid, sex hormone binding globulin (SHBG) levels, insulin sensitivity (hyperinsulinaemic-euglycaemic clamp) and body fat (DXA) were assessed in all individuals at baseline and day 28, and sex steroidogenesis-related enzyme expression in vastus lateralis biopsies was examined in a subset (n = 11). Body weight, fat mass and fasting insulin levels were increased by overfeeding (P < 0.01) and insulin was increased significantly more in FH+ men (P<0.01). Serum sex hormone binding globulin (SHBG) and 5α-dihydrotestosterone (DHT) were reduced with overfeeding (P < 0.05), and serum testosterone and DHT were reduced to a greater extent in FH+ men (P < 0.05). Overfeeding reduced mRNA expression of 3β-hydroxysteroid dehydrogenase (HSD) and 17βHSD (P ≤ 0.007), independently of group. 5α-Reductase (SRD5A1) mRNA expression was not changed overall, but a time by group interaction was observed (P = 0.04). Overfeeding reduced SHBG and muscle expression of enzymes involved in the formation of testosterone in skeletal muscle. Men with a family history of T2DM were more susceptible to deleterious outcomes of overfeeding with greater reductions in serum testosterone and DHT and greater increases in markers of insulin resistance, which may contribute to increased risk of developing type 2 diabetes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Renal expression of aminopeptidase A in rats with two-kidney, one-clip hypertension.

PubMed

Wolf, G; Wenzel, U; Assmann, K J; Stahl, R A

2000-12-01

Angiotensin II (ANG II) is a major factor involved in the progression of chronic renal disease. Although the generation of this vasoactive peptide has been investigated in great detail, only a few studies have hitherto addressed the metabolism of ANG II into fragments such as angiotensin III and IV (ANG III, IV) which may exert physiological effects independent of ANG II. Aminopeptidase A (APA) is the major enzyme degrading ANG II. The aim of the current study was to evaluate glomerular APA expression in rats with two-kidney, one-clip hypertension. The left renal artery was restricted with a 0.2-mm silver clip. Kidneys were harvested 1 and 4 weeks after surgery. APA enzyme and protein expression was evaluated in kidney sections. Total APA enzyme activity and mRNA expression was assessed in isolated glomeruli. Degradation of exogenous ANG II by isolated glomeruli was measured with reverse-phase high-performance liquid chromatography. APA enzyme activity, protein, and mRNA expression were stimulated in the clipped kidney 1 week after surgery compared with the contralateral kidney or normal controls. In contrast, 4 weeks after clipping APA activity and expression was higher in the contralateral kidney. In parallel to these findings, degradation of ANG II was greatest in isolated glomeruli obtained from the clipped kidney after 1 week. However, preparations from the contralateral kidney 4 weeks after surgery were more active in the metabolism of exogenous ANG II. The present study provides evidence that APA is complexly regulated in in vivo situations with an activated local renin-ANG II system. ANG II appears to play a direct role in this regulation. However, since conversion of ANG II to ANG III by APA is the initial step leading to the formation of ANG IV which may exert detrimental effects not mediated through classical ANG II receptors, a local increase in APA activity may contribute to the progression of chronic renal disease even during complete AT(1)-receptor blockade.

Mitochondrial MnSOD mRNA expression in human chorioamniotic membranes and its association with labor, inflammation and infection

PubMed Central

Than, Nandor Gabor; Romero, Roberto; Tarca, Adi L.; Draghici, Sorin; Erez, Offer; Chaiworapongsa, Tinnakorn; Kim, Yeon Mee; Kim, Sun Kwon; Vaisbuch, Edi; Tromp, Gerard

2010-01-01

Objective Human parturition is characterized by the activation of genes involved in acute inflammatory in the fetal membranes. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges reactive oxygen species (ROS). MnSOD is up-regulated in sites of inflammation and has an important role in the down-regulation of acute inflammatory processes. Therefore, the aim of this study was to determine the differences in MnSOD mRNA expression in the fetal membranes in patients with term and preterm labor as well as in acute chorioamnionitis. Study design Fetal membranes were obtained from patients in the following groups: 1) term not in labor (n=29); 2) term in labor (n=29); 3) spontaneous preterm labor with intact mebranes (n=16); 4) PTL with histological chorioamnionitis (n=12); 5) preterm prelabor rupture of membranes (PPROM; n=17); and 6) PPROM with histological chorioamnionitis (n=21). MnSOD mRNA expression in the membranes was determined by quantitative real-time RT-PCR. Results 1) MnSOD mRNA expression was higher in the fetal membranes of patients at term in labor than those not in labor (2.4-fold; p=0.02); 2) the amount of MnSOD mRNA in the fetal membranes was higher in PTL than in term labor or in PPROM (7.2-fold, p=0.03; 3.2-fold, p=0.03, respectively); 3) MnSOD mRNA expression was higher when histological chorioamnionitis was present both among patients with PPROM (3.8-fold, p=0.02) and with PTL (5.4-fold, p=0.02) than in patients with these conditions without histological chorioamnionitis; 4) expression of MnSOD mRNA was higher in PTL with chorioamnionitis than in PPROM with chorioamnionitis (4.3-fold, p=0.03); Conclusion The increase in MnSOD mRNA expression by fetal membranes in term labor and in histological chorioamnionitis in PTL and PPROM suggests that the fetus deploys anti-oxidant mechanisms to constrain the inflammatory processes in the chorioamniotic membranes. PMID:19900038

Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

2016-01-05

Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Deoxyribonucleic Acid Replication and Expression of Early and Late Bacteriophage Functions in Bacillus subtilis

PubMed Central

Pène, Jacques J.; Marmur, Julius

1967-01-01

The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with bacteriophage 2C has been studied. These phage-induced enzymes are synthesized at different times during the latent period. It was shown by actinomycin inhibition that the formation of the late enzyme (lysozyme) required messenger ribonucleic acid (mRNA) synthesized de novo after the initiation of translation of mRNA which specifies the early function (dCMP deaminase). The inhibition of phage DNA synthesis by mitomycin C prevented the synthesis of lysozyme only when added before the onset of phage DNA replication, but it did not affect the synthesis or action of dCMP deaminase when added at any time during the latent period. Treatment of infected cells with mitomycin C after phage DNA synthesis had reached 8 to 10% of its maximal rate resulted in the production of normal amounts of lysozyme. These observations suggest that mRNA specifying early enzymes can be transcribed from parental (and probably also from progeny) DNA, whereas late functional messengers can be transcribed only after the formation of progeny DNA. PMID:4990039

Reversal of high fat diet-induced obesity through modulating lipid metabolic enzymes and inflammatory markers expressions in rats.

PubMed

A, Kalaivani; Uddandrao, V V Sathibabu; Parim, Brahmanaidu; Ganapathy, Saravanan; P R, Nivedha; Kancharla, Sushma Chandulee; P, Rameshreddy; K, Swapna; Sasikumar, Vadivukkarasi

2018-03-19

In this study, we evaluated the ameliorative potential of Cucurbita maxima seeds oil (CSO (100 mg/kg body weight)) supplementation to high fat diet (HFD)-induced obese rats for 30 days on the changes in body weight, markers of lipid metabolism such as LDL, HDL, triglycerides, total cholesterol, adiponectin, leptin, amylase, and lipase. We also investigated the effects of CSO on the changes of lipid metabolic enzymes such as fatty-acid synthase, acetyl CoA carboxylase, carnitine palmitoyl transferase-1, HMG CoA reductase, and inflammatory markers (TNF-α and IL-6). Administration of CSO revealed significant diminution in body weight gain, altered the activity, expressions of lipid marker enzymes and inflammatory markers. It demonstrated that CSO had considerably altered these parameters when evaluated with HFD control rats. In conclusion, this study suggested that CSO might ameliorate the HFD-induced obesity by altering the enzymes and mRNA expressions important to lipid metabolism.

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

PubMed

Jayashree, R; Nazeem, P A; Rekha, K; Sreelatha, S; Thulaseedharan, A; Krishnakumar, R; Kala, R G; Vineetha, M; Leda, P; Jinu, U; Venkatachalam, P

2018-06-01

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH.

PubMed

Caballero, Francisco; Fernández, Anna; De Lacy, Antonio M; Fernández-Checa, Jose C; Caballería, Juan; García-Ruiz, Carmen

2009-04-01

Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains unknown. Due to the emerging role of free cholesterol (FC) in NAFLD, our aim was to examine the correlation between FC accumulation in patients with NAFLD and the expression of enzymes that regulate cholesterol homeostasis. Filipin staining, indicative of FC accumulation, and real-time PCR analyses were performed in 31 NAFLD patients and in seven controls. All NASH patients (n=14) and 4 out of 17 patients with steatosis exhibited filipin staining compared to controls (0 out of 7 subjects with normal liver histology and BMI). Sterol regulatory element-binding protein-2 (SREBP-2) mRNA levels were 7- and 3-fold higher in NASH and steatosis patients, respectively, compared to controls. Since hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterol synthesis and transcriptionally controlled by SREBP-2 we measured its mRNA levels, being 3- to 4-fold higher in NAFLD compared to controls, without any difference between NASH and steatosis patients. Fatty acid synthase (FAS) and SREBP-1c expression were not significantly induced in NAFLD, while ATP-binding cassette sub-family G member 1 (ABCG1), a transporter involved in cholesterol egress, and acyl-CoA-cholesterol acyltransferase mRNA levels were modestly increased (1.5- to 2.5-fold, p<0.05), regardless of fibrosis. Interestingly, mRNA levels of steroidogenic acute regulatory protein (StAR), a mitochondrial-cholesterol transporting polypeptide, increased 7- and 15-fold in steatosis and NASH patients, respectively, compared to controls. FC increases in NASH and correlates with SREBP-2 induction. Moreover, StAR overexpression in NASH suggests that mitochondrial FC may be a player in disease progression and a novel target for intervention.

Effect of different wavelengths of light on the antioxidant and immunity status of juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress

NASA Astrophysics Data System (ADS)

Choe, Jong Ryeol; Shin, Yoon Sub; Choi, Ji Yong; Kim, Tae Hwan; Jung, Min-Min; Choi, Cheol Young

2017-12-01

We investigated the effect of light wavelengths on antioxidant and immunity parameters in juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress (25 and 30°C). We exposed the fish to light emitting diodes (LEDs) emitting green (520 nm) and red light (630 nm) of 0.25 and 0.5 W/m2 intensity, and measured the activity, and mRNA and protein expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. We also determined the levels of plasma hydrogen peroxide (H2O2), melatonin, and lysozyme. Furthermore, the mRNA and protein levels of caspase-3 were measured and terminal transferase dUTP nick end labeling (TUNEL) assays were performed. We observed that mRNA expression and activities of antioxidant enzymes and plasma H2O2 levels were significantly higher after exposure to high temperatures. However, increases in these parameters were significantly lower after exposure to green LED light. The plasma melatonin and lysozyme levels were significantly lower in the different groups after exposure to high temperatures; however, in groups exposed to green LED light, their levels were significantly higher than those in the control group. The expression pattern of caspase-3 mRNA was similar to that of H2O2. The TUNEL assay showed that apoptosis was markedly higher at higher water temperatures than that at 20°C. These results indicate that high water temperatures induce oxidative stress and decrease the immunity in juvenile rock bream but green LED light inhibits the rise in oxidative stress and combats the decrease in immunity and should, thus, be useful in the culture of rock bream.

Protopine and allocryptopine increase mRNA levels of cytochromes P450 1A in human hepatocytes and HepG2 cells independently of AhR.

PubMed

Vrba, Jiri; Vrublova, Eva; Modriansky, Martin; Ulrichova, Jitka

2011-06-10

The isoquinoline alkaloids protopine and allocryptopine are present in phytopreparations from medicinal plants, such as Fumaria officinalis. Since nothing is known about effects of the alkaloids on the expression of xenobiotic-metabolizing enzymes, we examined whether protopine or allocryptopine affect the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells. In HepG2 cells, protopine and allocryptopine significantly increased CYP1A1 mRNA levels after 24h exposure at concentrations from 25 and 10 μM, respectively, as shown by real-time PCR. Both protopine and allocryptopine also dose-dependently increased CYP1A1 and CYP1A2 mRNA levels in human hepatocytes. However, the effects of the tested alkaloids on both cell models were much lower than the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. Using gene reporter assays performed in transiently transfected HepG2 cells, we demonstrated that the induction of CYP1A1 expression by either protopine or allocryptopine was associated with mild or negligible activation of the aryl hydrocarbon receptor. In contrast to TCDD, CYP1A mRNA levels induced by protopine or allocryptopine in both HepG2 cells and human hepatocytes did not result in elevated CYP1A protein or activity levels as shown by western blotting and EROD assays, respectively. We conclude that the use of products containing protopine and/or allocryptopine may be considered safe in terms of possible induction of CYP1A enzymes. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Effect of dietary α-lipoic acid on the mRNA expression of genes involved in drug metabolism and antioxidation system in rat liver.

PubMed

Ide, Takashi

2014-08-14

In the present study, the mRNA levels of hepatic proteins involved in the drug metabolism of rats fed α-lipoic acid were evaluated by DNA microarray and real-time PCR analyses. Experimental diets containing 0, 0·1, 0·25 and 0·5 % (w/w) α-lipoic acid were fed to four groups of rats consisting of seven animals each for 21 d. DNA microarray analysis revealed that the diet containing 0·5 % α-lipoic acid significantly (P< 0·05) increased the mRNA levels of various phase I drug-metabolising enzymes up to 15-fold and phase II enzymes up to 52-fold in an isoenzyme-specific manner. α-Lipoic acid also up-regulated the mRNA levels of some members of the ATP-binding cassette transporter superfamily, presumed to be involved in the exportation of xenobiotics, up to 6·6-fold. In addition, we observed that α-lipoic acid increased the mRNA levels of many proteins involved in antioxidation, such as members of the thiol redox system (up to 5·5-fold), metallothioneins (up to 12-fold) and haeme oxygenase 1 (1·5-fold). These results were confirmed using real-time PCR analysis, and α-lipoic acid dose dependently increased the mRNA levels of various proteins involved in drug metabolism and antioxidation. Consistent with these observations, α-lipoic acid dose dependently increased the hepatic concentration of glutathione and the activities of glutathione reductase and glutathione transferase measured using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, but decreased the hepatic and serum concentrations of malondialdehyde. In conclusion, the present study unequivocally demonstrated that α-lipoic acid increases the mRNA expression of proteins involved in drug metabolism and antioxidation in the liver.

Maternal nutrient deprivation induces sex-specific changes in thyroid hormone receptor and deiodinase expression in the fetal guinea pig brain

PubMed Central

Chan, Shiao Y; Andrews, Marcus H; Lingas, Rania; McCabe, Chris J; Franklyn, Jayne A; Kilby, Mark D; Matthews, Stephen G

2005-01-01

Thyroid hormone deprivation during fetal life has been implicated in neurodevelopmental morbidity. In humans, poor growth in utero is also associated with fetal hypothyroxinaemia. In guinea pigs, a short period (48 h) of maternal nutrient deprivation at gestational day (gd) 50 results in fetuses with hypothyroxinaemia and increased brain/body weight ratios. Thyroid hormone action is mediated by nuclear thyroid hormone receptors (TRs) and is dependent upon the prereceptor regulation of supply of triiodothyronine (T3) by deiodinase enzymes. Examination of fetal guinea pig brains using in situ hybridization demonstrated widespread expression of mRNAs encoding TRα1, α2 and β1, with regional colocalization of deiodinase type 2 (D2) mRNA in the developing forebrain, limbic structures, brainstem and cerebellum at gd52. With maternal nutrient deprivation, TRα1 and β1 mRNA expression was significantly increased in the male, but decreased in the female fetal hippocampus and cerebellum and other areas showing high TR expression under euthyroid conditions. Maternal nutrient deprivation resulted in elevated D2 mRNA expression in males and females. Deiodinase type 3 (D3) mRNA expression was confined to the shell of the nucleus accumbens, the posterior amygdalohippocampal area, brainstem and cerebellum, and did not change with maternal nutrient deprivation. In conclusion, maternal nutrient deprivation resulted in sex-specific changes in TR mRNA expression and a generalized increase in D2 mRNAs within the fetal brain. These changes may represent a protective mechanism to maintain appropriate thyroid hormone action in the face of fetal hypothyroxinaemia in order to optimize brain development. PMID:15878952

The relationship between deiodinase activity and inflammatory responses under the stimulation of uremic toxins.

PubMed

Xu, Gaosi; Tu, Weiping; Qin, Shulan

2014-08-31

It is unclear to what extent uremic toxins participate in inflammatory responses and the activities of deiodinases, as well as the effects of deiodinases on inflammatory cytokines. Hepatocellular carcinoma cell lines (HepG2) were transfected with small interfering ribonucleic acid (siRNA) specific for deiodinase type 1 (DIO1) and cultured with or without uremic toxins. The mRNA expression of DIO1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α was detected by quantitative real-time PCR. The presence of selenoprotein M (SelM) and DIO1 was assessed by western blotting. Sonicate deiodinase activities in HepG2 cells were measured by a dithiothreitol-stimulated assay. The NF-κB, AP-1 and CREB-1 inflammatory signal pathways were confirmed by EMSA. After culturing for 24 h, the mRNA expression of DIO1 was significantly decreased by the specific siRNA (reduced by 76%, P = 0.0002). Uremic toxins significantly increased the mRNA expression (P < 0.01) of IL-1β, IL-6 and TNF-α and inhibited DIO1 mRNA expression (P < 0.01) compared with controls. Suppression of DIO1 by siRNA significantly decreased the mRNA expression of IL-1β and IL-6 (P < 0.05) but not TNF-α (P = 0.093). Uremic toxins and specific siRNA synchronously reduced the protein expression of SelM and DIO1. Uremic toxins activate the expression of inflammatory cytokines. The major findings of this study indicate that the uremic toxins, more than inflammatory cytokines, play direct inhibitory roles in DIO1 enzyme activity, which then provides a negative feedback to the growing accumulation of inflammatory cytokines.

Variable liver fat concentration as a proxy for body fat mobilization postpartum has minor effects on insulin-induced changes in hepatic gene expression related to energy metabolism in dairy cows.

PubMed

Weber, C; Schäff, C T; Kautzsch, U; Börner, S; Erdmann, S; Bruckmaier, R M; Röntgen, M; Kuhla, B; Hammon, H M

2017-02-01

The liver plays a central role in adaptation for energy requirements around calving, and changes in the effects of insulin on hepatic energy metabolism contribute to metabolic adaptation in dairy cows. Hepatic insulin effects may depend on body fat mobilization. The objective of this study was to investigate the effects of insulin on the hepatic gene expression of enzymes involved in energy metabolism and factors related to nutrition partitioning in cows with high and low total liver fat concentration (LFC) after calving. Holstein cows were retrospectively grouped according to their LFC after calving as a proxy for body fat mobilization. Cows were classified as low (LLFC; LFC <24% fat/dry matter; n = 9) and high (HLFC; LFC >24.4% fat/dry matter; n = 10) fat-mobilizing after calving. Euglycemic-hyperinsulinemic clamps [6 mU/(kg × min) of insulin for 6 h] were performed in wk 5 antepartum (ap) and wk 3 postpartum (pp). Before and at the end of the euglycemic-hyperinsulinemic clamps, liver biopsies were taken to measure the mRNA abundance of enzymes involved in carbohydrate and lipid metabolism, expression related to the somatotropic axis, and adrenergic and glucocorticoid receptors. The mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK; PCK1), acyl-CoA-dehydrogenase very long chain (ACADVL), and hydroxyl-methyl-glutaryl-CoA-synthase 1 increased, but the mRNA abundance of solute carrier family 2 (SLC2A2 and SLC2A4), growth hormone receptor 1A (GHR1A), insulin-like growth factor 1 (IGF1), sterol regulatory element binding factor 1, adrenoceptor α 1A, and glucocorticoid receptor decreased from ap to pp. Insulin treatment was associated with decreased PCK1, mitochondrial PEPCK, glucose-6-phosphatase, propionyl-CoA-carboxylase α, carnitine-palmitoyl-transferase 1A, ACADVL, and insulin receptor mRNA, but increased IGF1 and SLC2A4 mRNA ap and pp and GHR1A mRNA pp. The mRNA abundance of SLC2A4 was greater, and the mRNA abundance of GHR1A and IGF1 tended to be lower in LLFC than in HLFC. Administration of insulin, albeit at a supraphysiological dose, was associated with inhibition of gene expression related to glucose production and β-oxidation, but we observed variable effects in the degree of insulin depression of individual genes. Insulin status is important for regulation of nutrient partitioning, but different LFC pp had very little influence on changes in hepatic gene expression following administration of insulin. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai.

PubMed

Yao, Zongli; Guo, Wenfei; Lai, Qifang; Shi, Jianquan; Zhou, Kai; Qi, Hongfang; Lin, Tingting; Li, Ziniu; Wang, Hui

2016-01-01

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, the full-length cDNA of the cytosolic carbonic anhydrase c isoform of G. przewalskii (GpCAc) was amplified and sequenced; mRNA levels and enzyme activity of GpCAc and blood chemistry were evaluated to understand the compensatory responses as the naked carp returned to the saline-alkaline lake after spawning. We found that GpCAc had a total length of 1400 bp and encodes a peptide of 260 amino acids. Comparison of the deduced amino acid sequences and phylogenetic analysis showed that GpCAc was a member of the cytosolic carbonic anhydrase II-like c family. Cytosolic-carbonic-anhydrase-c-specific primers were used to analyze the tissue distribution of GpCAc mRNA expression. Expression of GpCAc mRNA was found in brain, gill, liver, kidney, gut, and muscle tissues, but primarily in the gill and posterior kidney; however, none was evident in red blood cells. Transferring fish from river water to lake water resulted in a respiratory alkalosis, osmolality, and ion rise in the blood, as well as significant decreases in the expression and enzyme activity of GpCAc in both the gill and kidney within 96 h. These results indicate that GpCAc may play an important role in the acclimation to both high salinity and carbonate alkalinity. Specifically, G. przewalskii decreases cytosolic carbonic anhydrase c expression to compensate for a respiratory alkalosis and to aid in osmoregulation during the transition from river to saline-alkaline lake.

Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collart, F.R.; Chubb, C.B.; Mirkin, B.L.

1992-07-10

IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results aremore » consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.« less

High Intensity High Volume Interval Training Improves Endurance Performance and Induces a Nearly Complete Slow-to-Fast Fiber Transformation on the mRNA Level.

PubMed

Eigendorf, Julian; May, Marcus; Friedrich, Jan; Engeli, Stefan; Maassen, Norbert; Gros, Gerolf; Meissner, Joachim D

2018-01-01

We present here a longitudinal study determining the effects of two 3 week-periods of high intensity high volume interval training (HIHVT) (90 intervals of 6 s cycling at 250% maximum power, P max /24 s) on a cycle ergometer. HIHVT was evaluated by comparing performance tests before and after the entire training (baseline, BSL, and endpoint, END) and between the two training sets (intermediate, INT). The mRNA expression levels of myosin heavy chain (MHC) isoforms and markers of energy metabolism were analyzed in M. vastus lateralis biopsies by quantitative real-time PCR. In incremental tests peak power (P peak ) was increased, whereas V ˙ O 2peak was unaltered. Prolonged time-to-exhaustion was found in endurance tests with 65 and 80% P max at INT and END. No changes in blood levels of lipid metabolites were detected. Training-induced decreases of hematocrit indicate hypervolemia. A shift from slow MHCI/β to fast MHCIIa mRNA expression occurred after the first and second training set. The mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a master regulator of oxidative energy metabolism, decreased after the second training set. In agreement, a significant decrease was also found for citrate synthase mRNA after the second training set, indicating reduced oxidative capacity. However, mRNA expression levels of glycolytic marker enzyme glyceraldehyde-3-phosphate dehydrogenase did not change after the first and second training set. HIHVT induced a nearly complete slow-to-fast fiber type transformation on the mRNA level, which, however, cannot account for the improvements of performance parameters. The latter might be explained by the well-known effects of hypervolemia on exercise performance.

Expression of a GALACTINOL SYNTHASE Gene in Tomato Seeds Is Up-Regulated before Maturation Desiccation and Again after Imbibition whenever Radicle Protrusion Is Prevented1

PubMed Central

Downie, Bruce; Gurusinghe, Sunitha; Dahal, Petambar; Thacker, Richard R.; Snyder, John C.; Nonogaki, Hiroyuki; Yim, Kyuock; Fukanaga, Keith; Alvarado, Veria; Bradford, Kent J.

2003-01-01

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance. PMID:12644684

Effects of gestational hypertension and pre-eclampsia in mRNA expression of fibrinolysis genes in primary cultured human umbilical vein endothelial cells.

PubMed

Poblete-Naredo, Irais; Rodríguez-Yáñez, Yury; Corona-Núñez, Rogelio O; González-Monroy, Stuart; Salinas, Juan E; Albores, Arnulfo

2018-05-17

Hypertension disorders (HD) and pre-eclampsia (PRE) are leading causes of maternal deaths worldwide. PRE is associated with vascular endothelial dysfunction and with deregulation of the fibrinolysis pathway genes. Fibrinolysis is the fibrin clot hydrolysis process catalyzed by plasmin, a proteolytic enzyme formed from plasminogen. Plasminogen is cleaved by tissue-type (tPA) and urokinase-type (uPA) activators and inhibited by the plasminogen activator inhibitors type-1 (PAI-1) and type-2 (PAI-2). The whole process maintains blood hemostasis. This study aims to assess PAI-1, PAI-2, tPA and uPA mRNA expression in primary cultured human umbilical vein endothelial cells (HUVEC) isolated and cultured from healthy, HD and PRE women. Results show that PAI-1 and PAI-2 mRNA decreased in HD-HUVEC, whereas PAI-1 and uPA decreased in PRE-HUVEC cultures compared to control ones. Notably, the expression ratio between pro- and anti-fibrinolytic actors remained unchanged among the studied groups. It seems that newborn's hemostasis is maintained balanced probably by a compensatory mechanism that involves changes in the fibrinolysis gene expression profile. The real impact of these changes in mRNA expression is unknown, however, it is suggested that these changes could be associated with an increased predisposition to vascular disease development in the progeny. Copyright © 2018. Published by Elsevier Ltd.

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences.

PubMed

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-06-09

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences

PubMed Central

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-01-01

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics. PMID:27279482

Differential regulation of the lung endothelin system by urban particulate matter and ozone.

PubMed

Thomson, Errol; Kumarathasan, Prem; Goegan, Patrick; Aubin, Rémy A; Vincent, Renaud

2005-11-01

Periodic elevation of ambient particulate matter and ozone levels is linked to acute cardiac morbidity and mortality. Increased plasma levels of the potent vasoconstrictor endothelin (ET)-1, a prognostic indicator of cardiac mortality, have been detected in both animal models and humans after exposure to air pollutants. The lungs are the primary source of circulating ET-1, but the direct effects of individual air pollutants and their interaction in modulating the pulmonary endothelin system are unknown. Fischer-344 rats were exposed to particles (0, 5, 50 mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Changes in gene expression were measured using real-time reverse transcription polymerase chain reaction immediately after exposure and following 24 h recovery in clean air. Both pollutants individually increased preproET-1, endothelin converting enzyme-1, and endothelial nitric oxide synthase mRNA levels in the lungs shortly after exposure, consistent with the concomitant increase in plasma of the 21 amino acid ET-1[1-21] peptide measured by HPLC-fluorescence. PreproET-1 mRNA remained elevated 24 h after exposure to particles but not after ozone, in line with previously documented changes of the peptide in plasma. Both pollutants transiently increased endothelin-B receptor mRNA expression, while ozone decreased endothelin-A receptor mRNA levels. Coexposure to particles plus ozone increased lung preproET-1 mRNA but not plasma ET-1[1-21], suggesting alternative processing or degradation of endothelins. This coincided with an increase in the lungs of matrix metalloproteinase-2 (MMP-2), an enzyme that cleaves bigET-1 to ET-1[1-32]. Taken together, our data indicate that ozone and particulate matter independently regulate the expression of lung endothelin system genes, but show complex toxicological interaction with respect to plasma ET-1.

Star-PAP Control of BIK Expression and Apoptosis Is Regulated by Nuclear PIPKIα and PKCδ Signaling

PubMed Central

Li, Weimin; Laishram, Rakesh S.; Ji, Zhe; Barlow, Christy A.; Tian, Bin; Anderson, Richard A.

2012-01-01

SUMMARY BIK protein is an initiator of mitochondrial apoptosis and BIK expression is induced by pro-apoptotic signals including DNA damage. Here we demonstrate that 3′-end processing and expression of BIK mRNA are controlled by the nuclear PI4,5P2-regulated poly(A) polymerase Star-PAP downstream of DNA damage. Nuclear PKCδ is a key mediator of apoptosis and DNA damage stimulates PKCδ association with the Star-PAP complex where PKCδ is required for Star-PAP-dependent BIK expression. PKCδ binds the PI4,5P2-generating enzyme PIPKIα, which is essential for PKCδ interaction with the Star-PAP complex and PKCδ activity is directly stimulated by PI4,5P2. Features in the BIK 3′-UTR uniquely define Star-PAP specificity and may block canonical PAP activity toward BIK mRNA. This reveals a nuclear phosphoinositide signaling nexus where PIPKIα, PI4,5P2 and PKCδ regulate Star-PAP control of BIK expression and induction of apoptosis. This pathway is distinct from the Star-PAP-mediated oxidative stress pathway indicating signal-specific regulation of mRNA 3′-end processing. PMID:22244330

Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin and full thickness model from Episkin.

PubMed

Luu-The, Van; Duche, Daniel; Ferraris, Corinne; Meunier, Jean-Roch; Leclaire, Jacques; Labrie, Fernand

2009-09-01

Episkin and full thickness model from Episkin (FTM) are human skin models obtained from in vitro growth of keratinocytes into the five typical layers of the epidermis. FTM is a full thickness reconstructed skin model that also contains fibroblasts seeded in a collagen matrix. To assess whether enzymes involved in chemical detoxification are expressed in Episkin and FTM and how their levels compare with the human epidermis, dermis and total skin. Quantification of the mRNA expression levels of phases 1 and 2 metabolizing enzymes in cultured Episkin and FTM and human epidermis, dermis and total skin using Realtime PCR. The data show that the expression profiles of 61 phases 1 and 2 metabolizing enzymes in Episkin, FTM and epidermis are generally similar, with some exceptions. Cytochrome P450-dependent enzymes and flavin monooxygenases are expressed at low levels, while phase 2 metabolizing enzymes are expressed at much higher levels, especially, glutathione-S-transferase P1 (GSTP1) catechol-O-methyl transferase (COMT), steroid sulfotransferase (SULT2B1b), and N-acetyl transferase (NAT5). The present study also identifies the presence of many enzymes involved in cholesterol, arachidonic acid, leukotriene, prostaglandin, eicosatrienoic acids, and vitamin D3 metabolisms. The present data strongly suggest that Episkin and FTM represent reliable and valuable in vitro human skin models for studying the function of phases 1 and 2 metabolizing enzymes in xenobiotic metabolisms. They could be used to replace invasive methods or laboratory animals for skin experiments.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

PubMed

Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

PubMed

Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

2018-01-05

The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia.

PubMed

Tirone, Nelson R; Peghini, Bethanea C; Barcelos, Ana Cristina M; Murta, Eddie F C; Michelin, Marcia A

2009-12-01

The present study evaluated mRNA expression of interferon-alpha (IFN-alpha), IFN-alpha receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III). Uterine cervix biopsies were collected from women with CIN I, II and III (n = 28) and controls without CIN lesions or human papilloma virus (HPV) infection (n = 17). The presence of high and low-risk HPV DNA was determined using hybrid capture. The mRNA levels of IFNAR-1, IFNAR-2, IFN-alpha and 2'5'OAS were determined by RT-PCR with specific primers. The control group exhibited a greater frequency of IFNAR-1 expression (10/17; 58.3%) than the CIN samples (4/28; 14.2%) (P = 0.0018), while, the expression of IFNAR-2 was also greater in the control samples (11/17; 64.7%) than in the patients with lesions (2/28; 7.1%) (P = 0.0018). Importantly, simultaneous expression of both receptors was observed only in the control group (8/17; 47.0%) (P = 0.0001). Among the CIN samples, there was one case of low expression of mRNA of IFNAR-1 and IFNAR-2. IFN-alpha was present in 14.2% (4/28) of the CIN samples but was not expressed in the control group. mRNA 2'5'OAS were expressed in 28.5% (8/28) of the CIN samples and 11.7% (2/17) of the control samples (not statistically significant). Fifty percent (14/28) of the CIN samples were positive for HPV DNA. Cervical biopsy samples from control women or those without neoplasia or HPV infection displayed higher IFN-alpha receptor expression than those with CIN, while simultaneous expression of both IFN-alpha receptor subunits was found only in the control group. There was no significant difference in mRNA expression of IFN-alpha and 2'5'OAS between the control and CIN groups. Then we concluded that the samples obtained from patients with CIN present low levels of the IFN-alpha receptor mRNA.

Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

PubMed

Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

USDA-ARS?s Scientific Manuscript database

NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

Effect of Hammerhead Ribozyme against Human Thymidylate Synthase on the Cytotoxicity of Thymidylate Synthase Inhibitors

PubMed Central

Takemura, Yuzuru; Miyachi, Hayato; Skelton, Lorraine; Jackman, Ann L.

1995-01-01

One of the resistance mechanisms to folate‐based thymidylate synthase (TS) inhibitors is the increase in TS activity in tumor cells. Human B lymphoblastoid cell line (W1L2) was made resistant to a lipophilic non‐polyglutamatable TS inhibitor (ZM249148), and the subline (W1L2:R179) showed a 20‐fold increase in TS enzyme activity with concomitant overexpression of TS mRNA. To overcome the resistance, we designed a ribozyme that can cleave the CUC sequences in a triple tandemly repeated sequence of TS mRNA. Expression of this ribozyme in W1L2:R179 cells transfected with Epstein Barr virus‐based expression vector resulted in sensitization to TS inhibitors concomitantly with a decrease of TS expression. The ribozyme expressed in transfectants was shown to be functional in cleaving artificial TS RNA in vitro. PMID:8567390

Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis.

PubMed

Vohra, Bhupinder P S; Planer, William; Armon, Jennifer; Fu, Ming; Jain, Sanjay; Heuckeroth, Robert O

2007-01-01

Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.

Increased 8-hydroxy-2'-deoxyguanosine in plasma and decreased mRNA expression of human 8-oxoguanine DNA glycosylase 1, anti-oxidant enzymes, mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients with systemic lupus erythematosus.

PubMed

Lee, H-T; Lin, C-S; Lee, C-S; Tsai, C-Y; Wei, Y-H

2014-04-01

We measured plasma levels of the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and leucocyte mRNA expression levels of the genes encoding the 8-OHdG repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), the anti-oxidant enzymes copper/zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase-1 (GPx-1), GPx-4, glutathione reductase (GR) and glutathione synthetase (GS), the mitochondrial biogenesis-related proteins mtDNA-encoded ND 1 polypeptide (ND1), ND6, ATPase 6, mitochondrial transcription factor A (Tfam), nuclear respiratory factor 1(NRF-1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) and hypoxia inducible factor-1α (HIF-1α) and the glycolytic enzymes hexokinase-II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase A (LDHa). We analysed their relevance to oxidative damage in 85 systemic lupus erythematosus (SLE) patients, four complicated SLE patients undergoing rituximab treatment and 45 healthy individuals. SLE patients had higher plasma 8-OHdG levels (P < 0·01) but lower leucocyte expression of the genes encoding hOGG1(P < 0·01), anti-oxidant enzymes (P < 0·05), mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) than healthy individuals. The increase in plasma 8-OHdG was correlated positively with the elevation of leucocyte expression of the genes encoding hOGG1 (P < 0·05), anti-oxidant enzymes (P < 0·05), several mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) in lupus patients. The patients, whose leucocyte mtDNA harboured D310 heteroplasmy, exhibited a positive correlation between the mtDNA copy number and expression of ND1, ND6 and ATPase 6 (P < 0·05) and a negative correlation between mtDNA copy number and systemic lupus erythematosus disease activity index (SLEDAI) (P < 0·05), as well as plasma 8-OHdG (P < 0·05). In particular, four complicated SLE patients with increased expression of the genes encoding the anti-oxidant enzymes, GAPDH, Tfam and PDHA1, experienced better therapeutic outcomes after rituximab therapy. In conclusion, higher oxidative damage with suboptimal increases in DNA repair, anti-oxidant capacity, mitochondrial biogenesis and glucose metabolism may be implicated in SLE deterioration, and this impairment might be improved by targeted biological therapy. © 2013 British Society for Immunology.

Effect of LED light spectra on starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus.

PubMed

Choi, Cheol Young; Shin, Hyun Suk; Choi, Young Jae; Kim, Na Na; Lee, Jehee; Kil, Gyung-Suk

2012-11-01

The present study aimed to test starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus illuminated by light-emitting diodes (LEDs): red (peak at 630 nm), green (peak at 530 nm), and blue (peak at 450 nm) within a visible light. We investigated the oxidative stress induced by starvation for 12 days during illumination with 3 LED light spectra through measuring antioxidant enzyme (superoxide dismutase [SOD] and catalase [CAT]) mRNA expression and activity; CAT western blotting; and measuring lipid peroxidation [LPO]), plasma H(2)O(2), lysozyme, glucose, alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT), and melatonin levels. In green and blue lights, expression and activity of antioxidant enzyme mRNA were significantly lower than those of other light spectra, results that are in agreement with CAT protein expression level by western blot analysis. Also, in green and blue lights, plasma H(2)O(2), lysozyme, glucose, AlaAT, AspAT, and melatonin levels were significantly lower than those in other light spectra. These results indicate that green and blue LEDs inhibit oxidative stress and enhance immune function in starved cinnamon clownfish. Copyright © 2012 Elsevier Inc. All rights reserved.

Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

PubMed

Sun, Shuhong; Zhang, Xiaotian; Lyu, Lin; Li, Xixi; Yao, Siliang; Zhang, Junjie

2016-12-09

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.

PubMed

Papadopoulos, Emmanuel I; Fragoulis, Emmanuel G; Scorilas, Andreas

2015-01-10

l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target. Copyright © 2014. Published by Elsevier B.V.

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid.

PubMed

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation.

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid

PubMed Central

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation. PMID:26170556

Regulatory effect of acetyl-l-carnitine on expression of lenticular antioxidant and apoptotic genes in selenite-induced cataract.

PubMed

Elanchezhian, R; Sakthivel, M; Geraldine, P; Thomas, P A

2010-03-30

Differential expression of apoptotic genes has been demonstrated in selenite-induced cataract. Acetyl-l-carnitine (ALCAR) has been shown to prevent selenite cataractogenesis by maintaining lenticular antioxidant enzyme and redox system components at near normal levels and also by inhibiting lenticular calpain activity. The aim of the present experiment was to investigate the possibility that ALCAR also prevents selenite-induced cataractogenesis by regulating the expression of antioxidant (catalase) and apoptotic [caspase-3, early growth response protein-1 (EGR-1) and cytochrome c oxidase subunit I (COX-I)] genes. The experiment was conducted on 9-day-old Wistar rat pups, which were divided into normal, cataract-untreated and cataract-treated groups. Putative changes in gene expression in whole lenses removed from the rats were determined by measuring mRNA transcript levels of the four genes by RT-PCR analysis, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The expression of lenticular caspase-3 and EGR-1 genes appeared to be upregulated, as inferred by detecting increased mRNA transcript levels, while that of COX-I and catalase genes appeared to be downregulated (lowered mRNA transcript levels) in the lenses of cataract-untreated rats. However, in rats treated with ALCAR, the lenticular mRNA transcript levels were maintained at near normal (control) levels. These results suggest that ALCAR may prevent selenite-induced cataractogenesis by preventing abnormal expression of lenticular genes governing apoptosis.

[Piperine regulates glucose metabolism disorder in HepG2 cells of insulin resistance models via targeting upstream target of AMPK signaling pathway].

PubMed

Wan, Chun-Ping; Wei, Ya-Gai; Li, Xiao-Xue; Zhang, Li-Jun; Yang, Rui; Bao, Zhao-Ri-Ge-Tu

2017-02-01

To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway. Copyright© by the Chinese Pharmaceutical Association.

PROTEASOME INHIBITOR TREATMENT REDUCED FATTY ACID, TRIACYLGLYCEROL AND CHOLESTEROL SYNTHESIS

PubMed Central

Oliva, Joan; French, Samuel W.; Li, Jun; Bardag-Gorce, Fawzia

2014-01-01

In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade®), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly down regulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to down regulate the enzymes 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were down regulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly down regulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then down regulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP down regulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in down regulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used. PMID:22445925

An oxidative DNA “damage” and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression

PubMed Central

Pastukh, Viktor; Roberts, Justin T.; Clark, David W.; Bardwell, Gina C.; Patel, Mita; Al-Mehdi, Abu-Bakr; Borchert, Glen M.

2015-01-01

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation. PMID:26432868

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

PubMed Central

2011-01-01

Background Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. Methods We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. Results AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. Conclusions Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing. PMID:21453546

Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

PubMed Central

Zhu, Qingsong; Jin, Lihua; Casero, Robert A.

2013-01-01

Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N1-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer. PMID:22976807

Coupling corticotropin-releasing-hormone and angiotensin converting enzyme 2 dampens stress responsiveness in male mice.

PubMed

Wang, Lei A; de Kloet, Annette D; Smeltzer, Michael D; Cahill, Karlena M; Hiller, Helmut; Bruce, Erin B; Pioquinto, David J; Ludin, Jacob A; Katovich, Michael J; Raizada, Mohan K; Krause, Eric G

2018-05-01

This study used mice to evaluate whether coupling expression of corticotropin-releasing hormone (CRH) and angiotensin converting enzyme 2 (ACE2) creates central interactions that blunt endocrine and behavioral responses to psychogenic stress. Central administration of diminazene aceturate, an ACE2 activator, had no effect on restraint-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis; however, mice that ubiquitously overexpress ACE2 had reduced plasma corticosterone (CORT) and pituitary expression of POMC mRNA. The Cre-LoxP system was used to restrict ACE2 overexpression to CRH synthesizing cells and probe whether HPA axis suppression was the result of central ACE2 and CRH interactions. Within the paraventricular nucleus of the hypothalamus (PVN), mice with ACE2 overexpression directed to CRH had a ≈2.5 fold increase in ACE2 mRNA, which co-localized with CRH mRNA. Relative to controls, mice overexpressing ACE2 in CRH cells had a decreased CORT response to restraint as well as decreased CRH mRNA in the PVN and CEA and POMC mRNA in the pituitary. Administration of ACTH similarly increased plasma CORT, indicating that the blunted HPA axis activation that accompanies ACE2 overexpression in CRH cells is centrally mediated. Anxiety-like behavior was assessed to determine whether the decreased HPA axis activation was predictive of anxiolysis. Mice with ACE2 overexpression directed to CRH cells displayed decreased anxiety-like behavior in the elevated plus maze and open field when compared to that of controls. Collectively, these results suggest that exogenous ACE2 suppresses CRH synthesis, which alters the central processing of psychogenic stress, thereby blunting HPA axis activation and attenuating anxiety-like behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Mechanism of TNF-α in bone defect of chronic apical periodontitis].

PubMed

Yu, Ya-Qiong; Qu, Liu; Qiu, Li-Hong; Guo, Jia-Jie; Ma, Nan; Zhu, Li

2016-08-01

To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of tumor necrosis factor-α(TNF-α) mRNA in MC3T3-E1 cells and the role of NF-κB signaling on the expression of macrophage colony stimulating factor (M-CSF) induced by TNF-α in MC3T3-El cells． METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 10 mg/L P.e-LPS for different time (0-24 h). The expression of TNF-α mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR). MC3T3-E1 cells were treated with different concentrations of TNF-α(0-10 ng/L) for 6 h. The expression of M-CSF mRNA and protein was detected by RT-PCR and enzyme-linked immunoadsordent assay(ELISA).The expression of M-CSF protein was also detected in 10 ng/L TNF-α treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor . Statistical analysis was performed using Multi-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of TNF-α mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express TNF-α mRNA in dose dependent manners. Maximal induction of TNF-α mRNA expression was seen in the MC3T3-E1 cells treated with 10 mg/L P.e-LPS for 6 h. After 6 h, the expression of TNF-α mRNA decreased gradually .The expression of M-CSF mRNA and protein was increased in a does- dependent manner by different concentrations of TNF-α treatment（0-10 ng/L）. The expression of M-CSF protein increased from (37±2) ng/L(control group) to (301±8) ng/L(10 ng/L group).The protein of M-CSF decreased significantly after pretreatment with 10 μmol/L BAY 11-7082 for 1 h, and the expression of M-CSF proteins was reduced from (253±14) ng/L to (154±2) ng/L .BAY group had no significant difference from the control group. The expression of TNF-α mRNA was increased by P. endodontalis LPS treatment in osteoblast. TNF-α may induce the expression of M-CSF in MC3T3-E1 cells through the signaling of NF-κB. It suggests that TNF-α affect osteoblasts through autocrine way for bone destruction in chronic apical periodontitis induced by P.e-LPS.

Stimulation of synthesis and release of brain-derived neurotropic factor from intestinal smooth muscle cells by substance P and pituitary adenylate cyclase-activating peptide.

PubMed

Al-Qudah, M; Alkahtani, R; Akbarali, H I; Murthy, K S; Grider, J R

2015-08-01

Brain-derived neurotrophic factor (BDNF) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF. The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. The expression and secretion of BDNF from smooth muscle cultured from the rabbit intestinal longitudinal muscle layer in response to substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was measured by western blot and enzyme-linked immunosorbent assay. BDNF mRNA was measured by reverse-transcription polymerase chain reaction. The expression of BNDF protein and mRNA was greater in smooth muscle cells (SMCs) from the longitudinal muscle than from circular muscle layer. PACAP and SP increased the expression of BDNF protein and mRNA in cultured longitudinal SMCs. PACAP and SP also stimulated the secretion of BDNF from cultured longitudinal SMCs. Chelation of intracellular calcium with BAPTA (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) prevented SP-induced increase in BDNF mRNA and protein expression and SP-induced secretion of BDNF. Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in SMCs and stimulate the release of BDNF. Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease. © 2015 John Wiley & Sons Ltd.

MMP‐2 and MMP‐14 Silencing Inhibits VEGFR2 Cleavage and Induces the Differentiation of Porcine Adipose‐Derived Mesenchymal Stem Cells to Endothelial Cells

PubMed Central

Almalki, Sami G.; Llamas Valle, Yovani

2017-01-01

Abstract The molecular mechanisms that control the ability of adipose‐derived mesenchymal stem cells (AMSCs) to remodel three‐dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs) in vitro. MSCs were isolated from porcine abdominal adipose tissue, and characterized by immunopositivity to CD44, CD90, CD105, and immunonegativity to CD14 and CD45. Plasticity of AMSCs was confirmed by multilineage differentiation. The mRNA transcripts for MMPs and Tissue Inhibitor of Metalloproteinases (TIMPs), and protein expression of EC markers were analyzed. The enzyme activity and protein expression were analyzed by gelatin zymography, enzyme‐linked immunosorbent assay (ELISA), and Western blot. The differentiation of AMSCs to ECs was confirmed by mRNA and protein expressions of the endothelial markers. The mRNA transcripts for MMP‐2 and MMP‐14 were significantly increased during the differentiation of MSCs into ECs. Findings revealed an elevated MMP‐14 and MMP‐2 expression, and MMP2 enzyme activity. Silencing of MMP‐2 and MMP‐14 significantly increased the expression of EC markers, formation of capillary tubes, and acetylated‐low‐density lipoprotein uptake, and decreased the cleavage of vascular endothelial growth factor receptor type 2 (VEGFR2). Inhibition of VEGFR2 significantly decreased the expression of EC markers. These novel findings demonstrate that the upregulation of MMP2 and MMP14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibit the cleavage of VEGFR2 and stimulate the differentiation of AMSCs to ECs. These findings provide a potential mechanism for the regulatory role of MMP‐2 and MMP‐14 in the re‐endothelialization of coronary arteries following intervention. Stem Cells Translational Medicine 2017;6:1385–1398 PMID:28213979

Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes.

PubMed

Vassella, Erik; Probst, Matthias; Schneider, André; Studer, Erwin; Renggli, Christina Kunz; Roditi, Isabel

2004-09-01

In cycling between the mammalian host and the tsetse fly vector, trypanosomes undergo major changes in energy metabolism and surface coat composition. Early procyclic (insect) forms in the tsetse fly midgut are coated by glycoproteins known as EP and GPEET procyclins. EP expression continues in late procyclic forms, whereas GPEET is down-regulated. In culture, expression of GPEET is modulated by glycerol or glucose. Here, we demonstrate that a glycerol-responsive element of 25 nucleotides within the 3' untranslated region of GPEET mRNA also controls expression by glucose and during development in the fly. In trypanosomes, mitochondrial ATP is produced mainly by the acetate: succinate-CoA transferase/succinyl-CoA synthetase (ASCT) cycle, the citric acid cycle, and the cytochromes. Silencing of the pyruvate dehydrogenase or succinyl-CoA synthetase from the ASCT cycle by RNA interference induces reexpression of GPEET in late procyclic forms, whereas inhibition of the citric acid cycle or the cytochromes has no effect. In contrast, inhibition of the alternative oxidase, the second branch of the electron transport chain, with salicylhydroxamic acid overrides the effect of glucose or glycerol and causes a reduction in the level of GPEET mRNA. Our results reveal a new mechanism by which expression of a surface glycoprotein is controlled by the activity of mitochondrial enzymes.

Thyroid hormone stimulates myoglobin expression in soleus and extensorum digitalis longus muscles of rats: concomitant alterations in the activities of Krebs cycle oxidative enzymes.

PubMed

dos Santos, R A; Giannocco, G; Nunes, M T

2001-06-01

Myoglobin (Mb) gene expression, Citrate Synthase (CS) and Succinate Dehydrogenase (SDH) activities of Soleus (S) and Extensorum Digitalis Longus (EDL) muscles were studied in intact, thyroidectomized and T3-treated (25 microg/100g, BW, ip, 15 days) rats. The fiber type composition of S muscle was also evaluated and used as control of the T3-induced effects. In the S muscle, the T3 treatment increased the Mb mRNA and protein expression, as well as the CS and SDH activity. These changes occurred parallel to the expected increase in type II (fast) and decrease in type I (slow)-fibers in S muscle. In the hypothyroid state, the Mb mRNA was decreased, while the Mb expression and CS activity tended to decrease. In contrast the SDH activity was increased, probably due to the enhanced motor activity that occurs as a short-term response to the hypothermia induced by hypothyroidism. In the EDL, the alterations were milder than those in S muscle in both thyroid states. These findings show that Mb gene expression is induced by T3. This is concomitant with the enhancement of Krebs Cycle enzyme activities and provides additional evidence that thyroid hormone increases the aerobic potential of skeletal muscles, as well as the speed of muscle contraction.

Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs

PubMed Central

Wang, D; Guo, Y; Wrighton, SA; Cooke, GE; Sadee, W

2011-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes ~50% of all clinically used drugs. Although CYP3A4 expression varies widely between individuals, the contribution of genetic factors remains uncertain. In this study, we measured allelic CYP3A4 heteronuclear RNA (hnRNA) and mRNA expression in 76 human liver samples heterozygous for at least one of eight marker SNPs and found marked allelic expression imbalance (1.6–6.3-fold) in 10/76 liver samples (13%). This was fully accounted for by an intron 6 SNP (rs35599367, C>T), which also affected mRNA expression in cell culture on minigene transfections. CYP3A4 mRNA level and enzyme activity in livers with CC genotype were 1.7- and 2.5-fold, respectively, greater than in CT and TT carriers. In 235 patients taking stable doses of atorvastatin, simvastatin, or lovastatin for lipid control, carriers of the T allele required significantly lower statin doses (0.2–0.6-fold, P=0.019) than non-T carriers for optimal lipid control. These results indicate that intron 6 SNP rs35599367 markedly affects expression of CYP3A4 and could serve as a biomarker for predicting response to CYP3A4-metabolized drugs. PMID:20386561

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-11-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed Central

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-01-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla. Images PMID:2903502

Interleukin-1β induces tumor necrosis factor-α secretion from rat hepatocytes.

PubMed

Yoshigai, Emi; Hara, Takafumi; Inaba, Hiroyuki; Hashimoto, Iwao; Tanaka, Yoshito; Kaibori, Masaki; Kimura, Tominori; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2014-05-01

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF-α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS). Primary cultured rat hepatocytes were used to analyze TNF-α expression in response to the pro-inflammatory cytokine, interleukin-1β (IL-1β). Livers of rats subjected to LPS-induced endotoxemia were analyzed. Immunocytochemistry and enzyme-linked immunosorbent assays demonstrated that IL-1β-treated rat hepatocytes secreted TNF-α, and RNA analyses indicated that TNF-α mRNA was induced specifically by IL-1β. Northern blot analysis showed that not only mRNA, but also a natural antisense transcript (asRNA), was transcribed from the rat Tnf gene in IL-1β-treated hepatocytes. TNF-α was detected in the hepatocytes of LPS-treated rats. Both TNF-α mRNA and asRNA were expressed in the hepatocytes of LPS-treated rats, human hepatocellular carcinoma and human monocyte/macrophage cells. To disrupt the interaction between TNF-α asRNA and TNF-α mRNA, sense oligonucleotides corresponding to TNF-α mRNA were introduced into rat hepatocytes resulting in significantly increased levels of TNF-α mRNA. One of these sense oligonucleotides increased a half-life of TNF-α mRNA, suggesting that the TNF-α asRNA may reduce the stability of TNF-α mRNA. IL-1β-stimulated rat hepatocytes are a newly identified source of TNF-α in the liver. TNF-α mRNA and asRNA are expressed in rats and humans, and the TNF-α asRNA reduces the stability of the TNF-α mRNA. Hepatocytes and TNF-α asRNA may be therapeutic targets to regulate levels of TNF-α mRNA. © 2013 The Japan Society of Hepatology.

Expression of Leaf Nitrate Reductase Genes from Tomato and Tobacco in Relation to Light-Dark Regimes and Nitrate Supply

PubMed Central

Galangau, Fabienne; Daniel-Vedele, Françoise; Moureaux, Thérèse; Dorbe, Marie-France; Leydecker, Marie-Thérèse; Caboche, Michel

1988-01-01

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels. Images Fig. 3 Fig. 5 Fig. 6 PMID:16666313

DIFFERENTIAL EFFECTS OF SINGLE VERSUS REPEATED ALCOHOL WITHDRAWAL ON THE EXPRESSION OF ENDOCANNABINOID SYSTEM-RELATED GENES IN THE RAT AMYGDALA

PubMed Central

Serrano, Antonia; Rivera, Patricia; Pavon, Francisco J.; Decara, Juan; Suárez, Juan; de Fonseca, Fernando Rodriguez; Parsons, Loren H.

2011-01-01

Background Endogenous cannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity and affective control. Methods The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol-exposed rats were generated by maintenance on an ethanol liquid diet (10%): one group received continuous access to ethanol for 15 days, while the second group was given intermittent access to the ethanol diet (5 days/week for 3 weeks). Control subjects were maintained on an isocaloric ethanol-free liquid diet. To provide an initial profile of acute withdrawal amygdala tissue was harvested following either 6 or 24 hours of ethanol withdrawal. Results Acute ethanol withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2-AG (FAAH and MAGL, respectively) were evident, as were reductions in mRNA expression for CB1, CB2 and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent ethanol exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g. CB1, CB2, GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2-AG biosynthesis (e.g. DAGL-α/β) were found in any condition. Conclusions These findings suggest that ethanol dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission. PMID:22141465

Molecular Cloning, Characterization, and Expression of a Catalase Gene in the Japanese Scallop Mizuhopecten yessoensis Induced in the Presence of Cadmium

NASA Astrophysics Data System (ADS)

Gao, Jialong; Ishizaki, Shoichiro; Nagashima, Yuji

2016-03-01

Cadmium (Cd) is known to influence the oxidative status of marine organisms and can induce the formation of reactive oxygen species (ROS). Catalase (CAT) is one of the important enzymes involved in scavenging high levels of ROS. In present study, we cloned CAT cDNA and investigated the response of this enzyme at the transcriptional level in the Japanese scallop Mizuhopecten yessoensis exposed to Cd. The full-length CAT cDNA (MyCAT) of 1,870 nucleotides including a 57 bp 5'-UTR, a coding sequence of 1,500 bp and a 313 bp 3'-UTR were identified from the scallop. The deduced amino acid sequence of MyCAT corresponds to 499 amino acids with predicted molecular weight of 56.48 kDa and contains highly conserved motifs of the proximal heme-binding site RLFSYSTH, proximal active signature FNRERIPERVVHAKGGG and three catalytic amino acid residues His72, Asn145, and Tyr355. Its significant homology to CATs from multiple alignments revealed that MyCAT had a high identity with CATs from other mollusks. CAT mRNA expression analysis revealed that expression level was highest in the digestive gland ( p < 0.01) but weak in muscle. Following exposure to 200 and 400 µg/l of Cd, a high amount of Cd was found to have accumulated in the digestive gland and CAT mRNA expression had significantly increased in this organ among 7-day exposed scallops ( p < 0.001). The result demonstrated that antioxidant enzymes such as CAT play important roles in counteracting Cd stress in M. yessoensis.

Adaptation of oxidative phosphorylation to photoperiod-induced seasonal metabolic states in migratory songbirds.

PubMed

Trivedi, Amit Kumar; Malik, Shalie; Rani, Sangeeta; Kumar, Vinod

2015-06-01

Eukaryotic cells produce chemical energy in the form of ATP by oxidative phosphorylation of metabolic fuels via a series of enzyme mediated biochemical reactions. We propose that the rates of these reactions are altered, as per energy needs of the seasonal metabolic states in avian migrants. To investigate this, blackheaded buntings were photoperiodically induced with non-migratory, premigratory, migratory and post-migratory phenotypes. High plasma levels of free fatty acids, citrate (an intermediate that begins the TCA cycle) and malate dehydrogenase (mdh, an enzyme involved at the end of the TCA cycle) confirmed increased availability of metabolic reserves and substrates to the TCA cycle during the premigratory and migratory states, respectively. Further, daily expression pattern of genes coding for enzymes involved in the oxidative decarboxylation of pyruvate to acetyl-CoA (pdc and pdk) and oxidative phosphorylation in the TCA cycle (cs, odgh, sdhd and mdh) was monitored in the hypothalamus and liver. Reciprocal relationship between pdc and pdk expressions conformed with the altered requirements of acetyl-CoA for the TCA cycle in different metabolic states. Except for pdk, all genes had a daily expression pattern, with high mRNA expression during the day in the premigratory/migratory phenotypes, and at night (cs, odhg, sdhd and mdh) in the nonmigratory phenotype. Differences in mRNA expression patterns of pdc, sdhd and mdh, but not of pdk, cs and odgh, between the hypothalamus and liver indicated a tissue dependent metabolism in buntings. These results suggest the adaptation of oxidative phosphorylation pathway(s) at gene levels to the seasonal alternations in metabolism in migratory songbirds. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of UV radiation and its implications on carotenoid pathway in Bixa orellana L.

PubMed

Sankari, M; Hridya, H; Sneha, P; George Priya Doss, C; Ramamoorthy, Siva

2017-11-01

The current study was undertaken to analyse the effect of short-term UV-B and UV-C radiations in provoking carotenoid biosynthesis in Bixa orellana. Seeds of B. orellana were germinated and exposed to the short term UV pre-treatment under controlled environmental condition for 5days. The UV treated young seedlings response in pigment contents; antioxidant enzyme activity and mRNA gene expression level were analysed. The pigment content such as chlorophyll was increased in both UV-B and UV-C treated seedlings, but the total carotenoid level was decreased when compared to the control seedlings this can be attributed to the plant adaptability to survive in a stressed condition. The β-carotene level was increased in UV-B, and UV-C treated young seedlings. No significant changes have occurred in the secondary pigment such as bixin and ABA. The activity of the antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase was significantly increased in UV-B treated seedlings when compared to the UV-C treated seedlings and control. The mRNA expression of the genes involved in bixin biosynthesis pathways such as DXS, PSY, PDS, LCY-β, LCY-ε, CMT, LCD, ADH and CCD genes showed different expression pattern in UV-B and UV-C treated young seedlings. Further we analysed the gene co-expression network to identify the genes which are mainly involved in carotenoid/bixin biosynthesis pathway. Form our findings the CCD, LCY, PDS, ZDS and PSY showed a close interaction. The result of our study shows that the short term UV-B and UV-C radiations induce pigment content, antioxidant enzyme activity and different gene expression pattern allowing the plant to survive in the oxidative stress condition. Copyright © 2017 Elsevier B.V. All rights reserved.

Alterations in the endocannabinoid system in the rat valproic acid model of autism.

PubMed

Kerr, D M; Downey, L; Conboy, M; Finn, D P; Roche, M

2013-07-15

The endocannabinoid system plays a crucial role in regulating emotionality and social behaviour, however it is unknown whether this system plays a role in symptoms associated with autism spectrum disorders. The current study evaluated if alterations in the endocannabinoid system accompany behavioural changes in the valproic acid (VPA) rat model of autism. Adolescent rats prenatally exposed to VPA exhibited impaired social investigatory behaviour, hypoalgesia and reduced lococmotor activity on exposure to a novel aversive arena. Levels of the endocananbinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG) in the hippocampus, frontal cortex or cerebellum were not altered in VPA- versus saline-exposed animals. However, the expression of mRNA for diacylglycerol lipase α, the enzyme primarily responsible for the synthesis of 2-AG, was reduced in the cerebellum of VPA-exposed rats. Furthermore, while the expression of mRNA for the 2-AG-catabolising enzyme monoacylglycerol lipase was reduced, the activity of this enzyme was increased, in the hippocampus of VPA-exposed animals. CB1 or CB2 receptor expression was not altered in any of the regions examined, however VPA-exposed rats exhibited reduced PPARα and GPR55 expression in the frontal cortex and PPARγ and GPR55 expression in the hippocampus, additional receptor targets of the endocannabinoids. Furthermore, tissue levels of the fatty acid amide hydrolase substrates, AEA, oleoylethanolamide and palmitoylethanolamide, were higher in the hippocampus of VPA-exposed rats immediately following social exposure. These data indicate that prenatal VPA exposure is associated with alterations in the brain's endocannabinoid system and support the hypothesis that endocannabinoid dysfunction may underlie behavioural abnormalities observed in autism spectrum disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Vitamin D alters genes involved in follicular development and steroidogenesis in human cumulus granulosa cells.

PubMed

Merhi, Zaher; Doswell, Angela; Krebs, Kendall; Cipolla, Marilyn

2014-06-01

Vitamin D deficiency is common among reproductive-aged women and has a role in female reproduction. This study evaluated the role of 1,25-dihydroxyvitamin D3 (vit D3) in ovarian follicular development and steroidogenesis by using a human granulosa cell (GC) model. Fifty-four women who underwent in vitro fertilization were enrolled. Follicular fluid (FF) and mural and cumulus GCs were collected from small and large follicles. In separate experiments, primary cumulus GCs were cultured with or without vit D3 followed by RT-PCR for mRNA expression levels. The effect of recombinant anti-Mullerian hormone (AMH) on nuclear localization of phospho-Smad 1/5/8 was evaluated in the presence or absence of vit D3 by using immunofluorescence. 25-Hydroxyvitamin D levels in FF as well as cell culture media AMH, progesterone, and estradiol (E2) concentrations were determined by ELISA and RIA. The following were measured: 1) mRNA expression levels; 2) 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme activity; 3) FSH-induced aromatase mRNA and E2 production; and 4) nuclear localization of phospho-Smad 1/5/8. In a multivariate analysis, 25 OH-D levels in FF negatively correlated with AMH and AMH receptor (AMHR)-II mRNA levels in cumulus GCs of small follicles. Compared with women with replete 25-hydroxyvitamin D levels in FF, those with insufficient/deficient levels had a 2-fold increase in AMHR-II mRNA levels in cumulus GCs of small follicles (P = .02). Treatment with vit D3 caused a decrease in AMHR-II and FSH receptor mRNA but an increase in 3-βHSD mRNA levels compared with control (P < .05). Vit D3 enhanced 3-βHSD enzyme activity as assessed by increasing progesterone release; however, vit D3 did not affect FSH-induced aromatase mRNA and E2 production, but it decreased the phosphorylation of Smad 1/5/8 and its nuclear localization. These data suggest that vit D3 alters AMH signaling and steroidogenesis in human cumulus GCs, possibly reflecting a state of GC luteinization potentiation.

Fatty acid binding protein 5 promotes tumor angiogenesis and activates the IL6/STAT3/VEGFA pathway in hepatocellular carcinoma.

PubMed

Pan, Long; Xiao, Heng; Liao, Rui; Chen, Qingsong; Peng, Chong; Zhang, Yuchi; Mu, Tong; Wu, Zhongjun

2018-06-25

Tumor angiogenesis is an essential process for facilitating tumor growth and metastasis. Fatty acid binding protein 5(FABP5)is highly expressed in hepatocellular carcinoma (HCC). Thus, we investigated the role of FABP5 in tumor angiogenesis during HCC development. In this study, the protein and mRNA levels of FABP5 in matched HCC and adjacent noncancerous liver tissues from 43 patients were determined using immunohistochemistry and real-time quantitative PCR, respectively. Two HCC cell lines (Huh7 and SMMC-7721) and human umbilical vein endothelial cells (HUVECS) were used to investigate the pro-angiogenic effect of FABP5 by tube formation, CCK8 and Transwell migration assays. The expression levels of interleukin 6 (IL6) and vascular endothelial growth factor A (VEGFA) secreted from HCC cells were detected by enzyme-linked immunosorbent assay (ELISA). In 43 HCC patients, the expression of FABP5 mRNA was positively correlated with intratumoral VEGFA mRNA expression. FABP5 mRNA expression was also associated with adverse HCC characteristics. In vitro, cell viability, cell migration and tube formation in HUVECs were enhanced with increasing expression of FABP5 in HCC cells. Downregulation of FABP5 expression inhibited the IL6/STAT3/VEGFA pathway in HCC cells and inhibited tumor angiogenesis. FABP5 was shown to promote angiogenesis and activate the IL6/STAT3/VEGFA pathway in HCC. FABP5 may be a potential antiangiogenic target in the treatment of HCC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galloway, Chad A.; Smith, Harold C., E-mail: harold.smith@rochester.edu

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expressionmore » of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.« less

Effects of 17alpha-methyltestosterone exposure on steroidogenesis and cyclin-B mRNA expression in previtellogenic oocytes of Atlantic cod (Gadus morhua).

PubMed

Kortner, Trond M; Arukwe, Augustine

2007-11-01

Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Digestion of a single meal affects gene expression of ion and ammonia transporters and glutamine synthetase activity in the gastrointestinal tract of freshwater rainbow trout.

PubMed

Bucking, Carol; Wood, Chris M

2012-04-01

Experiments on freshwater rainbow trout, Oncorhynchus mykiss, demonstrated how digestion affected the transcriptional expression of gastrointestinal transporters following a single satiating meal (~3% body mass ration) after a 1-week fast. Quantitative real-time polymerase chain reaction was employed to measure the relative mRNA expression of three previously cloned and sequenced transporters [H(+)-K(+)-ATPase (HKA), Na(+)/HCO(3)(-) cotransporter (NBC), and the Rhesus glycoprotein (Rhbg1; an ammonia transporter)] over a 24-h time course following feeding. Plasma total ammonia increased about threefold from pre-feeding levels to 288 μmol l(-1), whereas total ammonia levels in chyme supernatant reached a sixfold higher value (1.8 mmol l(-1)) than plasma levels. Feeding did not appear to have a statistically significant effect on the relative mRNA expression of the gastric HKA or Rhbg1. However, the relative mRNA expression of gastric NBC was increased 24 h following the ingestion of a meal. Along the intestinal tract, feeding increased the relative mRNA expression of Rhbg1, but had no effect on the expression of NBC. Expression of the gastric HKA was undetectable in the intestinal tract of freshwater rainbow trout. Digestion increased the activity of glutamine synthetase in the posterior intestine at 12 and 24 h following feeding. This study is among the first to show that there are digestion-associated changes in gene expression and enzyme activity in the gastrointestinal tract of teleost fish illustrating the dynamic plasticity of this organ. These post-prandial changes occur over the relative short-term duration of digesting a single meal.

Short communication: expression of transporters and metabolizing enzymes in the female lower genital tract: implications for microbicide research.

PubMed

Zhou, Tian; Hu, Minlu; Cost, Marilyn; Poloyac, Samuel; Rohan, Lisa

2013-11-01

Topical vaginal microbicides have been considered a promising option for preventing the male-to-female sexual transmission of HIV; however, clinical trials to date have not clearly demonstrated robust and reproducible effectiveness results. While multiple approaches may help enhance product effectiveness observed in clinical trials, increasing the drug exposure in lower genital tract tissues is a compelling option, given the difficulty in achieving sufficient drug exposure and positive correlation between tissue exposure and microbicide efficacy. Since many microbicide drug candidates are substrates of transporters and/or metabolizing enzymes, there is emerging interest in improving microbicide exposure and efficacy through local modulation of transporters and enzymes in the female lower genital tract. However, no systematic information on transporter/enzyme expression is available for ectocervical and vaginal tissues of premenopausal women, the genital sites most relevant to microbicide drug delivery. The current study utilized reverse transcriptase polymerase chain reaction (RT-PCR) to examine the mRNA expression profile of 22 transporters and 19 metabolizing enzymes in premenopausal normal human ectocervix and vagina. Efflux and uptake transporters important for antiretroviral drugs, such as P-gp, BCRP, OCT2, and ENT1, were found to be moderately or highly expressed in the lower genital tract as compared to liver. Among the metabolizing enzymes examined, most CYP isoforms were not detected while a number of UGTs such as UGT1A1 were highly expressed. Moderate to high expression of select transporters and enzymes was also observed in mouse cervix and vagina. The implications of this information on microbicide research is also discussed, including microbicide pharmacokinetics, the utilization of the mouse model in microbicide screening, as well as the in vivo functional studies of cervicovaginal transporters and enzymes.

Short Communication: Expression of Transporters and Metabolizing Enzymes in the Female Lower Genital Tract: Implications for Microbicide Research

PubMed Central

Zhou, Tian; Hu, Minlu; Cost, Marilyn; Poloyac, Samuel

2013-01-01

Abstract Topical vaginal microbicides have been considered a promising option for preventing the male-to-female sexual transmission of HIV; however, clinical trials to date have not clearly demonstrated robust and reproducible effectiveness results. While multiple approaches may help enhance product effectiveness observed in clinical trials, increasing the drug exposure in lower genital tract tissues is a compelling option, given the difficulty in achieving sufficient drug exposure and positive correlation between tissue exposure and microbicide efficacy. Since many microbicide drug candidates are substrates of transporters and/or metabolizing enzymes, there is emerging interest in improving microbicide exposure and efficacy through local modulation of transporters and enzymes in the female lower genital tract. However, no systematic information on transporter/enzyme expression is available for ectocervical and vaginal tissues of premenopausal women, the genital sites most relevant to microbicide drug delivery. The current study utilized reverse transcriptase polymerase chain reaction (RT-PCR) to examine the mRNA expression profile of 22 transporters and 19 metabolizing enzymes in premenopausal normal human ectocervix and vagina. Efflux and uptake transporters important for antiretroviral drugs, such as P-gp, BCRP, OCT2, and ENT1, were found to be moderately or highly expressed in the lower genital tract as compared to liver. Among the metabolizing enzymes examined, most CYP isoforms were not detected while a number of UGTs such as UGT1A1 were highly expressed. Moderate to high expression of select transporters and enzymes was also observed in mouse cervix and vagina. The implications of this information on microbicide research is also discussed, including microbicide pharmacokinetics, the utilization of the mouse model in microbicide screening, as well as the in vivo functional studies of cervicovaginal transporters and enzymes. PMID:23607746

Regulation of cytochrome P450 mRNA expression in primary porcine hepatocytes by selected secondary plant metabolites from chicory (Cichorium intybus L.).

PubMed

Rasmussen, Martin Krøyer; Klausen, Christina Lindgaard; Ekstrand, Bo

2014-03-01

Chicory (Cichorium intybus) has been shown to induce enzymes of pharmacokinetic relevance (cytochrome P450; CYP). The aim of this study was to investigate the effects of selected secondary plant metabolites with a global extract of chicory root, on the expression of hepatic CYP mRNA (1A2, 2A19, 2C33, 2D25, 2E1 and 3A29), using primary porcine hepatocytes. Of the tested secondary plant metabolites, artemisinin, scoparone, lactucin and esculetin all induced increased expression of specific CYPs, while esculin showed no effect. In contrast, a global extract of chicory root decreased the expression of CYP1A2, 2C33, 2D25 and 3A29 at high concentrations. The results suggest that purified secondary metabolites from chicory affect CYP expression and thereby might affect detoxification in general, and that global extracts of plants can have effects different from individual components. Copyright © 2013 Elsevier Ltd. All rights reserved.

The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

PubMed Central

Deffit, Sarah N; Yee, Brian A; Manning, Aidan C; Rajendren, Suba; Vadlamani, Pranathi; Wheeler, Emily C; Domissy, Alain; Washburn, Michael C

2017-01-01

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression. PMID:28925356

Engineered external guide sequences are highly effective in inhibiting gene expression and replication of hepatitis B virus in cultured cells.

PubMed

Zhang, Zhigang; Vu, Gia-Phong; Gong, Hao; Xia, Chuan; Chen, Yuan-Chuan; Liu, Fenyong; Wu, Jianguo; Lu, Sangwei

2013-01-01

External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an in vitro selection procedure to generate EGS variants that efficiently induce human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy.

Endocrine disruptors and human reproductive failure: the in vitro effect of phthalates on human luteal cells.

PubMed

Romani, Federica; Tropea, Anna; Scarinci, Elisa; Federico, Alex; Dello Russo, Cinzia; Lisi, Lucia; Catino, Stefania; Lanzone, Antonio; Apa, Rosanna

2014-09-01

To evaluate the influence of phthalates on human luteal cell function. Laboratory study. University hospital. Twenty-three normally menstruating patients in the midluteal phase. Human luteal cells isolated from corpora lutea for primary cultures. Progesterone (P4) and prostaglandin release assayed by enzyme immunoassay, vascular endothelial growth factor (VEGF) secretion by enzyme-linked immunosorbent assay (ELISA), and VEGF mRNA expression by real-time polymerase chain reaction. We investigated the effect of di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) on basal and hCG-induced progesterone (P4) release, as well as DEHP effect on the balance between prostaglandin (PG) E2, vascular endothelial growth factor (VEGF)-luteotrophic factors, and the luteolitic PGF2α in isolated human steroidogenc cells. Phthalates influence on VEGF expression has been also evaluated. DEHP, DBP, and BBP were able to reduce both basal and hCG-stimulated P4 as well as PGE2 release. PGF2α release was reduced after DEHP incubation. VEGF protein release was decreased by the incubation with the tested phthalates. VEGF mRNA expression was not affected by DEHP, DBP, and BBP. As expected, both hCG and cobalt chloride were able to induce P4 release and VEGF release and mRNA expression in human luteal cells respectively. The results show the ability of phthalates to affect luteal steroidogenesis as well as the balance between luteotrophic and luteolytic factors suggesting an interference of phthalates in human luteal function. These data may contribute to clarify the classically known impaired reproductive health observed after phthalates exposure. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

MCD diet-induced steatohepatitis is associated with alterations in asymmetric dimethylarginine (ADMA) and its transporters.

PubMed

Di Pasqua, Laura G; Berardo, Clarissa; Rizzo, Vittoria; Richelmi, Plinio; Croce, Anna Cleta; Vairetti, Mariapia; Ferrigno, Andrea

2016-08-01

Using an experimental model of NASH induced by a methionine-choline-deficient (MCD) diet, we investigated whether changes occur in serum and tissue levels of asymmetric dimethylarginine (ADMA). Male Wistar rats underwent NASH induced by 8-week feeding with an MCD diet. Serum and hepatic biopsies at 2, 4 and 8 weeks were taken, and serum enzymes, ADMA and nitrate/nitrite (NOx), were evaluated. Hepatic biopsies were used for mRNA and protein expression analysis of dimethylarginine dimethylaminohydrolase-1 (DDAH-1) and protein methyltransferases (PRMT-1), enzymes involved in ADMA metabolism and synthesis, respectively, and ADMA transporters (CAT-1, CAT-2A and CAT-2B). Lipid peroxides (TBARS), glutathione, ATP/ADP and DDAH activity were quantified. An increase in serum AST and ALT was detected in MCD animals. A time-dependent decrease in serum and tissue ADMA and increase in mRNA expression of DDAH-1 and PRMT-1 as well as higher rates of mRNA expression of CAT-1 and lower rates of CAT-2A and CAT-2B were found after 8-week MCD diet. An increase in serum NOx and no changes in protein expression in DDAH-1 and CAT-1 and higher content in CAT-2 and PRMT-1 were found at 8 weeks. Hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high TBARS levels and low glutathione content. In conclusion, a decrease in serum and tissue ADMA levels in the MCD rats was found associated with a reduction in DDAH activity due to the marked oxidative stress observed. Changes in ADMA levels and its transporters are innovative factors in the onset and progression of hepatic alterations correlated with MCD diet-induced NASH.

Identification and gene expression of anaerobically induced enolase in Echinochloa phyllopogon and Echinochloa crus-pavonis.

PubMed Central

Fox, T C; Mujer, C V; Andrews, D L; Williams, A S; Cobb, B G; Kennedy, R A; Rumpho, M E

1995-01-01

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) has been identified as an anaerobic stress protein in Echinochloa oryzoides based on the homology of its internal amino acid sequence with those of enolases from other organisms, by immunological reactivity, and induction of catalytic activity during anaerobic stress. Enolase activity was induced 5-fold in anoxically treated seedlings of three flood-tolerant species (E. oryzoides, Echinochloa phyllopogon, and rice [Oryza sativa L.]) but not in the flood-intolerant species (Echinochloa crus-pavonis). A 540-bp fragment of the enolase gene was amplified by polymerase chain reaction from cDNAs of E. phyllopogon and maize (Zea mays L.) and used to estimate the number of enolase genes and to study the expression of enolase transcripts in E. phyllopogon, E. crus-pavonis, and maize. Southern blot analysis indicated that only one enolase gene is present in either E. phyllopogon or E. crus-pavonis. Three patterns of enolase gene expression were observed in the three species studied. In E. phyllopogon, enolase induction at both the mRNA and enzyme activity levels was sustained at all times with a further induction after 48 h of anoxia. In contrast, enolase was induced in hypoxically treated maize root tips only at the mRNA level. In E. crus-pavonis, enolase mRNA and enzyme activity were induced during hypoxia, but activity was only transiently elevated. These results suggest that enolase expression in maize and E. crus-pavonis during anoxia are similarly regulated at the transcriptional level but differ in posttranslational regulation, whereas enolase is fully induced in E. phyllopogon during anaerobiosis. PMID:7480340

Functional Significance of Single Nucleotide Polymorphisms in the Lactase Gene in Diverse United States Subjects and Evidence for a Novel Lactase Persistence Allele at -13909 in Those of European Ancestry

PubMed Central

Baffour-Awuah, Nana Yaa; Fleet, Sarah; Baker, Susan S.; Butler, Johannah L.; Campbell, Catarina; Tischfield, Samuel; Mitchell, Paul D.; Moon, Jennifer E.; Allende-Richter, Sophie; Fishman, Laurie; Bousvaros, Athos; Fox, Victor; Kuokkanen, Mikko; Montgomery, Robert K.; Grand, Richard J.; Hirschhorn, Joel N.

2014-01-01

Objectives Recent data from mainly homogeneous European and African populations implicate a 140 bp region 5′ to the transcriptional start site of LCT (the lactase gene) as a regulatory site for lactase persistence and non-persistence. As there are no studies of United States non-homogeneous populations, we performed genotype/phenotype analysis of the -13910 and -22018 LCT SNPs in New England children, mostly of European ancestry. Methods Duodenal biopsies were processed for disaccharidase activities, RNA quantification by RT-PCR, allelic expression ratios by PCR, and genotyping and SNP analysis. Results were compared to clinical information. Results Lactase activity and mRNA levels, as well as sucrase-to-lactase ratios of enzyme activity and mRNA, showed robust correlations with genotype. None of the other LCT SNPs showed as strong a correlation with enzyme or mRNA activities as did -13910. Data were consistent with the -13910 being the causal sequence variant rather than -22018. Four individuals heterozygous for -13910T/C had allelic expression patterns similar to individuals with -13910C/C genotypes; of these, 2 showed equal LCT expression from the 2 alleles and a novel variant (-13909C>A) associated with lactase persistence. Conclusion The identification of -13910C/C genotype is very likely to predict lactase non-persistence, consistent with prior published studies. A -13910T/T genotype will frequently, but not perfectly, predict lactase persistence in this mixed European-ancestry population; a -13910T/C genotype will not predict the phenotype. A long, rare haplotype in 2 individuals with -13910T/C genotype but equal allele-specific expression contains a novel lactase persistence allele present at -13909. PMID:25625576

Adipokinetic hormone-induced antioxidant response in Spodoptera littoralis.

PubMed

Večeřa, Josef; Krishnan, Natraj; Mithöfer, Axel; Vogel, Heiko; Kodrík, Dalibor

2012-03-01

The antioxidative potential of the Manduca sexta adipokinetic hormone (Manse-AKH) in the last instar larvae of Spodoptera littoralis (Noctuidae, Lepidoptera) was demonstrated after exposure to oxidative stress (OS) elicited by feeding on artificial diet containing tannic acid (TA). Determination of protein carbonyls (PCs) and reduced glutathione (GSH) levels, monitoring of activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferases (GSTs), as well as measuring of the mRNA expression of CAT and SOD were used as markers of the OS. Injection of the Manse-AKH (5 pmol per individual) reversed the OS status by mitigation of PCs formation and by stimulation of glutathione-S-transferases (GSTs) activity. The CAT and SOD mRNA expression was significantly suppressed after the Manse-AKH injection while activity of these enzymes was not affected. These results indicate that diminishing of OS after the AKH injection might be a result of activation of specific enzymatic pathway possibly at the post-translational level rather than a direct effect on regulation of antioxidant marker genes at the transcriptional level. Copyright © 2011 Elsevier Inc. All rights reserved.

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

PubMed

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Altered expression of genes involved in progesterone biosynthesis, metabolism and action in endometrial cancer.

PubMed

Sinreih, Maša; Hevir, Neli; Rižner, Tea Lanišnik

2013-02-25

Endometrial cancer (EC) is one of the most common gynecological malignancies worldwide. It is associated with prolonged exposure to estrogens that is unopposed by the protective effects of progesterone, which suggests that altered progesterone biosynthesis, metabolism and actions might be implicated in the development of EC. Our aim was to evaluate these processes through quantitative real-time PCR expression analysis in up to 47 pairs of EC tissue and adjacent control endometrium. First, we examined the expression of genes encoding proteins associated with progesterone biosynthesis: steroidogenic acute regulatory protein (STAR); a side chain cleavage enzyme (CYP11A1); and 3β-hydroxysteroid dehydrogenase/ketosteroid isomerase (HSD3B). There were 1.9- and 10.0-fold decreased expression of STAR and CYP11A1, respectively, in EC versus adjacent control endometrium, with no significant differences in the expression of HSD3B1 and HSD3B2. Next, we examined expression of genes encoding five progesterone metabolizing enzymes: the 3-keto and 20-ketosteroid reductases (AKR1C1-AKR1C3) and 5α-reductases (SRD5A1 and SRD5A2); and the opposing 20α-hydroxysteroid dehydrogenase (HSD17B2). These genes are expressed in EC and adjacent control endometrium. No statistically significant differences were seen in mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1. Expression of HSD17B2 was 3.0-fold increased, and expression of SRD5A2 was 3.7-fold decreased, in EC versus adjacent control endometrium. We also examined mRNA levels of progesterone receptors A and B (PGR), and separately the expression of progesterone receptor B (PR-B). Here we saw 1.8- and 2.0-fold lower mRNA levels of PGR and PR-B, respectively, in EC versus adjacent control endometrium. This down-regulation of STAR, CYP11A1 and PGR in endometrial cancer may lead to decreased progesterone biosynthesis and actions although the effects on progesterone levels should be further studied. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of macrophage colony stimulating factor in mouse osteoblasts].

PubMed

Yu, Yaqiong; Qiu, Lihong; Guo, Jiajie; Qu, Liu; Xu, Liya; Zhong, Ming

2014-09-01

To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of macrophage colony stimulating factor (M-CSF) mRNA and protein in MC3T3-E1 cells and the role of nucler factor-κB (NF-κB) in the process. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (0-50 mg/L) and 10 mg/L Pe-LPS for different hours (0-24 h). The expression of M-CSF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsordent assay (ELISA). The cells untreated by Pe-LPS served as control. The expression of M- CSF mRNA and protein was also detected in 10 mg/L Pe- LPS treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor. The groups were divided as follows, control group, BAY group (10 µmol/L BAY 11-7082 treated alone MC3T3-E1 cells), Pe-LPS group (10 mg/L Pe-LPS stimulated MC3T3-E1 cells for 6 h), BAY combine with Pe-LPS group (10 µmol/L BAY 11-7082 pretreated cells for 1 h and 10 mg/L of Pe-LPS stimulated MC3T3-E1 cells for 6 h). The level of M- CSF mRNA and protein increased significantly after treatment with different concentrations of Pe-LPS (0-50 mg/L), which indicated that Pe-LPS induced osteoblasts to express M-CSF mRNA and protein in dose dependent manners. The expression of M-CSF protein increased from (35 ± 2) ng/L (control group) to (170 ± 8) ng/L (50 mg/L group). Maximal induction of M-CSF mRNA expression was found in the MC3T3- E1 cells treated with 10 mg/L Pe-LPS for 6 h. After 6 h, the expression of M-CSF mRNA decreased gradually. The expression of M-CSF protein also increased with the treatment of 10 mg/L Pe-LPS for 10 h [(122 ± 4) ng/L]. After 10 h, the expression of M-CSF protein decreased gradually. The mRNA and proteins of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. There was no significant difference between BAY group and the control. Pe-LPS may induce the expression of M-CSF mRNA and protein in MC3T3-E1 cells through the signaling of NF-κB.

Prenatal arsenic exposure alters the programming of the glucocorticoid signaling system during embryonic development

PubMed Central

Caldwell, Katharine E.; Labrecque, Matthew T.; Solomon, Benjamin R.; Ali, Abdulmehdi; Allan, Andrea M.

2015-01-01

The glucocorticoid system, which plays a critical role in a host of cellular functions including mood disorders and learning and memory, has been reported to be disrupted by arsenic. In previous work we have developed and characterized a prenatal moderate arsenic exposure (50 ppb) model and identified several deficits in learning and memory and mood disorders, as well as alterations within the glucocorticoid receptor signaling system in the adolescent mouse. In these present studies we assessed the effects of arsenic on the glucocorticoid receptor (GR) pathway in both the placenta and the fetal brain in response at two critical periods, embryonic days 14 and 18. The focus of these studies was on the 11β-hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 11β-HSD2) which play a key role in glucorticoid synthesis, as well as the expression and set point of the GR negative feedback regulation. Negative feedback regulation is established early in development. At E14 we found arsenic exposure significantly decreased expression of both protein and message in brain of GR and the 11β-HSD1, while 11β-HSD2 enzyme protein levels were increased but mRNA levels were decreased in the brain. These changes in brain protein continued into the E18 time point, but mRNA levels were no longer significantly altered. Placental HSD11B2 mRNA was not altered by arsenic treatment but protein levels were elevated at E14. GR placental protein levels were decreased at E18 in the arsenic exposed condition. This suggests that arsenic exposure may alter GR expression levels as a consequence of a prolonged developmental imbalance between 11β-HSD1 and 11β-HSD2 protein expression despite decreased 11HSDB2 mRNA. The suppression of GR and the failure to turn down 11β-HSD2 protein expression during fetal development may lead to an altered set point for GR signaling throughout adulthood. To our knowledge, these studies are the first to demonstrate that gestational exposure to moderate levels of arsenic results in altered fetal programming of the glucocorticoid system. PMID:25459689

Differential expression of diacylglycerol acyltransferase (DGAT) genes in olive tissues.

PubMed

Giannoulia, K; Haralampidis, K; Poghosyan, Z; Murphy, D J; Hatzopoulos, P

2000-12-01

Fatty acids are accumulated in triacylglycerols (TAGs), in specialized organelles of seeds named oil bodies. The major site of TAG accumulation is detected in developing seed and mesocarp of certain species. We have isolated two cDNAs encoding DGAT enzymes from olives. The deduced polypeptides differ by 26 amino acids in size. However, they have high homology and almost identical hydropathy profiles. The DGAT gene is expressed in all tissues that synthesize TAGs. However, higher levels of DGAT transcripts have been detected in seed tissues of developing olive drupe. DGAT expression and mRNA accumulation in drupe tissues is developmentally regulated. Each DGAT transcript shows a distinct profile of accumulation. The existence of two different DGAT transcripts might reflect two different enzymes with discrete function and/or localization.

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in cervical cancer and associates with tumor growth.

PubMed

Liang, Jing; Nishi, Hirotaka; Bian, Mei-Lu; Higuma, Chinatsu; Sasaki, Toru; Ito, Hiroe; Isaka, Keiichi

2012-10-01

We found that the ubiquitin-conjugating enzyme E2-EPF mRNA is highly expressed in cervical squamous cancer relative to normal tissues and its expression levels positively correlate with clinical stage. Reduction of E2-EPF protein levels by >80% using shRNA decreases the expression levels of HIF-1α, and the proliferation, invasion and tumorigenicity of SiHa, a cervical squamous cancer cell line. E2-EPF knockdown also increases the chemosensitivity to topoisomerase I inhibitor (topotecan) and II (etoposide and doxorubicin). Our results suggest that E2-EPF is associated with the growth and aggressivity of cervical tumor cells. Targeting the E2-EPF pathway may have potential clinical applications for the treatment of cervical cancer.

Plant defense genes are regulated by ethylene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecker, J.R.; Davis, R.W.

One of the earliest detectable events during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. This gaseous plant stress hormone may be a signal for plants to activate defense mechanisms against invading pathogens such as bacteria, fungi, and viruses. The effect of ethylene on four plant genes involved in three separate plant defense response pathways was examined; these included (i and ii) genes that encode L-phenylalanine ammonia-lyase (EC 4.3.1.5) and 4-coumarate:CoA ligase (4-coumarate:CoA ligase (AMP-forming), EC 6.2.1.12), enzymes of the phenylpropanoid pathway, (iii) the gene encoding chalcone synthase, an enzyme of the flavonoid glycoside pathway, and (iv) the genesmore » encoding hydroxyproline-rich glycoprotein, a major protein component(s) of plant cell walls. Blot hybridization analysis of mRNA from ethylene-treated carrot roots reveals marked increases in the levels of phenylalanine ammonia-lyase mRNA, 4-coumarate CoA ligase mRNA, chalcone synthase mRNA, and certain hydroxyproline-rich glycoprotein transcripts. The effect of ethylene on hydroxyproline-rich glycoprotein mRNA accumulation was different from that of wounding. Ethylene induces two hydroxyproline-rich glycoprotein mRNAs (1.8 and 4.0 kilobases), whereas wounding of carrot root leads to accumulation of an additional hydroxyproline-rich mRNA (1.5 kilobases). These results indicate that at least two distinct signals, ethylene and a wound signal, can affect the expression of plant defense-response genes.« less

Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

PubMed

Gomez-Sanchez, Elise P; Ganjam, Venkataseshu; Chen, Yuan Jian; Liu, Ying; Zhou, Ming Yi; Toroslu, Cigdem; Romero, Damian G; Hughson, Michael D; de Rodriguez, Angela; Gomez-Sanchez, Celso E

2003-08-01

The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.

Conditional knock-out of lipoic acid protein ligase 1 reveals redundancy pathway for lipoic acid metabolism in Plasmodium berghei malaria parasite.

PubMed

Wang, Min; Wang, Qiong; Gao, Xiang; Su, Zhong

2017-06-27

Lipoic acid is a cofactor for α-keto acid dehydrogenase system that is involved in the central energy metabolism. In the apicomplexan parasite, Plasmodium, lipoic acid protein ligase 1 (LplA1) and LplA2 catalyse the ligation of acquired lipoic acid to the dehydrogenase complexes in the mitochondrion. The enzymes LipB and LipA mediate lipoic acid synthesis and ligation to the enzymes in the apicoplast. These enzymes in the lipoic acid metabolism machinery have been shown to play important roles in the biology of Plasmodium parasites, but the relationship between the enzymes is not fully elucidated. We used an anhydrotetracycline (ATc)-inducible transcription system to generate transgenic P. berghei parasites in which the lplA1 gene was conditionally knocked out (LplA1-cKO). Phenotypic changes and the lplA1 and lplA2 gene expression profiles of cloned LplA1-cKO parasites were analysed. LplA1-cKO parasites showed severely impaired growth in vivo in the first 8 days of infection, and retarded blood-stage development in vitro, in the absence of ATc. However, these parasites resumed viability in the late stage of infection and mounted high levels of parasitemia leading to the death of the hosts. Although lplA1 mRNA expression was regulated tightly by ATc during the whole course of infection, lplA2 mRNA expression was significantly increased in the late stage of infection only in the LplA1-cKO parasites that were not exposed to ATc. The lplA2 gene can be activated as an alternative pathway to compensate for the loss of LplA1 activity and to maintain lipoic acid metabolism.

Improving Escherichia coli FucO for furfural tolerance by saturation mutagenesis of individual amino acid positions.

PubMed

Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P; Geddes, Ryan D; Shanmugam, Keelnatham T; Ingram, Lonnie O

2013-05-01

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. In Escherichia coli, furfural tolerance can be increased by expressing the native fucO gene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form of fucO that confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-type fucO gene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutant fucO genes differed by less than 2-fold. The Km (furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides -7 to +37) were weak for the native gene (-4.1 kcal mol(-1)) but weaker still for the fucO mutant (-1.0 to -0.1 kcal mol(-1)). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.

Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions

PubMed Central

Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P.; Geddes, Ryan D.; Shanmugam, Keelnatham T.

2013-01-01

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. In Escherichia coli, furfural tolerance can be increased by expressing the native fucO gene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form of fucO that confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-type fucO gene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutant fucO genes differed by less than 2-fold. The Km (furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides −7 to +37) were weak for the native gene (−4.1 kcal mol−1) but weaker still for the fucO mutant (−1.0 to −0.1 kcal mol−1). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels. PMID:23475621

Impact of Fusarium mycotoxins on hepatic and intestinal mRNA expression of cytochrome P450 enzymes and drug transporters, and on the pharmacokinetics of oral enrofloxacin in broiler chickens.

PubMed

Antonissen, Gunther; Devreese, Mathias; De Baere, Siegrid; Martel, An; Van Immerseel, Filip; Croubels, Siska

2017-03-01

Cytochrome P450 (CYP450) drug biotransformation enzymes and multidrug resistance (MDR) proteins may influence drug disposition processes. The first part of the study aimed to evaluate the effect of mycotoxins deoxynivalenol (DON) and/or fumonisins (FBs), at contamination levels approaching European Union guidance levels, on intestinal and hepatic CYP450 enzymes and MDR proteins gene expression in broiler chickens. mRNA expression of genes encoding CYP450 enzymes (CYP3A37, CYP1A4 and CYP1A5) and drug transporters (MDR1/ABCB1 and MRP2/ABCC2) was determined using qRT-PCR. A significant up-regulation of CYP1A4 (P = 0.037) and MDR1 (P = 0.036) was observed in the jejunum of chickens fed a diet contaminated with FBs. The second part of this study aimed to investigate the impact of feeding a FBs contaminated diet on the oral absorption of enrofloxacin (10 mg/kg BW), a MDR1 substrate. A significant (P = 0.045), however small, decreased area under the plasma concentration-time curve (AUC 0-48  h, mean ± SD) was observed for enrofloxacin in chickens fed the FBs contaminated diet compared to the control group, 16.28 ± 1.82 h μg/mL versus 18.27 ± 1.79 h μg/mL. These findings suggest that concurrent administration of drugs with FBs contaminated feed might alter the pharmacokinetic characteristics of CYP1A4 substrate drugs and MDR1 substrates, such as enrofloxacin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pungent ginger components modulates human cytochrome P450 enzymes in vitro

PubMed Central

Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

2013-01-01

Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 μmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

Meta-analysis of expression and function of neprilysin in Alzheimer's disease.

PubMed

Zhang, Huifeng; Liu, Dan; Wang, Yixing; Huang, Huanhuan; Zhao, Yujia; Zhou, Hui

2017-09-14

Neprilysin (NEP) is one of the most important Aβ-degrading enzymes, and its expression and activity in Alzheimer's brain have been widely reported, but the results remain debatable. Thus, the meta-analysis was performed to elucidate the role of NEP in Alzheimer's disease (AD). The relevant case-control or cohort studies were retrieved according to our inclusion/exclusion criteria. Six studies with 123 controls and 141 AD cases, seven studies with 102 controls and 90 AD cases, and four studies with 93 controls and 132 AD cases were included in meta-analysis of NEP's protein, mRNA, and enzyme activity respectively. We conducted Meta regression to detect the sources of heterogeneity and further performed cumulative meta-analysis or subgroup analysis. Our meta-analysis revealed a significantly lower level of NEP mRNA (SMD=-0.44, 95%CI: -0.87, -0.00, p=0.049) in AD cases than in non-AD cases, and such pattern was not altered over time in the cumulative meta-analysis. However, the decrease of NEP protein (SMD=-0.18, 95%CI: -0.62, 0.25) and enzyme activity (SMD=-0.35, 95%CI: -1.03, 0.32) in AD cases did not pass the significance check, while the cumulative meta-analysis by average age showed the pooled effect became insignificant as adding the studies with younger subjects, which indicates that the protein expression and enzyme activity of NEP in the cortex are affected by age. Therefore, the present meta-analysis suggests the need of further investigation of roles of NEP in AD pathogenesis and treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

6-shogaol, a major compound in ginger, induces aryl hydrocarbon receptor-mediated transcriptional activity and gene expression.

PubMed

Yoshida, Kazutaka; Satsu, Hideo; Mikubo, Ayano; Ogiwara, Haru; Yakabe, Takafumi; Inakuma, Takahiro; Shimizu, Makoto

2014-06-18

Xenobiotics are usually detoxified by drug-metabolizing enzymes and excreted from the body. The expression of many of drug-metabolizing enzymes is regulated by the aryl hydrocarbon receptor (AHR). Some substances in vegetables have the potential to be AHR ligands. To search for vegetable components that exhibit AHR-mediated transcriptional activity, we assessed the activity of vegetable extracts and identified the active compounds using the previously established stable AHR-responsive HepG2 cell line. Among the hot water extracts of vegetables, the highest activity was found in ginger. The ethyl acetate fraction of the ginger hot water extract remarkably induced AHR-mediated transcriptional activity, and the major active compound was found to be 6-shogaol. Subsequently, the mRNA levels of AHR-targeting drug-metabolizing enzymes (CYP1A1, UGT1A1, and ABCG 2) and the protein level of CYP1A1 in HepG2 cells were shown to be increased by 6-shogaol. This is the first report that 6-shogaol can regulate the expression of detoxification enzymes by AHR activation.

Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, K.K.; Sanduja, R.; Tsai, A.L.

Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarlymore » low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.« less

Ethanol-induced changes in Poly (ADP ribose) Polymerase and neuronal developmental gene expression

PubMed Central

Gavin, David P.; Kusumo, Handojo; Sharma, Rajiv P.; Guizzetti, Marina

2016-01-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that elevated PARP enzymatic activity reduced PPARγ promoter binding, and this corresponded to decreased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. PMID:27497606

Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

PubMed

Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

2016-11-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. Published by Elsevier Ltd.

De novo steroid biosynthesis in human prostate cell lines and biopsies.

PubMed

Sakai, Monica; Martinez-Arguelles, Daniel B; Aprikian, Armen G; Magliocco, Anthony M; Papadopoulos, Vassilios

2016-05-01

Intratumoral androgen formation may be a factor in the development of prostate cancer (PCa), particularly castration-resistant prostate cancer (CRPC). To evaluate the ability of the human prostate to synthesize de novo steroids, we examined the expression of key enzymes and proteins involved in steroid biosynthesis and metabolism. Using TissueScan™ Cancer qPCR Arrays and quantitative RT-PCR, we performed comparative gene expression analyses between various prostate cell lines and biopsies, including normal, hyperplastic, cancerous, and androgen-deprived prostate cells lines, as well as normal, benign prostate hyperplasia (BPH), PCa, and CRPC human specimens. These studies were complemented with steroid biosynthesis studies in normal and BPH cells. Normal human prostate WPMY-1 and WPE1-NA22, benign prostate hyperplasia BPH-1, and cancer PC-3, LNCaP, and VCaP cell lines, as well as normal, BPH, PCa, and CRPC specimens, were used. Although all cell lines express mRNA encoding for hydroxymethylglutaryl-CoA reductase (HMGCR), the mitochondrial translocator protein TSPO and cholesterol side chain cleavage enzyme CYP11A1 were only observed in WPMY-1, BPH-1, and LNCaP cells. HSD3B1, HSD3B2, and CYP17A1 are involved in androgen formation and were not found in most cell lines. WPE1-NA22 and BPH-1 cells were unable to synthesize de novo steroids from mevalonate. Moreover, androgen-deprived cells did not have alterations in the expression of enzymes that could lead to de novo steroid formation. All prostate specimens expressed TSPO and CYP11A1. HSD3B1/2, CYP17A1, HSD17B5, and CYP19A1 mRNA expression was distinct to the profile observed in cells lines. The majority of BPH (90.9%) and PCa (83.1%) specimens contained CYP17A1, compared to control (normal) specimens (46.7%). BPH (82%), PCa (59%), normal (40%), and CRPC (34%) specimens expressed the four key enzymes that metabolize cholesterol to androgens. These studies question the use of prostate cell lines to study steroid biosynthesis and demonstrate that human prostate samples contain transcripts encoding for key steroidogenic enzymes and proteins indicating that they have the potential to synthesize de novo steroids. We propose CYP17A1 as a candidate enzyme that can be used for patient stratification and treatment in BPH and PCa. © 2016 Wiley Periodicals, Inc.

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

PubMed

Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-02-01

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

Low-level laser therapy (LLLT; 780 nm) acts differently on mRNA expression of anti- and pro-inflammatory mediators in an experimental model of collagenase-induced tendinitis in rat.

PubMed

Pires, Débora; Xavier, Murilo; Araújo, Tiago; Silva, José Antônio; Aimbire, Flavio; Albertini, Regiane

2011-01-01

Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Tendinopathies are directly related to unbalance in expression of pro- and anti-inflammatory cytokines which are responsible by degeneration process of tendinocytes. In the current study, we decided to investigate if LLLT could reduce mRNA expression for TNF-α, IL-1β, IL-6, TGF-β cytokines, and COX-2 enzyme. Forty-two male Wistar rats were divided randomly in seven groups, and tendinitis was induced with a collagenase intratendinea injection. The mRNA expression was evaluated by real-time PCR in 7th and 14th days after tendinitis. LLLT irradiation with wavelength of 780 nm required for 75 s with a dose of 7.7 J/cm(2) was administered in distinct moments: 12 h and 7 days post tendinitis. At the 12 h after tendinitis, the animals were irradiated once in intercalate days until the 7th or 14th day in and them the animals were killed, respectively. In other series, 7 days after tendinitis, the animals were irradiated once in intercalated days until the 14th day and then the animals were killed. LLLT in both acute and chronic phases decreased IL-6, COX-2, and TGF-β expression after tendinitis, respectively, when compared to tendinitis groups: IL-6, COX-2, and TGF-β. The LLLT not altered IL-1β expression in any time, but reduced the TNF-α expression; however, only at chronic phase. We conclude that LLLT administered with this protocol reduces one of features of tendinopathies that is mRNA expression for pro-inflammatory mediators.

Interacting influence of potassium and polychlorinated biphenyl on cortisol and aldosterone biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, L.-A.; Lin, Tsu-Chun Emma

Giving human adrenocortical H295R cells 14 mM KCl for 24 h significantly induced not only aldosterone biosynthesis but also cortisol biosynthesis. Pre-treating the cells with polychlorinated biphenyl 126 (PCB126) further increased potassium-induced aldosterone and cortisol productions in a dose-dependent manner, but all examined concentrations of PCB126 had little effect on the yields of precursor steroids progesterone and 17-OH-progesterone. Subsequent examinations revealed that CYP11B1 and CYP11B2 genes, responsible for the respective final steps of the cortisol and aldosterone biosynthetic pathways, exhibited increased responsiveness to PCB126 under high potassium. While 10{sup -5} M PCB126 was needed to induce a significant increase inmore » the basal mRNA abundance of either gene, PCB126 could enhance potassium-induced mRNA expression of CYP11B1 at 10{sup -7} M and CYP11B2 at 10{sup -9} M. Actually, potassium and PCB126 synergistically upregulated mRNA expression of both genes. Potassium raised the transcriptional rates of CYP11B1 and CYP11B2 probably through a conserved Ad5 cis-element, whereas PCB126 appeared to regulate these two genes at the post-transcriptional level. Positive potassium-PCB126 synergism was also detected in CYP11B2 enzyme activity estimated by aldosterone/progesterone ratio. In contrast, potassium and PCB126 increased CYP11B1 enzyme activity or cortisol/17-OH-progesterone ratio additively. Moreover, potassium improved the time effect of PCB126 on gene expression and enzyme activity of CYP11B2, but not the PCB126 time response of CYP11B1. These data demonstrated that potassium differentially enhanced the potency of PCB126 to induce CYP11B1- and CYP11B2-mediated steroidogenesis.« less

Dietary Tributyrin Supplementation Attenuates Insulin Resistance and Abnormal Lipid Metabolism in Suckling Piglets with Intrauterine Growth Retardation

PubMed Central

He, Jintian; Dong, Li; Xu, Wen; Bai, Kaiwen; Lu, Changhui; Wu, Yanan; Huang, Qiang; Zhang, Lili; Wang, Tian

2015-01-01

Intrauterine growth retardation (IUGR) is associated with insulin resistance and lipid disorder. Tributyrin (TB), a pro-drug of butyrate, can attenuate dysfunctions in body metabolism. In this study, we investigated the effects of TB supplementation on insulin resistance and lipid metabolism in neonatal piglets with IUGR. Eight neonatal piglets with normal birth weight (NBW) and 16 neonatal piglets with IUGR were selected, weaned on the 7th day, and fed basic milk diets (NBW and IUGR groups) or basic milk diets supplemented with 0.1% tributyrin (IT group, IUGR piglets) until day 21 (n = 8). Relative parameters for lipid metabolism and mRNA expression were measured. Piglets with IUGR showed higher (P < 0.05) concentrations of insulin in the serum, higher (P < 0.05) HOMA-IR and total cholesterol, triglycerides (TG), non-esterified fatty acid (NEFA) in the liver, and lower (P < 0.05) enzyme activities (hepatic lipase [HL], lipoprotein lipase [LPL], total lipase [TL]) and concentration of glycogen in the liver than the NBW group. TB supplementation decreased (P < 0.05) the concentrations of insulin, HOMA-IR, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in the serum, and the concentrations of TG and NEFA in the liver, and increased (P < 0.05) enzyme activities (HL, LPL, and TL) and concentration of glycogen in the liver of the IT group. The mRNA expression for insulin signal transduction pathway and hepatic lipogenic pathway (including transcription factors and nuclear factors) was significantly (P < 0.05) affected in the liver by IUGR, which was efficiently (P < 0.05) attenuated by diets supplemented with TB. TB supplementation has therapeutic potential for attenuating insulin resistance and abnormal lipid metabolism in IUGR piglets by increasing enzyme activities and upregulating mRNA expression, leading to an early improvement in the metabolic efficiency of IUGR piglets. PMID:26317832

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida 1

PubMed Central

Weiss, David; van Blokland, Rik; Kooter, Jan M.; Mol, Joseph N. M.; van Tunen, Arjen J.

1992-01-01

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA3). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA3 in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA3 in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA3 was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA3 controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA3 resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA3. The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA3 suggest that GA3 controls chs transcription in an indirect manner. Our data support a model in which GA3 induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668613

High Expression of Antiviral and Vitamin D Pathway Genes Are a Natural Characteristic of a Small Cohort of HIV-1-Exposed Seronegative Individuals.

PubMed

Aguilar-Jimenez, Wbeimar; Saulle, Irma; Trabattoni, Daria; Vichi, Francesca; Lo Caputo, Sergio; Mazzotta, Francesco; Rugeles, Maria T; Clerici, Mario; Biasin, Mara

2017-01-01

Natural resistance to HIV-1 infection is influenced by genetics, viral-exposure, and endogenous immunomodulators such as vitamin D (VitD), being a multifactorial phenomenon that characterizes HIV-1-exposed seronegative individuals (HESNs). We compared mRNA expression of 10 antivirals, 5 immunoregulators, and 3 VitD pathway genes by qRT-PCR in cells of a small cohort of 11 HESNs, 16 healthy-controls (HCs), and 11 seropositives (SPs) at baseline, in response to calcidiol (VitD precursor) and/or aldithriol-2-(AT2)-inactivated HIV-1. In addition, the expression of TIM-3 on T and NK cells of six HCs after calcidiol and calcitriol (active VitD) treatments was evaluated by flow cytometry. Calcidiol increased the mRNA expression of HAVCR2 (TIM-3; Th1-cells inhibitor) in HCs and HESNs. AT2-HIV-1 increased the mRNA expression of the activating VitD enzyme CYP27B1 , of the endogenous antiviral proteins MX2, TRIM22, APOBEC3G , and of immunoregulators ERAP2 and HAVCR2 , but reduced the mRNA expression of VitD receptor ( VDR ) and antiviral peptides PI3 and CAMP in all groups. Remarkably, higher mRNA levels of VDR, CYP27B1, PI3, CAMP, SLPI , and of ERAP2 were found in HESNs compared to HCs either at baseline or after stimuli. Furthermore, calcitriol increases the percentage of CD4+ T cells expressing TIM-3 protein compared to EtOH controls. These results suggest that high mRNA expression of antiviral and VitD pathway genes could be genetically determined in HESNs more than viral-induced at least in peripheral blood mononuclear cells. Moreover, the virus could potentiate bio-activation and use of VitD, maintaining the homeostasis of the immune system. Interestingly, VitD-induced TIM-3 on T cells, a T cell inhibitory and anti-HIV-1 molecule, requires further studies to analyze the functional outcomes during HIV-1 infection.

Coordination of gene expression of arachidonic and docosahexaenoic acid cascade enzymes during human brain development and aging.

PubMed

Ryan, Veronica H; Primiani, Christopher T; Rao, Jagadeesh S; Ahn, Kwangmi; Rapoport, Stanley I; Blanchard, Helene

2014-01-01

The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA) participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades. AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging. The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism. Expression patterns were split into Development (0 to 20 years) and Aging (21 to 78 years) intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2), cyclooxygenases (COX)-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA) and PTGS2 (COX-2) genes at 1q25, highly inter-correlated genes were at distant chromosomal loci. Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease.

Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

PubMed Central

Ames, EG; Lawson, MJ; Mackey, AJ; Holmes, JW

2013-01-01

Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are reexpressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P < 0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. PMID:23688780

Quantification and study of the L-DOPA decarboxylase expression in gastric adenocarcinoma cells treated with chemotherapeutic substances.

PubMed

Korbakis, Dimitrios; Fragoulis, Emmanuel G; Scorilas, Andreas

2013-03-01

3,4-Dihydroxy-L-phenylalanine decarboxylase (DDC) is an enzyme implicated in the biosynthetic pathways of the neurotransmitters dopamine and probably serotonin. DDC gene expression has been studied in numerous malignancies and the corresponding data have shown remarkable alterations in the mRNA and/or protein levels encoded by the gene. The aim of this study was to examine any modulations in the DDC mRNA levels in gastric cancer cells after their treatment with the chemotherapeutic agents 5-fluorouracil, leucovorin, irinotecan, etoposide, cisplatin, and taxol. The sensitivity of the AGS gastric adenocarcinoma cells to the antineoplastic drugs was evaluated using the MTT assay. Total RNA was extracted and reverse transcribed into cDNA. A highly sensitive quantitative real-time PCR methodology was developed for the quantification of DDC mRNA. GAPDH was used as a housekeeping gene. Relative quantification analysis was carried out using the comparative C T method ((Equation is included in full-text article.)). The treatment of AGS cells with several concentrations of various broadly used anticancer drugs resulted in significant modulations of the DDC mRNA levels compared with those in the untreated cells in a time-specific and drug-specific manner. Generally, DDC expression levels appeared to decrease after three time periods of exposure to the selected chemotherapeutic agents, suggesting a characteristic DDC mRNA expression profile that is possibly related to the mechanism of each drug. Our experimental data show that the DDC gene might serve as a new potential molecular biomarker predicting treatment response in gastric cancer cells.

Tissue-specific mRNA expression profiling in grape berry tissues

PubMed Central

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. Conclusion These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality. PMID:17584945

Single-nucleotide polymorphisms and mRNA expression for melatonin synthesis rate-limiting enzyme in recurrent depressive disorder.

PubMed

Gałecki, Piotr; Szemraj, Janusz; Bartosz, Grzegorz; Bieńkiewicz, Małgorzata; Gałecka, Elzbieta; Florkowski, Antoni; Lewiński, Andrzej; Karbownik-Lewińska, Małgorzata

2010-05-01

Depressive disorder (DD) is characterised by disturbances in blood melatonin concentration. It is well known that melatonin is involved in the control of circadian rhythms, sleep included. The use of melatonin and its analogues has been found to be effective in depression therapy. Melatonin synthesis is a multistage process, where the last stage is catalysed by acetylserotonin methyltransferase (ASMT), the reported rate-limiting melatonin synthesis enzyme. Taking into account the significance of genetic factors in depression development, the gene for ASMT may become an interesting focus for studies in patients with recurrent DD. The goal of the study was to evaluate two single-nucleotide polymorphisms (SNPs) (rs4446909; rs5989681) of the ASMT gene, as well as mRNA expression for ASMT in recurrent DD-affected patients. We genotyped two polymorphisms in a group of 181 recurrent DD patients and in 149 control subjects. The study was performed using the polymerase chain reaction/restriction fragment length polymorphism method. The distribution of genotypes in both studied SNPs in the ASMT gene differed significantly between DD and healthy subjects. The presence of AA genotype of rs4446909 polymorphism and of GG genotype of rs5989681 polymorphism was associated with lower risk for having recurrent DD. In turn, patients with depression were characterised by reduced mRNA expression for ASMT. In addition, ASMT transcript level in both recurrent DD patients and in healthy subjects depended significantly on genotype distributions in both polymorphisms. In conclusion, our results suggest the ASMT gene as a susceptibility gene for recurrent DD.

Kinetic analysis of the effects of target structure on siRNA efficiency

NASA Astrophysics Data System (ADS)

Chen, Jiawen; Zhang, Wenbing

2012-12-01

RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

Association of MAOA, 5-HTT, and NET promoter polymorphisms with gene expression and protein activity in human placentas

PubMed Central

Zhang, Huiping; Smith, Graeme N.; Liu, Xudong

2010-01-01

Monoamine oxidase A (MAOA) and the transporters for serotonin (5-HTT) and norepinephrine (NET) may play important roles in regulating maternal monoamine neurotransmitters transferred across the placenta to the fetus. We investigated whether promoter polymorphisms in MAOA (uVNTR), 5-HTT (5-HTTLPR), and NET (NETpPR AAGG4) could influence gene expression and protein activity in human placentas. Normal term human placentas (n = 73) were collected, and placental MAOA, 5-HTT, and NET mRNA levels and protein activity were determined. The mRNA levels or protein activities were compared between different genotype groups. Placentas hemizygous (male fetus) or homozygous (female fetus) for MAOA uVNTR 4-repeat allele had significantly higher MAOA mRNA levels than those hemizygous or homozygous for the 3-repeat allele (P = 0.001). However, no significant difference in MAOA enzyme activity was found for these two groups of genotypes (P = 0.161). Placentas with the 5-HTTLPR short (S)-allele (S/S+S/L) had significantly lower 5-HTT mRNA levels and serotonin uptake rate than those homozygous for the long (L)-allele (L/L) (mRNA: P < 0.001; serotonin transporting activity: P < 0.001). Placentas homozygous for the NET AAGG4 L4 allele had significantly higher NET mRNA levels, as well as dopamine and norepinephrine uptake rates, than those with the S4/L4 genotype (mRNA: P < 0.001; dopamine transporting activity: P = 0.012; norepinephrine transporting activity: P = 0.011). These findings suggest that the three promoter polymorphisms of MAOA, 5-HTT, and NET influence gene expression levels and protein activity of these genes in human placentas, potentially leading to different fetal levels of maternal monoamine neurotransmitters, which may have an impact on fetal neurodevelopment. PMID:20332182

Association of MAOA, 5-HTT, and NET promoter polymorphisms with gene expression and protein activity in human placentas.

PubMed

Zhang, Huiping; Smith, Graeme N; Liu, Xudong; Holden, Jeanette J A

2010-06-01

Monoamine oxidase A (MAOA) and the transporters for serotonin (5-HTT) and norepinephrine (NET) may play important roles in regulating maternal monoamine neurotransmitters transferred across the placenta to the fetus. We investigated whether promoter polymorphisms in MAOA (uVNTR), 5-HTT (5-HTTLPR), and NET (NETpPR AAGG(4)) could influence gene expression and protein activity in human placentas. Normal term human placentas (n = 73) were collected, and placental MAOA, 5-HTT, and NET mRNA levels and protein activity were determined. The mRNA levels or protein activities were compared between different genotype groups. Placentas hemizygous (male fetus) or homozygous (female fetus) for MAOA uVNTR 4-repeat allele had significantly higher MAOA mRNA levels than those hemizygous or homozygous for the 3-repeat allele (P = 0.001). However, no significant difference in MAOA enzyme activity was found for these two groups of genotypes (P = 0.161). Placentas with the 5-HTTLPR short (S)-allele (S/S+S/L) had significantly lower 5-HTT mRNA levels and serotonin uptake rate than those homozygous for the long (L)-allele (L/L) (mRNA: P < 0.001; serotonin transporting activity: P < 0.001). Placentas homozygous for the NET AAGG(4) L(4) allele had significantly higher NET mRNA levels, as well as dopamine and norepinephrine uptake rates, than those with the S(4)/L(4) genotype (mRNA: P < 0.001; dopamine transporting activity: P = 0.012; norepinephrine transporting activity: P = 0.011). These findings suggest that the three promoter polymorphisms of MAOA, 5-HTT, and NET influence gene expression levels and protein activity of these genes in human placentas, potentially leading to different fetal levels of maternal monoamine neurotransmitters, which may have an impact on fetal neurodevelopment.

Ventilation-induced increases in EGFR ligand mRNA are not altered by intra-amniotic LPS or ureaplasma in preterm lambs.

PubMed

Hillman, Noah H; Gisslen, Tate; Polglase, Graeme R; Kallapur, Suhas G; Jobe, Alan H

2014-01-01

Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.

Activation of Tyrosine Hydroxylase mRNA Translation by cAMP in Midbrain Dopaminergic Neurons

PubMed Central

Chen, Xiqun; Xu, Lu; Radcliffe, Pheona; Sun, Baoyong; Tank, A. William

2009-01-01

During prolonged stress or chronic treatment with neurotoxins, robust compensatory mechanisms occur which maintain sufficient levels of catecholamine neurotransmitters in terminal regions. One of these mechanisms is the up-regulation of tyrosine hydroxylase (TH), the enzyme that controls catecholamine biosynthesis. In neurons of the periphery and locus coeruleus, this up-regulation is associated with an initial induction of TH mRNA. In contrast, this induction either does not occur or is nominal in mesencephalic dopamine neurons. The reasons for this lack of compensatory TH mRNA induction remain obscure, because so little is known about the regulation of TH expression in these neurons. In this report we test whether activation of the cAMP signaling pathway regulates TH gene expression in two rodent models of midbrain dopamine neurons, ventral midbrain organotypic slice cultures and MN9D cells. Our results demonstrate that elevation of cAMP leads to induction of TH protein and TH activity in both model systems; however, TH mRNA levels are not up-regulated by cAMP. The induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH mRNA translation. This translational activation is mediated by sequences within the 3′UTR of TH mRNA. Our results support a model in which cAMP induces or activates trans-factors that interact with the TH mRNA 3′UTR to increase TH protein synthesis. An understanding of this novel regulatory mechanism may help to explain the control of TH gene expression and consequently dopamine biosynthesis in midbrain neurons under different physiological and pathological conditions. PMID:18349104

Bacterium-Induced CXCL10 Secretion by Osteoblasts Can Be Mediated in Part through Toll-Like Receptor 4

PubMed Central

Gasper, Nancy A.; Petty, Cynthia C.; Schrum, Laura W.; Marriott, Ian; Bost, Kenneth L.

2002-01-01

Two common pathogens known to cause bone infection, Salmonella and Staphylococcus aureus, were investigated to determine their abilities to induce chemokine expression in cultured mouse and human osteoblasts. While these cells are responsible for bone formation, we were surprised to find that they could respond to bacterial infection by upregulating expression of the chemokine CXCL10 (IP-10). However, there were significant differences in the abilities of the gram-negative bacterium Salmonella and the gram-positive bacterium S. aureus to induce expression of CXCL10. Reverse transcription-PCR and enzyme-linked immunosorbent assay analyses showed high levels of Salmonella-induced CXCL10 mRNA and protein expression, respectively, whereas the osteoblast response to S. aureus was significantly less. Consistent with these findings, Salmonella-derived lipopolysaccharide (LPS), but not S. aureus-derived peptidoglycan, could induce expression of CXCL10. An antibody against toll-like receptor 4 (TLR4) could block the LPS-induced CXCL10 production, demonstrating the functional expression of TLR4 by osteoblasts. Despite the inducible nature of TLR2 mRNA expression by bacterium-infected osteoblasts, peptidoglycan failed to stimulate CXCL10 secretion. Immunofluorescent staining of bacterium-infected calvaria (i.e., skull bone) demonstrated the presence of CXCL10 in osteoblasts. The fact that osteoblasts did not express CXCR3 mRNA, whereas T lymphocytes can express high levels of this receptor, suggests that osteoblast-derived CXCL10 may recruit T lymphocytes to the sites of bone infections. PMID:12117914

Up-regulation of cyclooxygenase-2 by product-prostaglandin E2

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Hughes-Fulford, M.

1997-01-01

The development of prostate cancer has been linked to high level of dietary fat intake. Our laboratory investigates the connection between cancer cell growth and fatty acid products. Studying human prostatic carcinoma PC-3 cells, we found that prostaglandin E2 (PGE2) increased cell growth and up-regulated the gene expression of its own synthesizing enzyme, cyclooxygenase-2 (COX-2). PGE2 increased COX-2 mRNA expression dose-dependently with the highest levels of stimulation seen at the 3-hour period following PGE2 addition. The NSAID flurbiprofen (5 microM), in the presence of exogenous PGE2, inhibited the up-regulation of COX-2 mRNA and cell growth. These data suggest that the levels of local intracellular PGE2 play a major role in the growth of prostate cancer cells through an activation of COX-2 gene expression.

Effects of heat stress on antioxidant defense system, inflammatory injury, and heat shock proteins of Muscovy and Pekin ducks: evidence for differential thermal sensitivities.

PubMed

Zeng, Tao; Li, Jin-jun; Wang, De-qian; Li, Guo-qin; Wang, Gen-lin; Lu, Li-zhi

2014-11-01

Rising temperatures are severely affecting the mortality, laying performance, and meat quality of duck. Our aim was to investigate the effect of acute heat stress on the expression of heat shock proteins (HSPs: HSP90, 70, 60, 40, and 10) and inflammatory factors (nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)) and antioxidant enzyme activity (superoxide dismutase (SOD), malondialdehybe (MDA), catalase (CAT), total antioxidant capacity (T-AOC)) in livers of ducks and to compare the thermal tolerance of Pekin and Muscovy ducks exposed to acute heat stress. Ducks were exposed to heat at 39 ± 0.5 °C for 1 h and then returned to 20 °C for 1 h followed by a 3-h recovery period. The liver and other tissues were collected from each individual for analysis. The mRNA levels of HSPs (70, 60, and 40) increased in both species, except for HSP10, which was upregulated in Muscovy ducks and had no difference in Pekin ducks after heat stress. Simultaneously, the mRNA level of HSP90 decreased in the stress group in both species. Morphological analysis indicated that heat stress induced tissue injury in both species, and the liver of Pekin ducks was severely damaged. The activities of several antioxidant enzymes increased in Muscovy duck liver, but decreased in Pekin duck. The mRNA levels of inflammatory factors were increased after heat stress in both duck species. These results suggested that heat stress could influence HSPs, inflammatory factors expression, and the activities of antioxidant enzymes. Moreover, the differential response to heat stress indicated that the Muscovy duck has a better thermal tolerance than does the Pekin duck.

The unique structural and biochemical development of single cell C4 photosynthesis along longitudinal leaf gradients in Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae)

PubMed Central

Koteyeva, Nuria K.; Voznesenskaya, Elena V.; Berry, James O.; Cousins, Asaph B.; Edwards, Gerald E.

2016-01-01

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica. Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis. PMID:26957565

Tissue-specific regulation of medium-chain acyl-CoA dehydrogenase gene by thyroid hormones in the developing rat.

PubMed

Djouadi, F; Riveau, B; Merlet-Benichou, C; Bastin, J

1997-05-15

During development, gene expression of medium-chain acyl-CoA dehydrogenase (MCAD), a nuclear-encoded mitochondrial enzyme that catalyses the first step of medium-chain fatty acid beta-oxidation, is highly regulated in tissues in accordance with fatty acid utilization, but the factors involved in this regulation are largely unknown. To investigate a possible role of thyroid hormones, rat pups were made hypothyroid by the administration of propylthiouracyl to the mother from day 12 of gestation, and their kidneys, heart and liver were removed on postnatal day 16 to determine MCAD mRNA abundance, protein level and enzyme activity. Similar experiments were run in 3,3',5-tri-iodothyronine (T3)-replaced hypothyroid (1 microg of T3/100 g body weight from postnatal day 5 to 15) and euthyroid pups. Hypothyroidism led to an increase in MCAD mRNA abundance in kidney and a decrease in abundance in heart, but had no effect in liver. The protein levels and enzyme activity were lowered in hypothyroid heart and kidney, suggesting that hypothyroidism affects post-transcriptional steps of gene expression in the kidney. All the effects of hypothyroidism were completely reversed in both heart and kidney by T3 replacement. Injection of a single T3 dose into 16-day-old euthyroid rats also led to tissue-specific changes in mRNA abundance. Nuclear run-on assays performed from hypothyroid and hypothyroid plus T3 rats showed that T3 stimulates MCAD gene transcription in heart and represses it in the kidney. These results indicate that the postnatal rise in circulating T3 is essential to the developmental regulation of the MCAD gene in vivo.

PEPCase Transcript Levels in Mesembryanthemum crystallinum Decline Rapidly upon Relief from Salt Stress 1

PubMed Central

Vernon, Daniel M.; Ostrem, James A.; Schmitt, Juergen M.; Bohnert, Hans J.

1988-01-01

Mesembryanthemum crystallinum plants respond to water stress by changing their pathway of carbon assimilation from C3 to Crassulacean acid metabolism (CAM). Stressed plants are characterized by elevated levels of phosphoenolpyruvate carboxylase (PEPCase) mRNA, protein, and enzyme activity. We wanted to determine whether CAM is a reversible response to environmental conditions or a developmentally programmed adaptation that is irreversibly expressed once induced. Plants were osmotically stressed by irrigation with 500 millimolar NaCl for 12 days to elicit CAM. Salt was then thoroughly flushed from the soil and PEPCase protein and transcript levels were monitored. PEPCase mRNA levels dropped by 77% within 2.5 hours after salt removal. PEPCase activity and polypeptide levels declined more slowly, with a half-life of 2 to 3 days. These results show that PEPCase expression in M. crystallinum is a reversible response to stress that is regulated at the level of transcription or stability of the PEPCase mRNA. Images Fig. 2 Fig. 3 PMID:16666021

Transcriptomic features associated with energy production in the muscles of Pacific bluefin tuna and Pacific cod.

PubMed

Shibata, Mami; Mekuchi, Miyuki; Mori, Kazuki; Muta, Shigeru; Chowdhury, Vishwajit Sur; Nakamura, Yoji; Ojima, Nobuhiko; Saitoh, Kenji; Kobayashi, Takanori; Wada, Tokio; Inouye, Kiyoshi; Kuhara, Satoru; Tashiro, Kosuke

2016-06-01

Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna.

Tissue- and cell-specific expression of mouse xanthine oxidoreductase gene in vivo: regulation by bacterial lipopolysaccharide.

PubMed Central

Kurosaki, M; Li Calzi, M; Scanziani, E; Garattini, E; Terao, M

1995-01-01

The expression of the xanthine oxidoreductase gene was studied in various mouse organs and tissues, under basal conditions and on treatment with bacterial lipopolysaccharide. Levels of xanthine oxidoreductase protein and mRNA were compared in order to understand the molecular mechanisms regulating the expression of this enzyme system. The highest amounts of xanthine oxidoreductase and the respective mRNA are observed in the duodenum and jejunum, where the protein is present in an unusual form because of a specific proteolytic cleavage of the primary translation product present in all locations. Under basal conditions, multiple tissue-specific mechanisms of xanthine oxidoreductase regulation are evident. Lipopolysaccharide increases enzyme activity in some, but not all tissues, mainly via modulation of the respective transcript, although translational and post-translational mechanisms are also active. In situ hybridization studies on tissue sections obtained from mice under control conditions or with lipopolysaccharide treatment demonstrate that xanthine oxidoreductase is present in hepatocytes, predominantly in the proximal tubules of the kidney, epithelial layer of the gastrointestinal mucosa, the alveolar compartment of the lung, the pulpar region of the spleen and the vascular component of the heart. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7864814

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haubenwallner, S.; Hoerl, G.; Hoefler, G.

A previously undescribed single missense mutation (C[yields]G) was detected within exon 5 of the LPL gene in two members of an Italian family affected with type I hyperlipoproteinemia. This mutation causes a highly conservative amino acid replacement (Asp[yields]Glu) at position 180 of the mature LPL protein resulting in a virtual absence of LPL enzyme activity and LPL enzyme mass in postheparin plasma. Adipose tissue mRNA concentrations and mRNA sizes were not affected. Both patients were homozygous for the mutation, whereas the parents were heterozygous. Comparison of the expression of the mutated cDNA and the wildtype cDNA in cos-7 cells revealedmore » proper transcription and translation of the mutated clone into an immunologically detectable protein. The mutated LPL protein was secreted from the cells in a manner similar to that of wild-type LPL and bound to heparin-Sepharose with identical properties. However, the mutated enzyme, in contrast to wildtype LPL, exhibited no detectable lipolytic activity against a triglyceride substrate. The results demonstrate that even a highly conservative amino acid replacement outside the proposed active site of LPL is incompatible with proper enzyme function. 16 refs., 3 figs.« less

Response of genes involved in lipid metabolism in rat epididymal white adipose tissue to different fasting conditions after long-term fructose consumption.

PubMed

Li, Jin-Xiu; Ke, Da-Zhi; Yao, Ling; Wang, Shang; Ma, Peng; Liu, Li; Zuo, Guo-Wei; Jiang, Li-Rong; Wang, Jian-Wei

2017-03-04

There has been much concern regarding the dietary fructose contributes to the development of metabolic syndrome. High-fructose diet changes the expression of genes involved in lipid metabolism. Levels of a number of hepatic lipogenic enzymes are increased by a high-carbohydrate diet in fasted-refed model rats/mice. Both the white adipose tissue (WAT) and the liver play a key role in the maintenance of nutrient homeostasis. Here, the aim of this study was to analyze the expression of key genes related to lipid metabolism in epididymal WAT (eWAT) in response to different fasting condition after long-term chronic fructose consumption. Rats were fed standard chow supplemented with 10% w/v fructose solution for 5 weeks, and killed after chow-fasting and fructose withdrawal (fasting) or chow-fasting and continued fructose (fructose alone) for 14 h. Blood parameters and the expression of genes involved in fatty acid synthesis (ChREBP, SREBP-1c, FAS, SCD1), triglyceride biosynthesis (DGAT-1, DGAT-2) and lipid mobilization (ATGL, HSL) in eWAT were analyzed. In addition, mRNA levels of PPAR-γ, CD36 and LPL were also detected. As expected, fructose alone increased the mRNA expression of FAS, SCD1, and correspondingly decreased ATGL and HSL mRNA levels. However, ChREBP, DGAT-2, ATGL and HSL mRNA levels restored near to normal while FAS and SCD1 tend to basic level under fasting condition. The mRNA expression of SREBP-1c, PPAR-γ and LPL did not changed at any situations but CD36 mRNA decreased remarkably in fructose alone group. In conclusion, these findings demonstrate that genes involved in lipid metabolism in rat eWAT are varied in response to different fasting conditions after long-term fructose consumption. Copyright © 2017. Published by Elsevier Inc.

[Effect of CD-14 and toll like receptors on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis].

PubMed

Jia, Ge; Qiu, Li-Hong; Li, Ren; Lü, You; Yu, Ya-Qiong; Zhong, Ming

2011-09-01

To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS). MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package. The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2. Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.

Angiotensin-converting enzyme inhibition and angiotensin AT1 receptor blockade downregulate angiotensin-converting enzyme expression and attenuate renal injury in streptozotocin-induced diabetic rats.

PubMed

Motawi, Tarek K; El-Maraghy, Shohda A; Senousy, Mahmoud A

2013-07-01

Angiotensin-converting enzyme (ACE) is upregulated in the diabetic kidney and contributes to renal injury. This study investigates the possible beneficial effects of the ACE inhibitor (ACEI), enalapril and the AT1 receptor blocker (ARB), valsartan, on renal ACE expression, renal structure, and function in streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were allocated into four groups: control, STZ-diabetic rats, and STZ-diabetic rats treated with either enalapril (10 mg/kg/day) or valsartan (50 mg/kg/day) for 8 weeks. Enalapril and valsartan reduced renal ACE mRNA and protein expression, Na(+) /K(+) -ATPase activity, oxidative stress, and serum transforming growth factor-β1 levels compared to the diabetic group. Both treatments normalized renal nitrate/nitrite levels and ameliorated the observed histopathological changes. In conclusion, ACE downregulation by ACEI and ARB indicates that angiotensin II upregulates ACE through AT1 receptor. Prevention of diabetes-induced changes in ACE expression and Na(+) /K(+) -ATPase activity could be a new explanation of the renoprotective effects of ACEIs and ARBs. © 2013 Wiley Periodicals, Inc.

Alternative splicing of iodothyronine deiodinases in pituitary adenomas. Regulation by oncoprotein SF2/ASF.

PubMed

Piekielko-Witkowska, Agnieszka; Kedzierska, Hanna; Poplawski, Piotr; Wojcicka, Anna; Rybicka, Beata; Maksymowicz, Maria; Grajkowska, Wieslawa; Matyja, Ewa; Mandat, Tomasz; Bonicki, Wieslaw; Nauman, Pawel

2013-06-01

Pituitary tumors belong to the group of most common neoplasms of the sellar region. Iodothyronine deiodinase types 1 (DIO1) and 2 (DIO2) are enzymes contributing to the levels of locally synthesized T3, a hormone regulating key physiological processes in the pituitary, including its development, cellular proliferation, and hormone secretion. Previous studies revealed that the expression of deiodinases in pituitary tumors is variable and, moreover, there is no correlation between mRNA and protein products of the particular gene, suggesting the potential role of posttranscriptional regulatory mechanisms. In this work we hypothesized that one of such mechanisms could be the alternative splicing. Therefore, we analyzed expression and sequences of DIO1 and DIO2 splicing variants in 30 pituitary adenomas and 9 non-tumorous pituitary samples. DIO2 mRNA was expressed as only two mRNA isoforms. In contrast, nine splice variants of DIO1 were identified. Among them, five were devoid of exon 3. In silico sequence analysis of DIO1 revealed multiple putative binding sites for splicing factor SF2/ASF, of which the top-ranked sites were located in exon 3. Silencing of SF2/ASF in pituitary tumor GH3 cells resulted in change of ratio between DIO1 isoforms with or without exon 3, favoring the expression of variants without exon 3. The expression of SF2/ASF mRNA in pituitary tumors was increased when compared with non-neoplastic control samples. In conclusion, we provide a new mechanism of posttranscriptional regulation of DIO1 and show deregulation of DIO1 expression in pituitary adenoma, possibly resulting from disturbed expression of SF2/ASF. Copyright © 2013 Elsevier B.V. All rights reserved.

The enteric serotonergic system is altered in patients with diverticular disease.

PubMed

Böttner, Martina; Barrenschee, Martina; Hellwig, Ines; Harde, Jonas; Egberts, Jan-Hendrik; Becker, Thomas; Zorenkov, Dimitri; Wedel, Thilo

2013-12-01

Disturbances of the enteric serotonergic system have been implicated in several intestinal motility disorders. Patients with diverticular disease (DD) have been reported to exhibit abnormal intestinal motility and innervation patterns. Gene expression profiles of the serotonergic system and distribution of the serotonin type 4 receptor (5HT-4R) were thus studied in patients with DD. Colonic specimens from patients with DD and controls were subjected to quantitative PCR for serotonin receptors 2B, 3A, 4, serotonin transporter and synthesising enzyme tryptophan hydroxylase. Localisation of 5HT-4R was determined by dual-label immunocytochemistry using smooth muscle actin (α-SMA) and pan-neuronal markers (PGP 9.5) and quantitative analysis was carried out. Site-specific gene expression analysis of 5HT-4R was assessed within myenteric ganglia and muscle layers. Correlation of 5HT-4R with muscarinic receptors 2 and 3 (M2R, M3R) messenger RNA expression was determined. 5HT-4R mRNA expression was downregulated in the tunica muscularis and upregulated in the mucosa of patients with DD, whereas the other components of the serotonergic system remained unchanged. 5HT-4R was detected in ganglia and muscle layers, but was decreased in the circular muscle layer and myenteric ganglia of patients with DD. 5HT-4R mRNA expression correlated with M2R/M3R mRNA expression in controls, but not in patients with DD. The serotonergic system is compromised in DD. Altered expression of 5HT-4R at mRNA and protein levels may contribute to intestinal motor disturbances reported in patients with DD. The findings support the hypothesis that DD is associated and possibly promoted by an enteric neuromuscular pathology.

Reversible effects of oxygen partial pressure on genes associated with placental angiogenesis and differentiation in primary-term cytotrophoblast cell culture.

PubMed

Debiève, F; Depoix, C; Gruson, D; Hubinont, C

2013-09-01

Timely regulated changes in oxygen partial pressure are important for placental formation. Disturbances could be responsible for pregnancy-related diseases like preeclampsia and intrauterine growth restriction. We aimed to (i) determine the effect of oxygen partial pressure on cytotrophoblast differentiation; (ii) measure mRNA expression and protein secretion from genes associated with placental angiogenesis; and (iii) determine the reversibility of these effects at different oxygen partial pressures. Term cytotrophoblasts were incubated at 21% and 2.5% O2 for 96 hr, or were switched between the two oxygen concentrations after 48 hr. Real-time PCR and enzyme-linked immunosorbent assays (ELISAs) were used to evaluate cell fusion and differentiation, measuring transcript levels for those genes involved in cell fusion and placental angiogenesis, including VEGF, PlGF, VEGFR1, sVEGFR1, sENG, INHA, and GCM1. Cytotrophoblasts underwent fusion and differentiation in 2.5% O2 . PlGF expression was inhibited while sVEGFR1 expression increased. VEGF and sENG mRNA expressions increased in 2.5% compared to 21% O2 , but no protein was detected in the cell supernatants. Finally, GCM1 mRNA expression increased during trophoblast differentiation at 21% O2 , but was inhibited at 2.5% O2 . These mRNA expression effects were reversed by returning the cells to 21% O2 . Thus, low-oxygen partial pressure does not inhibit term-cytotrophoblast cell fusion and differentiation in vitro. Lowering the oxygen partial pressure from 21% to 2.5% caused normal-term trophoblasts to reversibly modify their expression of genes associated with placental angiogenesis. This suggests that modifications observed in pregnancy diseases such as preeclampsia or growth retardation are probably due to an extrinsic effect on trophoblasts. Copyright © 2013 Wiley Periodicals, Inc.

Aberrant expression and distribution of enzymes of the urea cycle and other ammonia metabolizing pathways in dogs with congenital portosystemic shunts.

PubMed

van Straten, Giora; van Steenbeek, Frank G; Grinwis, Guy C M; Favier, Robert P; Kummeling, Anne; van Gils, Ingrid H; Fieten, Hille; Groot Koerkamp, Marian J A; Holstege, Frank C P; Rothuizen, Jan; Spee, Bart

2014-01-01

The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.

Aberrant Expression and Distribution of Enzymes of the Urea Cycle and Other Ammonia Metabolizing Pathways in Dogs with Congenital Portosystemic Shunts

PubMed Central

van Straten, Giora; van Steenbeek, Frank G.; Grinwis, Guy C. M.; Favier, Robert P.; Kummeling, Anne; van Gils, Ingrid H.; Fieten, Hille; Groot Koerkamp, Marian J. A.; Holstege, Frank C. P.; Rothuizen, Jan; Spee, Bart

2014-01-01

The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase. PMID:24945279

12- and 15-lipoxygenases in human carotid atherosclerotic lesions: Associations with cerebrovascular symptoms

USDA-ARS?s Scientific Manuscript database

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovasc...

Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

PubMed

Rohde, Kristian; Rovsing, Louise; Ho, Anthony K; Møller, Morten; Rath, Martin F

2014-08-01

The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

Betaine Supplementation in Maternal Diet Modulates the Epigenetic Regulation of Hepatic Gluconeogenic Genes in Neonatal Piglets

PubMed Central

Cai, Demin; Jia, Yimin; Song, Haogang; Sui, Shiyan; Lu, Jingyu; Jiang, Zheng; Zhao, Ruqian

2014-01-01

In this study, gestational sows were fed control or betaine-supplemented diets (3 g/kg) throughout the pregnancy, and the newborn piglets were used to elucidate whether maternal dietary betaine affected offspring hepatic gluconeogenic genes through epigenetic mechanisms. Neonatal piglets born to betaine-supplemented sows had significantly higher serum and hepatic betaine contents, together with significantly greater expression of methionine metabolic enzymes in the liver. Interestingly, significantly higher serum concentrations of lactic acid and glucogenic amino acids, including serine, glutamate, methionine and histidine, were detected in the piglets born to betaine-supplemented sows, which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity, as well as greater protein expression of gluconeogenic enzymes, pyruvate carboxylase (PC), cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK1), mitochondrional phosphoenolpyruvate carboxykinase (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1). Moreover, maternal betaine significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes. The lower PEPCK1 mRNA was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3, while the up-regulated PEPCK2 and FBP1 mRNA was associated with DNA hypomethylation and more enriched activation histone mark H3K4me3. Furthermore, the expression of two miRNAs predicted to target PC and 6 miRNAs predicted to target PEPCK1 was dramatically suppressed in the liver of piglets born to betaine-supplemented sows. Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through enhanced hepatic methionine metabolism and epigenetic regulations, which involve DNA and histone methylations, and possibly miRNAs-mediated post-transcriptional mechanism. PMID:25153319

Increased PI3-kinase in presympathetic brain areas of the spontaneously hypertensive rat.

PubMed

Veerasingham, Shereeni J; Yamazato, Masanobu; Berecek, Kathleen H; Wyss, J Michael; Raizada, Mohan K

2005-02-18

Existing evidence led us to hypothesize that increases in p85alpha, a regulatory subunit of PI3-kinase, in presympathetic brain areas contribute to hypertension. PI3-kinase p85alpha, p110alpha, and p110delta mRNA was 1.5- to 2-fold higher in the paraventricular nucleus (PVN) of spontaneously hypertensive rats (SHR) compared with their controls, Wistar Kyoto rats (WKY). The increase in p85alpha/p110delta was attenuated in SHR treated with captopril, an angiotensin (Ang)-converting enzyme inhibitor, from in utero to 6 months of age. In the rostral ventrolateral medulla (RVLM), p110delta mRNA was approximately 2-fold higher in SHR than in WKY. Moreover, the increases in mRNA were associated with higher PI3-kinase activity in both nuclei. The functional relevance was studied in neuronal cultures because SHR neurons reflect the augmented p85alpha mRNA and PI3-kinase activity. Expression of a p85 dominant-negative mutant decreased norepinephrine (NE) transporter mRNA and [3H]NE uptake by approximately 60% selectively in SHR neurons. In summary, increased p85alpha/p110delta expression in the PVN and RVLM is associated with increased PI3-kinase activity in the SHR. Furthermore, normalized PI3-kinase p85alpha/p110delta expression within the PVN might contribute to the overall effect of captopril, perhaps attributable to a consequent decrease in NE availability.

Effects of tumour necrosis factor α upon the metabolism of the endocannabinoid anandamide in prostate cancer cells.

PubMed

Karlsson, Jessica; Gouveia-Figueira, Sandra; Alhouayek, Mireille; Fowler, Christopher J

2017-01-01

Tumour necrosis factor α (TNFα) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase-2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. TNFα treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acyl-phosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon γ treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE2-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFα treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.

Estrogen receptor beta regulates the expression of tryptophan-hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe nuclei

PubMed Central

Donner, Nina C; Handa, Robert J

2009-01-01

Dysfunctions of the brain serotonin (5-HT) system are often associated with affective disorders, such as depression. The raphe nuclei target the limbic system and most forebrain areas and constitute the main source of 5-HT in the brain. All 5-HT neurons express tryptophan hydroxylase-2 (TPH2), the brain specific, rate-limiting enzyme for 5-HT synthesis. ERbeta agonists have been shown to attenuate anxiety-and despair-like behaviors in rodent models. Therefore, we tested the hypothesis that ERbeta may contribute to the regulation of gene expression in 5-HT neurons of the dorsal raphe nuclei (DRN) by examining the effects of systemic and local application of the selective ERbeta agonist diarylpropionitrile (DPN) on tph2 mRNA expression. Ovariectomized (OVX) female rats were injected subcutaneously (s.c.) with DPN or vehicle once daily for 8 days. In situ hybridization revealed that systemic DPN-treatment elevated basal tph2 mRNA expression in the caudal and mid-dorsal DRN. Behavioral testing of all animals in the open field (OF) and on the elevated plus maze (EPM) on days 6 and 7 of treatment confirmed the anxiolytic nature of ERbeta activation. Another cohort of female OVX rats was stereotaxically implanted bilaterally with hormone-containing wax pellets flanking the DRN. Pellets contained either 17-beta-estradiol (E), DPN, or no hormone. Both DPN and E significantly enhanced tph2 mRNA expression in the mid-dorsal DRN. DPN also increased tph2 mRNA in the caudal DRN. DPN- and E-treated rats displayed a more active stress-coping behavior in the forced-swim test (FST). No behavioral differences were found in the OF or on the EPM. These data indicate that ERbeta acts at the level of the rat DRN to modulate tph2 mRNA expression and thereby influence 5-HT synthesis in DRN subregions. Our results also suggest that local activation of ERbeta neurons in the DRN may be sufficient to decrease despair-like behavior, but not anxiolytic behaviors. PMID:19559077

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

PubMed

Yan, Ke; Zhong, Zheng-rong; Xu, Yun-xia; Ding, Shu-qin; Hu, Jian-guo; Xu, Yuan-hong; Luo, Qing-lie; Shen, Ji-long

2015-06-01

To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Fructose-induced increases in expression of intestinal fructolytic and gluconeogenic genes are regulated by GLUT5 and KHK

PubMed Central

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Tharabenjasin, Phuntila; Gao, Nan

2015-01-01

Marked increases in fructose consumption have been tightly linked to metabolic diseases. One-third of ingested fructose is metabolized in the small intestine, but the underlying mechanisms regulating expression of fructose-metabolizing enzymes are not known. We used genetic mouse models to test the hypothesis that fructose absorption via glucose transporter protein, member 5 (GLUT5), metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein in brain 11a (Rab11a)-dependent endosomes are required for the regulation of intestinal fructolytic and gluconeogenic enzymes. Fructose feeding increased the intestinal mRNA and protein expression of these enzymes in the small intestine of adult wild-type (WT) mice compared with those gavage fed with lysine or glucose. Fructose did not increase expression of these enzymes in the GLUT5 knockout (KO) mice. Blocking intracellular fructose metabolism by KHK ablation also prevented fructose-induced upregulation. Glycolytic hexokinase I expression was similar between WT and GLUT5- or KHK-KO mice and did not vary with feeding solution. Gavage feeding with the fructose-specific metabolite glyceraldehyde did not increase enzyme expression, suggesting that signaling occurs before the hydrolysis of fructose to three-carbon compounds. Impeding GLUT5 trafficking to the apical membrane using intestinal epithelial cell-specific Rab11a-KO mice impaired fructose-induced upregulation. KHK expression was uniformly distributed along the villus but was localized mainly in the basal region of the cytosol of enterocytes. The feedforward upregulation of fructolytic and gluconeogenic enzymes specifically requires GLUT5 and KHK and may proactively enhance the intestine's ability to process anticipated increases in dietary fructose concentrations. PMID:26084694

The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdulla, Dalya; Goralski, Kerry B.; College of Pharmacy, Burbidge Building, Dalhousie University, Halifax, Nova Scotia, B3H 3J5

2006-10-01

It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo,more » an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.« less

Progesterone treatment modulates mRNA OF neurosteroidogenic enzymes in a murine model of multiple sclerosis.

PubMed

Garay, Laura; Gonzalez Giqueaux, Paula; Guennoun, Rachida; Schumacher, Michael; Gonzalez Deniselle, Maria Claudia; De Nicola, Alejandro F

2017-01-01

Previous studies of experimental autoimmune encephalomyelitis (EAE) have shown that progesterone decreases inflammatory cell infiltration and proinflammatory factors, increases myelination and attenuates clinical grade of EAE mice. To elucidate potential mediators of these effects, we analyzed the mRNA expression of neurosteroidogenic enzymes in the spinal cord, in view of the protective role of steroids in EAE. We also analyzed mitochondrial morphology and dynamics (fusion and fission proteins), considering the role of mitochondria in neurosteroidogenesis. EAE was induced in C57Bl6 mice using MOG 40-54 and killed on day 16 after induction. Using qPCR, we found in steroid-untreated EAE mice decreased mRNAs for the steroidogenic acute regulatory protein (Star), voltage-dependent anion channel (VDAC), P450scc (cholesterol side-chain cleavage), 5α-reductase, 3α-hydroxysteroid dehydrogenase (3α-HSD) and aromatase, whereas levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) showed a large intra-group variance. We also found increased mRNA expression of 18Kd translocator protein (TSPO), which likely resulted from the reactive microgliosis in this model. EAE mice also showed pathological mitochondrial morphology and reduced expression of fission and fusion protein mRNAs. Most importantly, pretreatment with progesterone a week before EAE induction increased Star,VDAC, P450scc, 5α-reductase type I, 3α-HSD and aromatase mRNAs and did not modify 3β-HSD. TSPO mRNA was decreased, consequent with the inhibition of microgliosis. Mitochondrial morphology was improved and fission/fusion protein mRNAs were enhanced by progesterone treatment. Furthermore, progesterone protective effects on mitochondrial and endoplasmic reticulum may allow the recovery of neurosteroidogenesis. In this way, endogenously synthesized neurosteroids may reinforce the beneficial effects of exogenous progesterone previously shown in MS mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells.

PubMed

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla; Hansson, Stefan; Casslén, Bertil

2009-02-01

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

PubMed

Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Effects of dietary probiotic supplementation on LXRα and CYP7α1 gene expression, liver enzyme activities and fat metabolism in ducks.

PubMed

Huang, Z; Mu, C; Chen, Y; Zhu, Z; Chen, C; Lan, L; Xu, Q; Zhao, W; Chen, G

2015-04-01

1. The objective of this study was to investigate the effects of dietary probiotic supplementation on liver X receptor alpha (LXRα) and cholesterol 7α-hydroxylase (CYP7α1) mRNA levels, protein enzymatic activities and fat metabolism in Cherry Valley Pekin ducks. 2. A total of 750 one-day-old Cherry Valley Pekin ducks were randomly divided into 5 groups with three replicates of 50 ducks each in a completely randomised experiment. Each group was fed on a basal diet supplemented with 0, 500, 1000, 1500 or 2000 mg probiotics/kg. 3. Body rate and feed conversion ratio were highest and abdominal subcutaneous fat % was lowest at 1000 mg probiotic/kg. 4. The mRNA levels of LXRα and CYP7α1 in liver tissue was estimated by RT-PCR; serum triglyceride (TG) and total cholesterol (TC) concentrations were measured by ELISA. 5. The expression levels and enzyme activity of LXRα and CYP7α1 increased in conjunction with decreases in TG and TC concentrations following probiotic supplementation to a maximum at 1000 mg probiotics/kg and decreased thereafter. 6. It is concluded that dietary probiotics can enhance LXRα and CYP7α1 enzyme activities in the liver and reduce lipid concentrations and fat deposition in ducks.

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Inhibition of small-intestinal sugar absorption mediated by sodium orthovanadate Na3VO4 in rats and its mechanisms

PubMed Central

Ai, Jing; Du, Jie; Wang, Ning; Du, Zhi-Min; Yang, Bao-Feng

2004-01-01

AIM: To investigate the inhibitory effects of sodium orthovanadate on small-intestinal glucose and maltose absorption in rats and its mechanism. METHODS: Normal Wistar rats were lavaged with sodium orthovanadate (16 mg/kg, 4 mg/kg and 1 mg/kg) for 6 d. Blood glucose values were measured after fasting and 0.5, 1, 1.5 and 2 h after glucose and maltose feeding with oxidation-enzyme method. α-glucosidase was abstracted from the upper small intestine, and its activity was examined. mRNA expression of α-glucosidase and glucose-transporter 2 (GLUT2) in epithelial cells of the small intestine was observed by in situ hybridization. RESULTS: Sodium orthovanadate could delay the increase of plasma glucose concentration after glucose and maltose loading, area under curve (AUC) in these groups was lower than that in control group. Sodium orthovanadate at dosages of 10 μmol/L, 100 μmol/L and 1000 μmol/L could suppress the activity of α-glucosidase in the small intestine of normal rats, with an inhibition rate of 68.18%, 87.22% and 91.91%, respectively. Sodium orthovanadate reduced mRNA expression of α-glucosidase and GLUT2 in epithelial cells of small intestine. CONCLUSION: Sodium orthovanadate can reduce and delay the absorption of glucose and maltose. The mechanism may be that it can inhibit the activity and mRNA expression of α-glucosidase, as well as mRNA expression of GLUT2 in small intestine. PMID:15534916

Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus.

PubMed

Urbatzka, R; Galante-Oliveira, S; Rocha, E; Lobo-da-Cunha, A; Castro, L F C; Cunha, I

2015-07-01

Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors. Copyright © 2015 Elsevier B.V. All rights reserved.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

PubMed

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of dietary biotin supplementation on glucagon production, secretion, and action.

PubMed

Lazo-de-la-Vega-Monroy, Maria-Luisa; Larrieta, Elena; Tixi-Verdugo, Wilma; Ramírez-Mondragón, Rafael; Hernández-Araiza, Ileana; German, Michael S; Fernandez-Mejia, Cristina

Despite increasing evidence that pharmacologic concentrations of biotin modify glucose metabolism, to our knowledge there have not been any studies addressing the effects of biotin supplementation on glucagon production and secretion, considering glucagon is one of the major hormones in maintaining glucose homeostasis. The aim of this study was to investigate the effects of dietary biotin supplementation on glucagon expression, secretion, and action. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 wk postweaning. Glucagon gene mRNA expression was measured by the real-time polymerase chain reaction. Glucagon secretion was assessed in isolated islets and by glucagon concentration in plasma. Glucagon action was evaluated by glucagon tolerance tests, phosphoenolpyruvate carboxykinase (Pck1) mRNA expression, and glycogen degradation. Compared with the control group, glucagon mRNA and secretion were increased from the islets of the biotin-supplemented group. Fasting plasma glucagon levels were higher, but no differences between the groups were observed in nonfasting glucagon levels. Despite the elevated fasting glucagon levels, no differences were found in fasting blood glucose concentrations, fasting/fasting-refeeding glucagon tolerance tests, glycogen content and degradation, or mRNA expression of the hepatic gluconeogenic rate-limiting enzyme, Pck1. These results demonstrated that dietary biotin supplementation increased glucagon expression and secretion without affecting fasting blood glucose concentrations or glucagon tolerance and provided new insights into the effect of biotin supplementation on glucagon production and action. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of zinc deficiency and supplementation on leptin and leptin receptor expression in pregnant mice.

PubMed

Ueda, Hidenori; Nakai, Taketo; Konishi, Tatsuya; Tanaka, Keiichi; Sakazaki, Fumitoshi; Min, Kyong-Son

2014-01-01

Leptin is an adipose-derived hormone that primarily regulates energy balance in response to nutrition. Human placental cells produce leptin, whereas murine placental cells produce soluble leptin receptors (Ob-R). However, the roles of these proteins during pregnancy have not been elucidated completely. As an essential metal, zinc (Zn) is central to insulin biosynthesis and energy metabolism. In the present study, the effects of Zn deficiency and supplementation on maternal plasma leptin and soluble Ob-R regulation in pregnant mice placentas were examined using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blotting. Nutritional Zn deficiency significantly reduced plasma insulin concentrations and fetal and placental weights in pregnant mice. Plasma leptin concentrations in pregnant mice also increased 20- to 40-fold compared with those in non-pregnant mice. Although dietary Zn deficiency and supplementation did not affect plasma leptin concentrations in non-pregnant mice, Zn-deficient pregnant mice had significantly reduced plasma leptin concentrations and adipose leptin mRNA expression. In contrast, Zn-supplemented pregnant mice had increased plasma leptin concentrations without increased adipose leptin mRNA expression. Placental soluble Ob-R mRNA expression also decreased in Zn-deficient mice and tended to increase in Zn-supplemented mice. These results indicate that Zn influences plasma leptin concentrations by modulating mRNA expression of soluble Ob-R in the placenta, and leptin in visceral fat during pregnancy. These data suggest that both adipose and placenta-derived leptin system are involved in the regulation of energy metabolism during fetal growth.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

PubMed

Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

2016-08-15

The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia.

PubMed

Witsch, Thilo J; Turowski, Pawel; Sakkas, Elpidoforos; Niess, Gero; Becker, Simone; Herold, Susanne; Mayer, Konstantin; Vadász, István; Roberts, Jesse D; Seeger, Werner; Morty, Rory E

2014-02-01

Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-β increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-β signaling in the experimental animal model of BPD, TGF-β was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD.

Silibinin down-regulates expression of secreted phospholipase A2 enzymes in cancer cells.

PubMed

Hagelgans, Albert; Nacke, Brit; Zamaraeva, Maria; Siegert, Gabriele; Menschikowski, Mario

2014-04-01

Silibinin, a naturally-occurring flavonoid produced by milk thistle, possesses antioxidant, anti-inflammatory and cancer-preventive activities. In the current study, we examined the effects of silibinin on the expression of secreted phospholipase A2 (sPLA2) enzymes, especially those of group IIA (hGIIA), which play a crucial role in inflammation and carcinogenesis. The effects of silibinin on sPLA2 expressions in human HepG2 hepatoma and PC-3 prostate cancer cells were analyzed using quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay technique. Silibinin inhibited the expression of hGIIA in unstimulated and cytokine-primed HepG2 and PC-3 cells. The mRNA levels of sPLA2 of groups IB, III and V were also significantly decreased by silibinin. Analyses of transcription factor activation suggest that nuclear factor-κB, but not specificity protein 1 (SP1) is implicated in the silibinin-mediated down-regulation of hGIIA. Silibinin exhibits inhibitory effects on basal and cytokine-induced expression of sPLA2s in cancer cells and therefore, may have the potential to protect against up-regulation of hGIIA and other sPLA2 isoforms during inflammation and cancer.

Novel Fatty Acid Lipoxygenases in the Development of Human and Murine Prostate Cancer

DTIC Science & Technology

2000-10-01

expression of COX-2 mRNA in benign and malignant prostate samples by RNase protection assays in collaboration with Dr. Matthew Breyer at Vanderbilt. COX-2...Shappell,* William E. Boeglin,t prostate adenocarcinomas. (Am J Pathol 1999, Sandy J. Olson,* Susan Kasper,f and 155:235-245) Alan R. Brasht From the...possible role of this novel enzyme in secretory function, 33157-331605. Samuelssor,- B. Dahlen SE, Lindgren JA, Rouzer CA, Serhan CN: Reduced expression in

Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

PubMed Central

Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

2014-01-01

Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol delta8-isomerase, a cholesterogenic enzyme with multiple functions.

PubMed Central

Bae, S; Seong, J; Paik, Y

2001-01-01

Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hünermann syndrome in humans. To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts. The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH). The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a characteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbuten-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A; IC(50)=4.2 microM), two well known potent inhibitors of SI. Northern-blot analysis of 3-week-old rats compared with 2-year-old rats showed that SI mRNA expression in both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats. The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Treatment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein-1 mRNA. Interestingly, this effect was not seen in emopamil-treated cells. The in vivo experiments also indicate that both mRNA expression and enzymic activity of SI in liver were induced approx. 3-fold in rats fed 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in normal chow for 2 weeks. With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases. PMID:11171067

Betaine Aldehyde Dehydrogenase expression during physiological cardiac hypertrophy induced by pregnancy.

PubMed

Rosas-Rodríguez, Jesús Alfredo; Soñanez-Organis, José Guadalupe; Godoy-Lugo, José Arquimides; Espinoza-Salazar, Juan Alberto; López-Jacobo, Cesar Jeravy; Stephens-Camacho, Norma Aurora; González-Ochoa, Guadalupe

2017-08-26

Betaine Aldehyde Dehydrogenase (betaine aldehyde: NAD(P) + oxidoreductase, (E.C. 1.2.1.8; BADH) catalyze the irreversible oxidation of betaine aldehyde (BA) to glycine betaine (GB) and is essential for polyamine catabolism, γ-aminobutyric acid synthesis, and carnitine biosynthesis. GB is an important osmolyte that regulates the homocysteine levels, contributing to a vascular risk factor reduction. In this sense, distinct investigations describe the physiological roles of GB, but there is a lack of information about the GB novo synthesis process and regulation during cardiac hypertrophy induced by pregnancy. In this work, the BADH mRNA expression, protein level, and activity were quantified in the left ventricle before, during, and after pregnancy. The mRNA expression, protein content and enzyme activity along with GB content of BADH increased 2.41, 1.95 and 1.65-fold respectively during late pregnancy compared to not pregnancy, and returned to basal levels at postpartum. Besides, the GB levels increased 1.53-fold during pregnancy and remain at postpartum. Our results demonstrate that physiological cardiac hypertrophy induced BADH mRNA expression and activity along with GB production, suggesting that BADH participates in the adaptation process of physiological cardiac hypertrophy during pregnancy, according to the described GB role in cellular osmoregulation, osmoprotection and reduction of vascular risk. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

PubMed

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named "hTERT messenger ribonucleic acid (mRNA)" the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0-160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control-untreated group. The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent ( P < 0.05) and not in time-dependent manner ( P > 0.05), in vitro . Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

miR-128 inhibits telomerase activity by targeting TERT mRNA

PubMed Central

Guzman, Herlinda; Sanders, Katie; Idica, Adam; Bochnakian, Aurore; Jury, Douglas; Daugaard, Iben; Zisoulis, Dimitrios G; Pedersen, Irene Munk

2018-01-01

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes. PMID:29568354

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basavarajappa, Mallikarjuna S., E-mail: mbasava2@illinois.edu; Craig, Zelieann R., E-mail: zelieann@illinois.edu; Hernandez-Ochoa, Isabel, E-mail: mihernandez@cinvestav.mx

2011-06-15

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E{sub 2}) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition,more » sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E{sub 2} metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E{sub 2}, testosterone, androstenedione, and progesterone (P{sub 4}) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17{beta}-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17{alpha}-hydroxylase/17,20-lyase (Cyp17a1), 3{beta} hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels. - Highlights: > MXC inhibits steroidogenesis > MXC inhibits steroidogenic enzymes > MXC induces metabolic enzymes« less

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

PubMed

Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

1985-10-01

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

Tissue-specific changes in OGG1 and SOD mRNA expression caused by NaOCl exposure in black seabream ( Acanthopagrus schlegelii)

NASA Astrophysics Data System (ADS)

Park, Ho-Ra; Kim, Yong; Yeo, Won-Jun; Kim, Ji-Hye; Han, Kyung-Nam

2017-09-01

The DNA-damage defense mechanism was studied in black seabreams after oxidative stress caused by exposure to sodium hypochlorite (NaOCl). Liver, muscle, and brain tissues were obtained after different NaOCl-exposure times (0, 24, 48, 72, and 96 h) and concentrations (0.5, 1, 1.5, 2, and 3 mg/L), after which oxoguanine glycosylase (OGG1) and superoxide dismutase (SOD) mRNA-expression levels were analyzed. At all NaOCl concentrations tested, liver OGG1 expression increased to a maximum in a time-dependent manner after NaOCl exposure and then decreased. In muscles, OGG1 expression increased over time following exposure to a low concentration of NaOCl (0.5, 1, and 1.5 mg/L), whereas it showed a mixed pattern (both increases and decreases observed) in the high-concentration groups (2 and 3 mg/L). SOD mRNA expression increased over time, both in the liver and muscles. In the brain, both OGG1 and SOD mRNA expression levels were highest after exposure to the lowest NaOCl concentration (0.5 mg/L), whereas basal levels were maintained over time at higher concentrations. These results indicate that OGG1 and SOD provide resistance to oxidative stress in black seabreams. In addition, continuous exposure to oxidative stress can suppress enzyme expression, suggesting a risk for long-term exposure to NaOCl.

Nitrous oxide production and mRNA expression analysis of nitrifying and denitrifying bacterial genes under floodwater disappearance and fertilizer application.

PubMed

Riya, Shohei; Takeuchi, Yuki; Zhou, Sheng; Terada, Akihiko; Hosomi, Masaaki

2017-06-01

A pulse of nitrous oxide (N 2 O) emission has been observed following the disappearance of floodwater by drainage. However, its mechanism is not well understood. We conducted a column study to clarify the mechanism for N 2 O production during floodwater disappearance by using a microsensor and determining the bacterial gene expression. An increase in N 2 O flux was observed following floodwater disappearance after the addition of NH 4 + , with a corresponding increase in the concentrations of NO 3 - and dissolved N 2 O in the oxic and anoxic soil layers, respectively. The transcription level of the bacterial amoA mRNA did not change, while that of nirK mRNA increased sharply after an hour of floodwater disappearance. An additional anoxic soil slurry experiment demonstrated that the addition of NO 3 - induced the expression of nirK gene and caused a concomitant increase in N 2 O production. These findings suggest that NO 3 - production in the oxic layers is important as it provides a substrate and induces the synthesis of denitrification enzymes in the anoxic layer during N 2 O production.

Molecular cloning of a catalase cDNA from Nicotiana glutinosa L. and its repression by tobacco mosaic virus infection.

PubMed

Yi, S Y; Yu, S H; Choi, D

1999-06-30

Recent reports revealed that catalase has a role in the plant defense mechanism against a broad range of pathogens through being inhibited by salicylic acid (SA). During an effort to clone disease resistance-responsive genes, a cDNA encoding catalase (Ngcat1; Nicotiana glutinosa cat1) was isolated from a tobacco cDNA library. In N. glutinosa, catalase is encoded by a small gene family. The deduced amino acid sequence of the Ngcat1 cDNA has 98% homology with the cat1 gene of N. plumbaginifolia. The Ngcat1 expression is controlled by the circadian clock, and its mRNA level is the most abundant in leaves. Both the expression of Ngcat1 mRNA and its enzyme activity in the tobacco plant undergoing a hypersensitive response (HR) to TMV infection were repressed. The repression of the mRNA level was also observed following treatment with SA. These results imply that SA may act as an inhibitor of catalase transcription during the HR of tobacco. Cloning and expression of the Ngcat1 in tobacco following pathogen infection and SA treatment are presented.

Coordination of Gene Expression of Arachidonic and Docosahexaenoic Acid Cascade Enzymes during Human Brain Development and Aging

PubMed Central

Ryan, Veronica H.; Primiani, Christopher T.; Rao, Jagadeesh S.; Ahn, Kwangmi; Rapoport, Stanley I.; Blanchard, Helene

2014-01-01

Background The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA) participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades. Hypothesis AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging. Methods The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism. Results Expression patterns were split into Development (0 to 20 years) and Aging (21 to 78 years) intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2), cyclooxygenases (COX)-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA) and PTGS2 (COX-2) genes at 1q25, highly inter-correlated genes were at distant chromosomal loci. Conclusions Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease. PMID:24963629

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Bloch, S. R.; Fiacco, G. J.; Partridge, N. C.

1999-01-01

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast. Copyright 1999 Wiley-Liss, Inc.

Triptolide Upregulates Myocardial Forkhead Helix Transcription Factor p3 Expression and Attenuates Cardiac Hypertrophy

PubMed Central

Ding, Yuan-Yuan; Li, Jing-Mei; Guo, Feng-Jie; Liu, Ya; Tong, Yang-Fei; Pan, Xi-Chun; Lu, Xiao-Lan; Ye, Wen; Chen, Xiao-Hong; Zhang, Hai-Gang

2016-01-01

The forkhead/winged helix transcription factor (Fox) p3 can regulate the expression of various genes, and it has been reported that the transfer of Foxp3-positive T cells could ameliorate cardiac hypertrophy and fibrosis. Triptolide (TP) can elevate the expression of Foxp3, but its effects on cardiac hypertrophy remain unclear. In the present study, neonatal rat ventricular myocytes (NRVM) were isolated and stimulated with angiotensin II (1 μmol/L) to induce hypertrophic response. The expression of Foxp3 in NRVM was observed by using immunofluorescence assay. Fifty mice were randomly divided into five groups and received vehicle (control), isoproterenol (Iso, 5 mg/kg, s.c.), one of three doses of TP (10, 30, or 90 μg/kg, i.p.) for 14 days, respectively. The pathological morphology changes were observed after Hematoxylin and eosin, lectin and Masson’s trichrome staining. The levels of serum brain natriuretic peptide (BNP) and troponin I were determined by enzyme-linked immunosorbent assay and chemiluminescence, respectively. The mRNA and protein expressions of α- myosin heavy chain (MHC), β-MHC and Foxp3 were determined using real-time PCR and immunohistochemistry, respectively. It was shown that TP (1, 3, 10 μg/L) treatment significantly decreased cell size, mRNA and protein expression of β-MHC, and upregulated Foxp3 expression in NRVM. TP also decreased heart weight index, left ventricular weight index and, improved myocardial injury and fibrosis; and decreased the cross-scetional area of the myocardium, serum cardiac troponin and BNP. Additionally, TP markedly reduced the mRNA and protein expression of myocardial β-MHC and elevated the mRNA and protein expression of α-MHC and Foxp3 in a dose-dependent manner. In conclusion, TP can effectively ameliorate myocardial damage and inhibit cardiac hypertrophy, which is at least partly related to the elevation of Foxp3 expression in cardiomyocytes. PMID:27965581

Effects of dietary zinc on gene expression of antioxidant enzymes and heat shock proteins in hepatopancreas of abalone Haliotis discus hannai.

PubMed

Wu, Chenglong; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhong, Xiaoli

2011-06-01

The expression patterns of different genes encoding antioxidant enzymes and heat shock proteins were investigated, in present study, by real-time quantitative PCR in the hepatopancreas of abalone Haliotis discus hannai fed with different levels of dietary zinc (6.69, 33.8, 710.6 and 3462.5 mg/kg) for 20 weeks. The antioxidant enzymes include Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT), mu-glutathione-s-transferase (mu-GST) and thioredoxin peroxidase (TPx). The results showed that the mRNA expression of these antioxidant enzymes increased and reached the maximum at the dietary zinc level of 33.8 mg/kg, and then dropped progressively. Expression levels of the heat shock proteins (HSP26, HSP70 and HSP90) firstly increased at 33.8 mg/kg dietary Zn level, and reached to the maximum at 710.6 mg/kg, then dropped at 3462.5 mg/kg (p<0.05). Excessive dietary Zn (710.6 and 3462.5 mg/kg) significantly increases the Zn content and significantly decreases the total antioxidant capacity (T-AOC) in hepatopancreas (p<0.05). These findings showed that dietary Zn (33.8 mg/kg) could highly trigger the expression levels of antioxidant enzymes and heat shock proteins, but excessive dietary Zn (710.6 and 3462.5 mg/kg) induces a high oxidative stress in abalone. Copyright © 2011 Elsevier Inc. All rights reserved.

Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eisenstein, R.S.; Rosen, J.M.

The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNAmore » was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.« less

Induction of Proteinases in the Human Preovulatory Follicle of the Menstrual Cycle by hCG

PubMed Central

Rosewell, Katherine L.; Al-Alem, Linah; Zakerkish, Farnosh; McCord, Lauren; Akin, James W.; Chaffin, Charles L.; Brännström, Mats; Curry, Thomas E.

2014-01-01

Objective To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women in order to gain insight into their possible roles during ovulation and early luteinization. Design Experimental prospective clinical study and laboratory-based investigation. Setting University Medical Center and private IVF center. Animal and Patient(s) Thirty eight premenopausal women undergoing surgery for tubal ligation and 6 premenopausal women undergoing ART. Intervention(s) Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. Main Outcome Measure(s) Expression of mRNA for MMPs and ADAMTSs in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. Result(s) Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS 9 mRNA increased in granulosa cells after hCG treatment, however thecal cell expression for ADAMTS1 was unchanged while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1 and ADAMTS9 to the granulosa and thecal cell layers. Conclusion(s) The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte. PMID:25516084

Induction of proteinases in the human preovulatory follicle of the menstrual cycle by human chorionic gonadotropin.

PubMed

Rosewell, Katherine L; Al-Alem, Linah; Zakerkish, Farnosh; McCord, Lauren; Akin, James W; Chaffin, Charles L; Brännström, Mats; Curry, Thomas E

2015-03-01

To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women to gain insight into their possible roles during ovulation and early luteinization. Experimental prospective clinical study and laboratory-based investigation. University medical center and private IVF center. Thirty-eight premenopausal women undergoing surgery for tubal ligation and six premenopausal women undergoing assisted reproductive techniques. Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. Expression of mRNA for matrix metalloproteinase (MMPs) and the A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS9 mRNA increased in granulosa cells after hCG treatment, however, thecal cell expression for ADAMTS1 was unchanged, while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1, and ADAMTS9 to the granulosa and thecal cell layers. The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Alleviative effects of s-allyl cysteine and s-ethyl cysteine on MCD diet-induced hepatotoxicity in mice.

PubMed

Lin, Chun-che; Yin, Mei-chin; Liu, Wen-hu

2008-11-01

Alleviative effects of s-allyl cysteine (SAC) and s-ethyl cysteine (SEC) upon methionine and choline deficient (MCD) diet-induced hepatotoxicity in mice were examined. SAC or SEC at 1g/L was added into drinking water for 7 weeks with MCD diet. MCD feeding significantly increased hepatic triglyceride and cholesterol levels, and elevated the activity of glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (P < 0.05). However, the intake of SAC or SEC significantly decreased hepatic triglyceride accumulation, and reduced G6PDH and FAS activities (P < 0.05). MCD feeding significantly lowered serum and hepatic glutathione (GSH) levels, increased malondialdehyde (MDA) and oxidized glutathione (GSSG) formation, and suppressed the activity and mRNA expression of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (P < 0.05). The intake of SAC or SEC significantly increased serum and hepatic GSH levels, decreased MDA and GSSG formation, restored the activity and mRNA expression of GPX, SOD and catalase (P < 0.05). MCD feeding significantly enhanced the mRNA expression of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, matrix metalloproteinases-9 (MMP-9) and collagen-alpha1 (P < 0.05). The intake of SAC and SEC significantly blunted the mRNA expression of IL-1beta, IL-6, TNF-alpha, TGF-beta1 and collagen-alpha1 (P < 0.05). SEC was greater than SAC in suppressing IL-6 and TNF-alpha expression (P < 0.05), but SAC was greater than SEC in suppressing collagen-alpha1 and TGF-beta1 expression (P < 0.05). These data suggest that SAC and SEC are potent agents against MCD-induced hepatotoxicity.

Sodium Meta-Arsenite Ameliorates Hyperglycemia in Obese Diabetic db/db Mice by Inhibition of Hepatic Gluconeogenesis

PubMed Central

Lee, Eun-Kyu; Oh, Hyun-Hee; Choi, Cheol Soo; Kim, Sujong; Jun, Hee-Sook

2014-01-01

Sodium meta-arsenite (SA) is implicated in the regulation of hepatic gluconeogenesis-related genes in vitro; however, the effects in vivo have not been studied. We investigated whether SA has antidiabetic effects in a type 2 diabetic mouse model. Diabetic db/db mice were orally intubated with SA (10 mg kg−1 body weight/day) for 8 weeks. We examined hemoglobin A1c (HbA1c), blood glucose levels, food intake, and body weight. We performed glucose, insulin, and pyruvate tolerance tests and analyzed glucose production and the expression of gluconeogenesis-related genes in hepatocytes. We analyzed energy metabolism using a comprehensive animal metabolic monitoring system. SA-treated diabetic db/db mice had reduced concentrations of HbA1c and blood glucose levels. Exogenous glucose was quickly cleared in glucose tolerance tests. The mRNA expressions of genes for gluconeogenesis-related enzymes, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly reduced in the liver of SA-treated diabetic db/db mice. In primary hepatocytes, SA treatment decreased glucose production and the expression of G6Pase, PEPCK, and hepatocyte nuclear factor 4 alpha (HNF-4α) mRNA. Small heterodimer partner (SHP) mRNA expression was increased in hepatocytes dependent upon the SA concentration. The expression of Sirt1 mRNA and protein was reduced, and acetylated forkhead box protein O1 (FoxO1) was induced by SA treatment in hepatocytes. In addition, SA-treated diabetic db/db mice showed reduced energy expenditure. Oral intubation of SA ameliorates hyperglycemia in db/db mice by reducing hepatic gluconeogenesis through the decrease of Sirt1 expression and increase in acetylated FoxO1. PMID:25610880

Markers of hepatic regeneration associated with surgical attenuation of congenital portosystemic shunts in dogs.

PubMed

Tivers, Michael S; Lipscomb, Victoria J; Smith, Kenneth C; Wheeler-Jones, Caroline P D; House, Arthur K

2014-05-01

Dogs with congenital portosystemic shunts (CPSS) have liver hypoplasia and hepatic insufficiency. Surgical CPSS attenuation results in liver growth associated with clinical improvement. The mechanism of this hepatic response is unknown, although liver regeneration is suspected. This study investigated whether markers of liver regeneration were associated with CPSS attenuation. Dogs treated with CPSS attenuation were prospectively recruited. Residual liver tissue was collected for gene expression analysis (seven genes) from 24 CPSS dogs that tolerated complete attenuation, 25 dogs that tolerated partial attenuation and seven control dogs. Relative gene expression was measured using quantitative polymerase chain reaction (qPCR). Blood samples were collected before, 24 h and 48 h post-surgery from 36 CPSS dogs and from 10 control dogs. Serum hepatocyte growth factor (HGF) concentration was measured using a canine specific enzyme-linked immunosorbent assay (ELISA). HGF mRNA expression was significantly decreased in CPSS compared with control dogs (P = 0.046). There were significant increases in HGF (P = 0.050) and methionine adenosyltransferase 2 A (MAT2A; P = 0.002) mRNA expression following partial CPSS attenuation. Dogs with complete attenuation had significantly greater MAT2A (P = 0.024) mRNA expression compared with dogs with partial attenuation. Serum HGF concentration significantly increased 24 h following CPSS attenuation (P < 0.001). Hepatic mRNA expression of two markers of hepatocyte proliferation (HGF and MAT2A) was associated with the response to surgery in dogs with CPSS, and serum HGF significantly increased following surgery, suggesting hepatocyte proliferation. These findings support the concept that hepatic regeneration is important in the hepatic response to CPSS surgery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular cloning and sequence analysis of two carbonic anhydrase in the swimming crab Portunus trituberculatus and its expression in response to salinity and pH stress.

PubMed

Pan, Luqing; Hu, Dongxu; Liu, Maoqi; Hu, Yanyan; Liu, Shengnan

2016-01-15

Carbonic anhydrase (CA) is involved in ion transport, acid-base balance and pH regulation by catalyzing the interconversion of CO2 and HCO3(-). In this study, full-length cDNA sequences of two CA isoforms were identified from Portunus trituberculatus. One was Portunus trituberculatus cytoplasmic carbonic anydrase (PtCAc) and the other one was Portunus trituberculatus glycosyl-phosphatidylinositol-linked carbonic anhydrase (PtCAg). The sequence of PtCAc was formed by an ORF of 816 bp, encoding a protein of 30.18 kDa. The PtCAg was constituted by an ORF of 927 bp, encoding a protein of 34.09 kDa. The deduced amino acid sequences of the two CA isoforms were compared to other crustacean' CA sequences. Both of them reflected high conservation of the residues and domains essential to the function of the two enzymes. The tissue expression analysis of PtCAc and PtCAg were detected in gill, muscle, hepatopancreas, hemocytes and gonad. PtCAc and PtCAg gene expressions were studied under salinity and pH challenge. The results showed that when salinity decreased (30 to 20 ppt), the mRNA expression of PtCAc increased significantly at 24 and 48 h, and the highest value appeared at 24h. The mRNA expression of PtCAg had the same situation with PtCAc. However, when salinity increased (30 to 35 ppt), only the mRNA expression of PtCAc increased significantly at 48 h. When pH changed, only the mRNA expression of PtCAc increased significantly at 12h, which was under low pH situation. The mRNA expression of PtCAg increased significantly at 12-48 h, and there was no significant difference of the expression between the pH challenged group and the control group in other experimental time. The results provided the base of understanding CA' function and the underlying mechanism in response to environmental changes in crustaceans. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of 11beta-hydroxysteroid dehydrogenase 1 and 2 in subcutaneous adipose tissue of lean and obese women with and without polycystic ovary syndrome.

PubMed

Svendsen, P F; Madsbad, S; Nilas, L; Paulsen, S K; Pedersen, S B

2009-11-01

To investigate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and 2 and hexose-6-phosphate dehydrogenase (H6PDH) mRNA in subcutaneous abdominal tissue from lean and obese women with and without polycystic ovary syndrome (PCOS), and to investigate the association between these enzymes and different measures of insulin sensitivity. Cross-sectional study. A total of 60 women, 36 women with PCOS, 17 lean (lean PCOS, LP) and 19 obese (obese PCOS, OP) and 24 age- and weight-matched control women, 8 lean (lean controls, LC) and 16 obese (obese controls, OC). Subcutaneous adipose tissue was collected from the abdomen. Peripheral insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp and determined as glucose disposal rate and insulin sensitivity index. Whole-body insulin sensitivity was calculated using homeostasis model assessment insulin resistance index. Body composition was evaluated by dual X-ray absorptiometry. Adipose mRNA expression of leptin and adiponectin were determined by real-time PCR. Polycystic ovary syndrome (P<0.05) and obesity (P<0.05) were independently associated with increased expression of 11beta-HSD1 mRNA. The subgroups LP and OC had increased 11beta-HSD1 and 11beta-HSD2 mRNA expression compared with LC (P<0.05, P<0.05). There were no effects of PCOS or obesity on11beta-HSD2 or H6PDH mRNA expression. Decreased peripheral insulin sensitivity (P<0.001) and increased upper body fat distribution (P<0.01) were associated with increased expression of 11beta-HSD1, but neither 11beta-HSD2 nor H6PDH. Polycystic ovary syndrome and obesity are independently associated with increased expression of 11beta-HSD1. This may lead to increased conversion of cortisone to cortisol in the peripheral adipose tissue and subsequently increased glucocorticoid activity. Decreased peripheral insulin sensitivity and central obesity was associated with increased expression of 11beta-HSD1.

Vitamin D Alters Genes Involved in Follicular Development and Steroidogenesis in Human Cumulus Granulosa Cells

PubMed Central

Doswell, Angela; Krebs, Kendall; Cipolla, Marilyn

2014-01-01

Context: Vitamin D deficiency is common among reproductive-aged women and has a role in female reproduction. Objective: This study evaluated the role of 1,25-dihydroxyvitamin D3 (vit D3) in ovarian follicular development and steroidogenesis by using a human granulosa cell (GC) model. Design, Setting, and Participants: Fifty-four women who underwent in vitro fertilization were enrolled. Intervention: Follicular fluid (FF) and mural and cumulus GCs were collected from small and large follicles. In separate experiments, primary cumulus GCs were cultured with or without vit D3 followed by RT-PCR for mRNA expression levels. The effect of recombinant anti-Mullerian hormone (AMH) on nuclear localization of phospho-Smad 1/5/8 was evaluated in the presence or absence of vit D3 by using immunofluorescence. 25-Hydroxyvitamin D levels in FF as well as cell culture media AMH, progesterone, and estradiol (E2) concentrations were determined by ELISA and RIA. Main Outcome Measures: The following were measured: 1) mRNA expression levels; 2) 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme activity; 3) FSH-induced aromatase mRNA and E2 production; and 4) nuclear localization of phospho-Smad 1/5/8. Results: In a multivariate analysis, 25 OH-D levels in FF negatively correlated with AMH and AMH receptor (AMHR)-II mRNA levels in cumulus GCs of small follicles. Compared with women with replete 25-hydroxyvitamin D levels in FF, those with insufficient/deficient levels had a 2-fold increase in AMHR-II mRNA levels in cumulus GCs of small follicles (P = .02). Treatment with vit D3 caused a decrease in AMHR-II and FSH receptor mRNA but an increase in 3-βHSD mRNA levels compared with control (P < .05). Vit D3 enhanced 3-βHSD enzyme activity as assessed by increasing progesterone release; however, vit D3 did not affect FSH-induced aromatase mRNA and E2 production, but it decreased the phosphorylation of Smad 1/5/8 and its nuclear localization. Conclusion: These data suggest that vit D3 alters AMH signaling and steroidogenesis in human cumulus GCs, possibly reflecting a state of GC luteinization potentiation. PMID:24628555

Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue

PubMed Central

CAVAZZOLA, LUCIANE ROSTIROLA; CARVALHAL, GUSTAVO FRANCO; DEVES, CANDIDA; RENCK, DAIANA; ALMEIDA, RICARDO; SANTOS, DIóGENES SANTIAGO

2015-01-01

Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. P<0.05 was considered to indicate a statistically significant difference. The results revealed that PDEF, ETV4, UP-1 and TP were expressed in 85.7, 90.5, 95.2 and 100% of the prostate cancer samples, and in 90.3, 96.8, 90.3 and 96.8% of the benign samples, respectively. PDEF and ETV4 exhibited a significantly higher relative expression level in the tumor samples compared with their benign counterparts. The relative expression of TP and UP-1 did not differ significantly between benign and cancerous prostate tissues. The relative expression of TP was moderately and significantly correlated with the expression of ETV4 in the benign tissues. The relative expression of UP-1 was significantly lower in T3 compared with T1 and T2 cancers. These findings indicate that PDEF, ETV4, TP and UP-1 are typically expressed in benign and malignant prostatic tissues. Further studies are necessary to define the role of these proteins as therapeutic targets in prostate cancer. PMID:26137165

Identification of a cytochrome P450 gene in the earthworm Eisenia fetida and its mRNA expression under enrofloxacin stress.

PubMed

Li, Yinsheng; Zhao, Chun; Lu, Xiaoxu; Ai, Xiaojie; Qiu, Jiangping

2018-04-15

Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. Earthworms have been used as a bioindicator of soil pollution in numerous studies, but no CYP450 gene has so far been cloned. RT-PCR and RACE-PCR were employed to construct and sequence the CYP450 gene DNA from the extracted mRNA in the earthworm Eisenia fetida. The cloned gene (EW1) has an open reading frame of 477bp. The 3'-terminal region contained both the consensus and the signature sequences characteristic of CYP450. It was closely related to the CYP450 gene from the flatworm genus Opisthorchis felineus with 87% homology. The predicted structure of the putative protein was 97% homologous to human CYP450 family 27. This gene has been deposited in GenBank (accession no. KM881474). Earthworms (E. fetida) were then exposed to 1, 10, 100, and 500mgkg -1 enrofloxacin in soils to explore the mRNA expression by real time qPCR. The effect of enrofloxacin on mRNA expression levels of EW1 exhibited a marked hormesis pattern across the enrofloxacin dose range tested. This is believed to be the first reported CYP450 gene in earthworms, with reference value for molecular studies on detoxification processes in earthworms. Copyright © 2017 Elsevier Inc. All rights reserved.

The post-menopausal ovary displays a unique pattern of steroidogenic enzyme expression.

PubMed

Havelock, Jon C; Rainey, William E; Bradshaw, Karen D; Carr, Bruce R

2006-01-01

While menopause results in the loss of cyclic steroid production, evidence exists for persistent, albeit reduced, ovarian androgen production. In order to continue to synthesize ovarian androgens, the steroidogenic enzymes necessary for androgen biosynthesis must be present. Few studies have selectively analysed some of the steroidogenic enzymes present in the post-menopausal ovary (PMO), and a comprehensive study of this matter has never been undertaken. RNA and protein were obtained from PMO, pre-menopausal ovarian stroma, corpora lutea (CL), ovarian follicles, placenta, and myometrium. Oligonucleotide microarray analysis was performed to compare the gene expression profiles of PMO with pre-menopausal ovarian stroma. Real-time RT-PCR was performed for LH/HCG receptor (LHCGR), steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase type I (HSD3B1) and type II (HSD3B2, 3betaHSD), 17a-hydroxylase (CYP17), cytochrome b5 (CytB5), and aromatase (CYP19). Western blot analysis was performed for StAR, CYP11A, CYP17,and 3betaHSD. The PMO and pre-menopausal ovarian stroma had a similar pattern of steroidogenic enzyme expression. The PMO had persistent, but reduced, levels of LHCGR and most steroidogenic enzymes. CYP19 and HSD3B2 mRNA were greatly reduced in PMO in comparison with CL (50-fold and 2000-fold less respectively). HSD3B2 was not detectable in PMO by western analysis. This study supports the idea that the PMO retains some steroidogenic capacity. However, based on steroidogenic enzyme expression, the PMO has a unique pattern of steroidogenic enzyme expression that favors Delta5 steroid formation over Delta4 steroid formation.

Omeprazole induces NAD(P)H quinone oxidoreductase 1 via aryl hydrocarbon receptor-independent mechanisms: Role of the transcription factor nuclear factor erythroid 2–related factor 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Shaojie; Patel, Ananddeep; Moorthy, Bhagavatula

2015-11-13

Activation of the aryl hydrocarbon receptor (AhR) transcriptionally induces phase I (cytochrome P450 (CYP) 1A1) and phase II (NAD(P)H quinone oxidoreductase 1 (NQO1) detoxifying enzymes. The effects of the classical and nonclassical AhR ligands on phase I and II enzymes are well studied in human hepatocytes. Additionally, we observed that the proton pump inhibitor, omeprazole (OM), transcriptionally induces CYP1A1 in the human adenocarcinoma cell line, H441 cells via AhR. Whether OM activates AhR and induces the phase II enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1), in fetal primary human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis thatmore » OM will induce NQO1 in HPMEC via the AhR. The concentrations of OM used in our experiments did not result in cytotoxicity. OM activated AhR as evident by increased CYP1A1 mRNA expression. However, contrary to our hypothesis, OM increased NQO1 mRNA and protein via an AhR-independent mechanism as AhR knockdown failed to abrogate OM-mediated increase in NQO1 expression. Interestingly, OM activated Nrf2 as evident by increased phosphoNrf2 (S40) expression in OM-treated compared to vehicle-treated cells. Furthermore, Nrf2 knockdown abrogated OM-mediated increase in NQO1 expression. In conclusion, we provide evidence that OM induces NQO1 via AhR-independent, but Nrf2-dependent mechanisms. - Highlights: • We investigated whether omeprazole induces NQO1 in human fetal lung cells. • Omeprazole induces the phase II enzyme, NQO1, in human fetal lung cells. • AhR deficiency fails to abrogate omeprazole-mediated induction of NQO1. • Omeprazole increases phosphoNrf2 (S40) protein expression in human fetal lung cells. • Nrf2 knockdown abrogates the induction of NQO1 by omeprazole in human lung cells.« less

Abrupt salinity stress induces oxidative stress via the Nrf2-Keap1 signaling pathway in large yellow croaker Pseudosciaena crocea.

PubMed

Zeng, Lin; Ai, Chun-Xiang; Wang, Yong-Hong; Zhang, Jian-She; Wu, Chang-Wen

2017-08-01

The aim of the present study was to evaluate the effects of abrupt salinity stress (12, 26 (control), and 40) on lipid peroxidation, activities and mRNA levels of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx, and GR), and gene expression of the Nrf2-Keap1 signaling molecules at different times (6, 12, 24, and 48 h) in the liver of large yellow croaker Pseudosciaena crocea. The results showed that lipid peroxidation was sharply reduced at 6 h and increased at 12 h before returning to control levels in the hypo-salinity group. Similarly, lipid peroxidation was significantly decreased at 6 h followed by a sharp increase towards the end of the exposure in the hyper-salinity group. Negative relationships between lipid peroxidation and antioxidant enzyme activities and positive relationships between activities and gene expression of antioxidant enzymes were observed, suggesting that the changes at molecular levels and enzyme activity levels may provide protective roles against damage from salinity stress. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for regulating these genes. Furthermore, there was a positive relationship between the mRNA levels of Nrf2 and Keap1, indicating that Keap1 plays an important role in switching off the Nrf2 response. In conclusion, this is the first study to elucidate effects of salinity stress on antioxidant responses in large yellow croaker through the Keap1-Nrf2 pathway.

Pycnogenol® inhibits lipid accumulation in 3T3-L1 adipocytes with the modulation of reactive oxygen species (ROS) production associated with antioxidant enzyme responses.

PubMed

Lee, Ok-Hwan; Seo, Min-Jung; Choi, Hyeon-Son; Lee, Boo-Yong

2012-03-01

Pycnogenol® is a group of flavonoids with antioxidant effects. Adipogenesis is the process of adipocyte differentiation. It causes the increase of lipids as well as ROS (reactive oxygen species). Lipid accumulation and ROS production were determined in 3 T3-L1 adipocyte, and the effect of Pycnogenol® was evaluated. Lipid accumulation was elevated in adipocyte treated with hydrogen peroxide, one of the ROS. Pycnogenol® showed an inhibitory effect on the lipid accumulation and ROS production during the adipogenesis. We also investigated the molecular events associated with ROS production and lipid accumulation. Our results showed that Pycnogenol® inhibited the mRNA expression of pro-oxidant enzymes, such as NOX4 (NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase 4), and the NADPH-producing G6PDH (glucose-6-phosphate dehydrogenase) enzyme. In addition, Pycnogenol® suppressed the mRNA abundance of adipogenic transcription factors, PPAR-γ (peroxisome proliferator-activated receptor γ) and C/EBP-α (CCAAT/enhancer binding protein α), and their target gene, aP2 (adipocyte protein 2) responsible for fatty acid transportation. On the other hand, Pycnogenol® increased the abundance of antioxidant proteins such as Cu/Zn-SOD (copper-zinc superoxide dismutase), Mn-SOD (manganese superoxide dismutase), GPx (glutathione peroxidase) and GR (glutathione reductase). Our results suggest that Pycnogenol® inhibits lipid accumulation and ROS production by regulating adipogenic gene expression and pro-/antioxidant enzyme responses in adipocytes. Copyright © 2011 John Wiley & Sons, Ltd.

Alteration of the Expression of Pesticide-Metabolizing Enzymes in Pregnant Mice: Potential Role in the Increased Vulnerability of the Developing Brain

PubMed Central

Fortin, Marie C.; Aleksunes, Lauren M.

2013-01-01

Studies on therapeutic drug disposition in humans have shown significant alterations as the result of pregnancy. However, it is not known whether pesticide metabolic capacity changes throughout pregnancy, which could affect exposure of the developing brain. We sought to determine the effect of pregnancy on the expression of hepatic enzymes involved in the metabolism of pesticides. Livers were collected from virgin and pregnant C57BL/6 mice at gestational days (GD)7, GD11, GD14, GD17, and postpartum days (PD)1, PD15, and PD30. Relative mRNA expression of several enzymes involved in the metabolism of pesticides, including hepatic cytochromes (Cyp) P450s, carboxylesterases (Ces), and paraoxonase 1 (Pon1), were assessed in mice during gestation and the postpartum period. Compared with virgin mice, alterations in the expression occurred at multiple time points, with the largest changes observed on GD14. At this time point, the expression of most of the Cyps involved in pesticide metabolism in the liver (Cyp1a2, Cyp2d22, Cyp2c37, Cyp2c50, Cyp2c54, and Cyp3a11) were downregulated by 30% or more. Expression of various Ces isoforms and Pon1 were also decreased along with Pon1 activity. These data demonstrate significant alterations in the expression of key enzymes that detoxify pesticides during pregnancy, which could alter exposure of developing animals to these chemicals. PMID:23223497

Identification and Function Analysis of enolase Gene NlEno1 from Nilaparvata lugens (Stål) (Hemiptera:Delphacidae)

PubMed Central

Wang, Wei-Xia; Li, Kai-Long; Chen, Yang; Lai, Feng-Xiang; Fu, Qiang

2015-01-01

The enolase [EC 4.2.1.11] is an essential enzyme in the glycolytic pathway catalyzing the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP). In this study, a full-length cDNA encoding α-enolase was cloned from rice brown planthopper (Nilaparvata lugens) and is provisionally designated as NlEno1. The cDNA sequence of NlEno1 was 1,851 bp with an open reading frame (ORF) of 1,305 bp and encoding 434 amino acids. The deduced protein shares high identity of 80–87% with ENO1-like protein from Hemiptera, Diptera, and Lepidoptera speices. The NlEno1 showed the highest mRNA expression level in hemolymph, followed by fat body, salivary gland, ovaries and egg, and showed trace mRNA levels in testis. The mRNA of NlEno1 showed up-regulated level in virulent N. lugens population Mudgo, IR56 and IR42 when compared with TN1 population. Injection of double-stranded RNA (dsRNA) of NlEno1 into the adults significantly down-regulated the NlEno1 mRNA level along with decreased eggs and offspring. Moreover, injection of NlEno1-dsRNA decreased mRNA level of Vitellogenin (Vg) gene. These results showed that the NlEno1, as a key glycolytic enzyme, may play roles in regulation of fecundity and adaptation of N. lugens to resistant rice varieties. PMID:26056319

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE PAGES

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

2018-04-18

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes.

PubMed

Standaert, Robert F; Giannone, Richard J; Michener, Joshua K

2018-06-01

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI , from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA , duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI , growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

PubMed

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

PubMed

Gorné, Lucas D; Acosta-Rodríguez, Victoria A; Pasquaré, Susana J; Salvador, Gabriela A; Giusto, Norma M; Guido, Mario Eduardo

2015-02-01

The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

Hemorrhagic Shock-Induced Vascular Hyporeactivity in the Rat: Relationship to Gene Expression of Nitric Oxide Synthase, Endothelin-1, and Select Cytokines in Corresponding Organs

DTIC Science & Technology

2005-01-01

the Selected Genes Sense Antisense Product length (bp) G3PDH 5=-TCCTGCACCACCAACTGCTTAG-3= 5=-TGCTTCACCACCTTCTTGATGTC-3= 341 iNOS 5...GAPDH, as a housekeeping gene, was not affected significantly by the hemorrhage protocol. The results showed that mRNA levels of all enzymes and

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

PubMed

Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Preadipocyte 11beta-hydroxysteroid dehydrogenase type 1 is a keto-reductase and contributes to diet-induced visceral obesity in vivo.

PubMed

De Sousa Peixoto, R A; Turban, S; Battle, J H; Chapman, K E; Seckl, J R; Morton, N M

2008-04-01

Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) within adipocytes might explain this paradox, the potential role of 11beta-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11beta-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11beta-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11beta-HSD1 activity in mice. 11beta-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11beta-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11beta-HSD1 was an 11beta-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11beta-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11beta-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11beta-HSD1(-/-) mice. The results suggest that 11beta-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.

Role of angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis in the hypotensive effect of azilsartan.

PubMed

Iwanami, Jun; Mogi, Masaki; Tsukuda, Kana; Wang, Xiao-Li; Nakaoka, Hirotomo; Ohshima, Kousei; Chisaka, Toshiyuki; Bai, Hui-Yu; Kanno, Harumi; Min, Li-Juan; Horiuchi, Masatsugu

2014-07-01

The possible counteracting effect of angiotensin (Ang)-converting enzyme (ACE)2/Ang-(1-7)/Mas axis against the ACE/Ang II/Ang II type 1 (AT1) receptor axis in blood pressure control has been previously described. We examined the possibility that this pathway might be involved in the anti-hypertensive effect of a newly developed AT1 receptor blocker (ARB), azilsartan, and compared azilsartan's effects with those of another ARB, olmesartan. Transgenic mice carrying the human renin and angiotensinogen genes (hRN/hANG-Tg) were given azilsartan or olmesartan. Systolic and diastolic blood pressure, as determined by radiotelemetry, were significantly higher in hRN/hANG-Tg mice than in wild-type (WT) mice. Treatment with azilsartan or olmesartan (1 or 5 mg kg(-1) per day) significantly decreased systolic and diastolic blood pressure, and the blood pressure-lowering effect of azilsartan was more marked than that of olmesartan. The urinary Na concentration decreased in an age-dependent manner in hRN/hANG-Tg mice. Administration of azilsartan or olmesartan increased urinary Na concentration, and this effect was weaker with olmesartan than with azilsartan. Azilsartan decreased ENaC-α mRNA expression in the kidney and decreased the ratio of heart to body weight. Olmesartan had a similar but less-marked effect. ACE2 mRNA expression was lower in the kidneys and hearts of hRN/hANG-Tg mice than in WT mice. This decrease in ACE2 mRNA expression was attenuated by azilsartan, but not by olmesartan. These results suggest that the hypotensive and anti-hypertrophic effects of azilsartan may involve activation of the ACE2/Ang-(1-7)/Mas axis with AT1 receptor blockade.

Effects of bis(guanylhydrazones) on the activity and expression of ornithine decarboxylase.

PubMed Central

Nikula, P; Alhonen-Hongisto, L; Jänne, J

1985-01-01

Derivatives of glyoxal bis(guanylhydrazone) (GBG), such as methylglyoxal bis(guanylhydrazone) and ethylglyoxal bis(guanylhydrazone), are potent inhibitors of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the key enzyme required for the synthesis of spermidine and spermine. These compounds, but not the parent compound, induce a massive accumulation of putrescine, partly by blocking the conversion of putrescine into spermidine, but also by strikingly stimulating ornithine decarboxylase (ODC; EC 4.1.1.17) activity. The mechanism of the stimulation of ODC activity and enhanced accumulation of the enzyme protein apparently involved a distinct stabilization of the enzyme against intracellular degradation. However, although the parent compound GBG also stabilized ODC, it powerfully inhibited the enzyme activity and the accumulation of immunoreactive protein in cultured L1210 leukaemia cells. Kinetic considerations indicated that, in addition to the stabilization, all three compounds, GBG in particular, inhibited the expression of ODC. It is unlikely that the decreased rate of synthesis of ODC was attributable to almost unaltered amounts of mRNA in drug-treated cells, thus supporting the view that especially GBG apparently depressed the expression of ODC at some post-transcriptional level. Images PMID:4062886

Expression of arginine kinase enzymatic activity and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus.

PubMed

Kotlyar, S; Weihrauch, D; Paulsen, R S; Towle, D W

2000-08-01

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.

First echinoderm trehalase from a tropical sea cucumber (Holothuria leucospilota): Molecular cloning and mRNA expression in different tissues, embryonic and larval stages, and under a starvation challenge.

PubMed

Huo, Da; Jiang, Xiao; Wu, Xiaofen; Ren, Chunhua; Yu, Zonghe; Liu, Jinshang; Li, Hongmei; Ruan, Yao; Wen, Jin; Chen, Ting; Hu, Chaoqun

2018-04-29

Trehalases are a group of enzymes that catalyse the conversion of trehalose to glucose, and they are observed in most organisms. In this study, the first echinoderm trehalase, designated Hl-Tre, was identified from a tropical sea cucumber, Holothuria leucospilota. The full-length cDNA of H. leucospilota trehalase (Hl-Tre) is 2461 bp in length with an open reading frame (ORF) of 1788 bp that encodes a 595-amino-acid protein with a deduced molecular weight of 67.95 KDa. The Hl-Tre protein contains a signal peptide at the N-terminal and a functional trehalase domain, which includes the signature motifs 1 and 2. The mRNA expression of Hl-Tre was ubiquitously detected in all selected tissues, with the highest level being detected in the intestine. By in situ hybridization (ISH), the positive Hl-Tre signals were observed in the brush borders of the intestinal mucosa. In embryonic and larval stages, the transcript levels of Hl-Tre decreased during embryonic development and increased after the pentactula stage. After a challenge of starvation, the intestinal Hl-Tre mRNA levels were observed to be first decreased and partially recovered thereafter. Overall, our study provided the first evidence for trehalase in echinoderms and showed that this enzyme was potentially linked to a trehalose metabolic pathway in sea cucumbers. Copyright © 2017. Published by Elsevier B.V.

Assessment of RNAi-induced silencing in banana (Musa spp.).

PubMed

Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge

2014-09-18

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt). RNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

RNA therapeutics: Beyond RNA interference and antisense oligonucleotides

PubMed Central

Kole, Ryszard; Krainer, Adrian R.; Altman, Sidney

2016-01-01

Here we discuss three RNA therapeutic technologies exploiting various oligonucleotides that bind RNA by base-pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by enzyme-dependent degradation of targeted mRNA. Steric blocking oligonucleotides block access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or also downregulate gene expression. Moreover, they can be extensively chemically modified, resulting in more drug-like properties. The ability of RNA blocking oligonucleotides to restore gene function makes them suited for treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to realizing its clinical potential. PMID:22262036

The regulated in development and DNA damage response 2 (REDD2) gene mediates human monocyte cell death through a reduction in thioredoxin-1 expression.

PubMed

Imen, Jguirim-Souissi; Billiet, Ludivine; Cuaz-Pérolin, Clarisse; Michaud, Nadège; Rouis, Mustapha

2009-05-15

In a previous study, we identified the regulated in development and DNA damage response 2 (REDD2) gene as a highly expressed gene in human atherosclerotic lesions in comparison to normal artery, as well as in cultured human macrophages, and showed its implication in oxidized low-density lipoprotein (LDL)-induced macrophage death sensitivity. In this article, we attempt to identify the mechanism by which REDD2 induces such a phenomenon. Transient transfection of U-937 monocytic cells with a pCI.CMV.REDD2 expression vector increased by approximately twofold the mRNA levels of REDD2 in comparison to control cells transfected with pCI.CMV.GFP. Reactive oxygen species (ROS) production was significantly induced in REDD2-transfected cells compared with control cells (157+/-48 and 100+/-8 arbitrary units/mg cell protein, respectively; p<0.05). Moreover, a significant increase in parameters known to reflect the oxidative modifications of LDL was observed. Among enzymes involved in ROS production or degradation, we found a specific reduction in thioredoxin-1 (Trx-1) mRNA ( approximately 52+/-7% decrease, p<0.01 vs control cells) and protein ( approximately 60+/-4% decrease, p<0.001 vs control cells) levels in cells overexpressing REDD2 in comparison to control cells. In contrast, transfection of U-937 cells with siRNA against REDD2 decreased the mRNA levels of REDD2 by approximately 60% and increased Trx-1 mRNA and protein levels. Moreover, we observed no or a moderate increase in Bax (proapoptotic) and a significant decrease in Bcl2 (antiapoptotic) gene expression in cells that overexpress REDD2 compared to control cells. In addition, we showed that Trx-1 mRNA and protein levels were increased at low H(2)O(2) doses and decreased at higher doses. Interestingly, macrophages isolated from human atherosclerotic lesions differentially express REDD2 and Trx-1. Indeed, in certain patients, levels of REDD2 mRNA were low and those of Trx-1 mRNA were high. In contrast, in other patients, levels of REDD2 were high and levels of Trx-1 mRNA were low.

Follicular thyroglobulin induces cathepsin H expression and activity in thyrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oda, Kenzaburo; Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama, Tokyo 189-0002; Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Toho University, 5-21-16 Omorinishi, Ota, Tokyo 143-8540

Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tgmore » can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways. - Highlights: • Follicular Tg increases cathepsin H mRNA and protein levels in rat thyroid cells. • Follicular Tg increases cathepsin H enzyme activity in rat thyroid cells. • After Tg stimulation cathepsin H co-localizes to lysosomes with follicular Tg. • Cathepsin H promotes hormone secretion by lysosome-mediated mechanisms.« less

Sex steroid hormone metabolism takes place in human ocular cells.

PubMed

Coca-Prados, Miguel; Ghosh, Sikha; Wang, Yugang; Escribano, Julio; Herrala, Annakaisa; Vihko, Pirkko

2003-08-01

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.

Aromatase mRNA in the brain of adult green anole lizards: effects of sex and season.

PubMed

Cohen, R E; Wade, J

2011-03-01

Neural testosterone metabolism, particularly the synthesis of oestradiol (E(2)) via the aromatase enzyme, is important for sexual behaviours in many vertebrates. In green anole lizards, E(2) metabolised from testosterone facilitates female receptivity and increases sexual motivation in males. Testosterone treatment increases aromatase activity in the whole brain homogenates of gonadectomised male, but not female, anoles, which is an effect limited to the breeding season (BS). To investigate the potential for local effects of this enzyme in reproductive behaviour, we used in situ hybridisation for aromatase mRNA to examine expression during the BS and nonbreeding season (NBS) in areas of the brain that control male sexual behaviours [preoptic area (POA) and amygdala (AMY)], as well as one regulating female reproductive behaviours ventromedial hypothalamus (VMH). Males had a greater total number of aromatase-expressing cells in the POA than females, and the density of aromatase-expressing cells (number per unit volume) was greater in the VMH and AMY of females. This density was also higher during the BS than NBS in the POA. Expression of aromatase in the AMY appeared to be lateralised because trends were detected for the left side to have more total cells and more cells per unit volume than the right. These results suggest that, similar to other vertebrates, regional aromatisation of testosterone may be important for the control of sex-specific reproductive behaviours. © 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.

Selective induction of phospholipase D1 in pathogen-activated human monocytes.

PubMed

Locati, M; Riboldi, E; Bonecchi, R; Transidico, P; Bernasconi, S; Haribabu, B; Morris, A J; Mantovani, A; Sozzani, S

2001-08-15

Phospholipase D (PLD) activation is part of the complex signalling cascade induced during phagocyte activation. Two PLD isoforms have been cloned, but their role in phagocyte functions is still poorly defined. We report that resting fresh circulating human monocytes expressed PLD1. PLD1 protein expression was rapidly down-regulated during cell culture. Lipopolysaccharide and pathogen-derived agonists (Candida albicans, arabinoside-terminated lipoarabinomannan and Gram-positive bacteria, but not mannose-capped lipoarabinomannan or double-stranded RNA) strongly induced PLD1 expression at both the mRNA and protein levels. Pro-inflammatory cytokines [interleukin (IL)-1beta and tumour necrosis factor alpha] had only a weak effect, whereas immune cytokines (IL-6 and interferon gamma), anti-inflammatory cytokines (IL-13 and IL-10) and chemoattractants (fMet-Leu-Phe and macrophage chemoattractant protein 1) were inactive. None of the agonists tested induced significant changes in the basal expression of PLD2 mRNA. Consistent with PLD1 up-regulation was the observation that PLD enzymic activity was higher in monocytes treated with active-pathogen-derived agonists than in control cells, when stimulated with PMA or with chemotactic agonists (fMet-Leu-Phe and C5a). Thus PLD2 seems to be a constitutive enzyme in circulating monocytes. Conversely, PLD1 is an inducible protein, rapidly regulated during culture conditions and selectively induced during cell activation. Therefore PLD1 might have a relevant role in immune responses against pathogens and in chronic inflammation.

Rapid Induction of Hypothalamic Iodothyronine Deiodinase Expression by Photoperiod and Melatonin in Juvenile Siberian Hamsters (Phodopus sungorus)

PubMed Central

Prendergast, Brian J.; Pyter, Leah M.; Kampf-Lassin, August; Patel, Priyesh N.

2013-01-01

Production of T3 in the mediobasal hypothalamus is critical for regulation of seasonal reproductive physiology. Type 2 iodothyronine deiodinase (DIO2) and DIO3 enzymes catalyze the prohormone T4 into biologically-active T3 and biologically-inactive rT3, respectively. In several seasonally-breeding vertebrates, DIO2 and DIO3 expression is implicated in photoperiod signal transduction in adulthood. These experiments tested the hypothesis that juvenile Siberian hamsters, which are highly responsive to photoperiod at weaning (postnatal day [PND]18), exhibit rapid and sustained changes in hypothalamic dio3 mRNA expression during photoperiod-induced and photoperiod-inhibited puberty. Hypothalamic dio2 and dio3 expression was measured via quantitative PCR in hamsters born and reared in a long-day photoperiod (15L:9D) and weaned on PND18 into short-day photoperiods (9L:15D). In SD males, hypothalamic dio3 mRNA was elevated 2.5-fold within 3 days (PND21) and continued to increase (>20-fold) through PND32; changes in dio3 mRNA preceded inhibition of gonadotropin (FSH) secretion and gonadal regression in SD. Females exhibited comparable dio3 responses to SD. In LD males, dio3 remained low and invariant from PND18–PND32. In contrast, dio2 mRNA rose conspicuously on PND21, independent of photoperiod, returning to basal levels thereafter. In LD, a single afternoon melatonin (MEL) injection on PND18 or PND20 was sufficient to increase hypothalamic dio3 mRNA, and dio3 increased in proportion to the number of successive days of MEL treatment. SD photoperiods and MEL exert rapid, sustained, and additive effects on hypothalamic dio3 mRNA, which may play a central role in inhibiting maturation of the peripubertal hypothalamo-pituitary-gonadal axis. PMID:23295738

Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

PubMed

Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

2013-04-14

The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA.

Structural and kinetic insights into stimulation of RppH-dependent RNA degradation by the metabolic enzyme DapF.

PubMed

Gao, Ang; Vasilyev, Nikita; Luciano, Daniel J; Levenson-Palmer, Rose; Richards, Jamie; Marsiglia, William M; Traaseth, Nathaniel J; Belasco, Joel G; Serganov, Alexander

2018-05-04

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5' termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5'-end-dependent mRNA degradation begins with the generation of monophosphorylated 5' termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH-DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

[Effects of lipopolysaccharides from Porphyromonas endodontalis on the expression of MIP-1α in mouse osteoblasts].

PubMed

Yu, Ya-Qiong; Li, Xiao-Lin; Qiu, Li-Hong; Guo, Jia-Jie; Yang, Di; Guo, Yan

2017-06-01

To investigate the effects of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophageinflammatoryprotein-1α (MIP-1α) mRNA and protein levels in MC3T3-E1 cells and the influence of curcumin in the process. MC3T3-E1 cells were treated with 20 mg/L P.e-LPS for different times (0-48 h). The expression of MIP-1α mRNA and protein was detected by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and enzyme linked immunosorbent assay(ELISA). MC3T3-E1 cells were pretreated with inhibitor of (curcumin) for 1 h, and then treated with 20 mg/L P.e-LPS. The expression of MIP-1α was also detected by real-time RT-PCR and ELISA.Statistical analysis was performed using one-way ANOVA and Dunnett's t test with SPSS 13.0 software package. In the observation time (0-48 h), the impact of 20 P.e-LPS mg/L on induction of MIP-1α in MC3T3-El cells exhibited a time-dependent manner. The expression of MIP-1α mRNA and protein decreased significantly after pretreatment with 10 μmol/L curcumin for 1 h. The results suggest that P.e-LPS may mediate MIP-1α expression in MC3T3-E1 cells, and curcumin has a significant inhibitory effect on this process.

Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney

PubMed Central

Rodríguez-Fuentes, Gabriela; Coburn, Cary; Currás-Collazo, Margarita; Guillén, Gabriel; Schlenk, Daniel

2010-01-01

Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm3/100 g to 0.6 cm3/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats. PMID:19429252

Magnesium Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

PubMed Central

Swift, Robert V.; Ong, Chau D.; Amaro, Rommie E.

2012-01-01

The messenger RNA guanylyltransferase, or mRNA capping enzyme, co-transcriptionally caps the 5′-end of nascent mRNA with GMP during the second in a set of three enzymatic reactions that result in the formation of an N7-methyl guanosine cap during mRNA maturation. The mRNA capping enzyme is characterized, in part, by a conserved lysine nucleophile that attacks the alpha-phosphorous atom of GTP, forming a lysine-GMP intermediate. Experiments have firmly established that magnesium is required for efficient intermediate formation, but have provided little insight into the requirement’s molecular origins. Using empirical and thermodynamic integration pKa estimates, along with conventional MD simulations, we show that magnesium binding likely activates the lysine nucleophile by increasing its acidity and by biasing the deprotonated nucleophile into conformations conducive to intermediate formation. These results provide additional functional understanding of an important enzyme in the mRNA transcript life cycle and allow functional analogies to be drawn that affect our understanding of the metal dependence of related superfamily members. PMID:23205906

Light response, oxidative stress management and nucleic acid stability in closely related Linderniaceae species differing in desiccation tolerance.

PubMed

Dinakar, Challabathula; Bartels, Dorothea

2012-08-01

In the present study, three closely related Linderniaceae species which differ in their sensitivity to desiccation are compared in response to light and oxidative stress defence. Lindernia brevidens, a desiccation-tolerant plant, displayed intense purple pigmentation in leaves under long-day conditions in contrast to Craterostigma plantagineum (desiccation tolerant) and Lindernia subracemosa (desiccation sensitive). The intense pigmentation in leaves does not affect the desiccation tolerance behaviour but seems to be related to oxidative stress protection. Green leaves of short-day and purple leaves of long-day plants provided suitable material for comparing basic photosynthetic parameters. An increase in non-photochemical quenching in purple leaves appears to prevent photoinhibition. Treatment with methyl viologen decreased the photochemical activities in both long-day and short-day plants but long-day plants which accumulate anthocyanins maintained a higher non-photochemical quenching than short-day plants. No differences were seen in the expression of desiccation-induced proteins and proteins involved in carbohydrate metabolism in short-day and long-day grown plants, whereas differences were observed in the expression of transcripts encoding chloroplast-localised stress proteins and transcripts encoding antioxidant enzymes. While the expression of genes encoding antioxidant enzymes were either constitutive or up-regulated during desiccation in C. plantagineum, the expression was down-regulated in L. subracemosa. RNA expression analysis indicated degradation of mRNA during desiccation in L. subracemosa but not in desiccation tolerant species. These results indicate that a better oxidative stress management and mRNA stability are correlated with desiccation tolerance.

Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle Abnormalities, Cachexia and Exercise Intolerance in Mice with Congestive Heart Failure

PubMed Central

Okutsu, Mitsuharu; Call, Jarrod A.; Lira, Vitor A.; Zhang, Mei; Donet, Jean A.; French, Brent A.; Martin, Kyle S.; Peirce-Cottler, Shayn M.; Rembold, Christopher M.; Annex, Brian H.; Yan, Zhen

2014-01-01

Background Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide (NO)-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of the NO-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. Methods and Results We demonstrated that systemic administration of endogenous nitric oxide donor S-Nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis [muscle creatine kinase (MCK)-EcSOD] in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF [α-myosin heavy chain (MHC)-calsequestrin] MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced heart failure. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria and vascular rarefaction in skeletal muscle. Conclusions EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF. PMID:24523418

A Novel Dark-Inducible Protein, LeDI-2, and Its Involvement in Root-Specific Secondary Metabolism in Lithospermum erythrorhizon1

PubMed Central

Yazaki, Kazufumi; Matsuoka, Hideaki; Shimomura, Koichiro; Bechthold, Andreas; Sato, Fumihiko

2001-01-01

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities. PMID:11299363

The effect of dietary carbohydrate on genes for fatty acid synthase and inflammatory cytokines in adipose tissues from lean and obese subjects.

PubMed

Hudgins, Lisa C; Baday, Aline; Hellerstein, Marc K; Parker, Thomas S; Levine, Daniel M; Seidman, Cynthia E; Neese, Richard A; Tremaroli, Jolanta D; Hirsch, Jules

2008-04-01

Hepatic de novo lipogenesis (DNL) is markedly stimulated in humans by low-fat diets enriched in simple sugars. However, the dietary responsiveness of the key enzyme controlling DNL in human adipose tissue, fatty acid synthase (FAS), is uncertain. Adipose tissue mRNA for FAS is increased in lean and obese subjects when hepatic DNL is elevated by a eucaloric, low-fat, high-sugar diet. Twelve lean and seven obese volunteers were given two eucaloric diets (10% vs. 30% fat; 75% vs. 55% carbohydrate; sugar/starch 60/40) each for 2 weeks by a random-order cross-over design. FAS mRNA in abdominal and gluteal adipose tissues was compared to hepatic DNL measured in serum by isotopic and nonisotopic methods. Adipose tissue mRNA for tumor necrosis factor-alpha and IL-6, which are inflammatory cytokines that modulate DNL, was also assayed. The low-fat high-sugar diet induced a 4-fold increase in maximum hepatic DNL (P<.001) but only a 1.3-fold increase in adipose tissue FAS mRNA (P=.029) and no change in cytokine mRNA. There was a borderline significant positive correlation between changes in FAS mRNA and hepatic DNL (P=.039). Compared to lean subjects, obese subjects had lower levels of FAS mRNA and higher levels of cytokine mRNA (P<.001). The results suggest that key elements of human adipose tissue DNL are less responsive to dietary carbohydrate than is hepatic DNL and may be regulated by diet-independent factors. Irrespective of diet, there is reduced expression of the FAS gene and increased expression of cytokine genes in adipose tissues of obese subjects.

Upregulation of NLRP3 Inflammasome in the Tears and Ocular Surface of Dry Eye Patients

PubMed Central

Wu, Jihong; Chen, Ling; Wang, Yan

2015-01-01

Purpose To evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye. Methods We recruited 54 patients with Sjögren’s syndrome dry eye (SSDE), 50 patients with non-Sjögren’s syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting. Results NLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups. Conclusions In the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye. PMID:25962072

Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

PubMed

Suzuki, Dai; Saito-Hakoda, Akiko; Ito, Ryo; Shimizu, Kyoko; Parvin, Rehana; Shimada, Hiroki; Noro, Erika; Suzuki, Susumu; Fujiwara, Ikuma; Kagechika, Hiroyuki; Rainey, William E; Kure, Shigeo; Ito, Sadayoshi; Yokoyama, Atsushi; Sugawara, Akira

2017-01-01

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

Dietary vitamin E deficiency does not affect global and specific DNA methylation patterns in rat liver.

PubMed

Fischer, Alexandra; Gaedicke, Sonja; Frank, Jan; Döring, Frank; Rimbach, Gerald

2010-10-01

The aim of the present study was to determine the effects of a 6-month dietary vitamin E (VE) deficiency on DNA methylation and gene expression in rat liver. Two enzymes, 5-α-steroid reductase type 1 (SRD5A1) and the regulatory subunit of γ-glutamylcysteinyl synthetase (GCLM), which are differentially expressed on the mRNA level, were analysed for promoter methylation in putative cytosine-phospho-guanine (CpG) island regions located at the 5' end using base-specific cleavage and matrix-assisted laser desorption ionisation time-of-flight MS. A twofold increase in the mRNA level of SRD5A1 gene and a twofold decrease in the mRNA level of GCLM gene in VE-deficient animals were not associated with different CpG methylation of the analysed promoter region. Furthermore, global DNA methylation was not significantly different in these two groups. Thus, the present results indicate that the VE-induced regulation of SRD5A1 and GCLM in rat liver is not directly mediated by changes in promoter DNA methylation.

The Co-Induced Effects of Molybdenum and Cadmium on the Trace Elements and the mRNA Expression Levels of CP and MT in Duck Testicles.

PubMed

Xia, Bing; Chen, Hua; Hu, Guoliang; Wang, Liqi; Cao, Huabin; Zhang, Caiying

2016-02-01

To investigate the chronic toxicity of molybdenum (Mo) and cadmium (Cd) on the trace elements and the mRNA expression levels of ceruloplasmin (CP) and metallothionein (MT) in duck testicles, 120 healthy 11-day-old male ducks were randomly divided into six groups with 20 ducks in each group. Ducks were treated with the diet containing different dosages of Mo or Cd. The source of Mo and Cd was hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) and cadmium sulfate (3CdSO4·8H2O), respectively, in this study. After being treated for 60 and 120 days, ten male birds in each group were randomly selected and euthanized and then testicles were aseptically collected for determining the mRNA expression levels of MT and CP, antioxidant indexes, and contents of trace elements in the testicle. In addition, testicle tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that co-exposure to Mo and Cd resulted in an increase in malondialdehyde (MDA) level while decrease in xanthine oxidase (XOD) and catalase (CAT) activities. The mRNA expression level of MT gene was upregulated while CP was decreased in combination groups. Contents of Mo, copper (Cu), iron (Fe), and zinc (Zn) decreased in combined groups while Cd increased in Cd and combined groups at 120 days. Furthermore, severe congestion, low sperm count, and malformation were observed in low dietary of Mo combined with Cd group and high dietary of Mo combined with Cd group. Our results suggested that Mo and Cd might aggravate testicular degeneration synergistically through altering the mRNA expression levels of MT and CP, increasing lipid peroxidation through inhibiting related enzyme activities and disturbing homeostasis of trace elements in testicles. Interaction of Mo and Cd may have a synergistic effect on the testicular toxicity.

High animal fat intake enhances prostate cancer progression and reduces glutathione peroxidase 3 expression in early stages of TRAMP mice.

PubMed

Chang, Seo-Na; Han, Juhee; Abdelkader, Tamer Said; Kim, Tae-Hyoun; Lee, Ji Min; Song, Juha; Kim, Kyung-Sul; Park, Jong-Hwan; Park, Jae-Hak

2014-09-01

Prostate cancer is the most frequently diagnosed cancer in Western men, and more men have been diagnosed at younger ages in recent years. A high-fat Western-style diet is a known risk factor for prostate cancer and increases oxidative stress. We evaluated the association between dietary animal fat and expression of antioxidant enzymes, particularly glutathione peroxidase 3 (GPx3), in the early stages of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Six-week-old male nontransgenic and TRAMP mice were placed on high animal fat (45% Kcal fat) or control (10% Kcal fat) diets and sacrificed after 5 or 10 weeks. The histopathological score increased with age and high-fat diet consumption. The histopathological scores in dorsal and lateral lobes increased in the 10-week high-fat diet group (6.2±0.2 and 6.2±0.4, respectively) versus the 10-week control diet group (5.3±0.3 and 5.2±0.2, respectively). GPx3 decreased both at the mRNA and protein levels in mouse prostate. GPx3 mRNA expression decreased (∼36.27% and ∼23.91%, respectively) in the anterior and dorsolateral prostate of TRAMP mice fed a high-fat diet compared to TRAMP mice fed a control diet. Cholesterol treatment increased PC-3 human prostate cancer cell proliferation, decreased GPx3 mRNA and protein levels, and increased H2 O2 levels in culture medium. Moreover, increasing GPx3 mRNA expression by troglitazone in PC-3 cells decreased cell proliferation and lowered H2 O2 levels. Dietary fat enhances prostate cancer progression, possibly by suppressing GPx3 expression and increasing proliferation of prostate intraepithelial neoplasia (PIN) epithelial cells. © 2014 Wiley Periodicals, Inc.

Effects of Intermittent Alcohol Exposure on Emotion and Cognition: A Potential Role for the Endogenous Cannabinoid System and Neuroinflammation

PubMed Central

Sanchez-Marin, Laura; Pavon, Francisco J.; Decara, Juan; Suarez, Juan; Gavito, Ana; Castilla-Ortega, Estela; Rodriguez de Fonseca, Fernando; Serrano, Antonia

2017-01-01

Intermittent alcohol exposure is a common pattern of adolescent alcohol use that can lead to binge drinking episodes. Alcohol use is known to modulate the endocannabinoid system (ECS), which is involved in neuronal communication, neuroplasticity, neuroinflammation and behavior. Adolescent male Wistar rats were exposed to 4-week intermittent alcohol intoxication (3 g/kg injections for 4 days/week) or saline (N = 12 per group). After alcohol deprivation, adult rats were assessed for emotionality and cognition and the gene expression of the ECS and other factors related to behavior and neuroinflammation was examined in the brain. Alcohol-exposed rats exhibited anxiogenic-like responses and impaired recognition memory but no motor alterations. There were brain region-dependent changes in the mRNA levels of the ECS and molecular signals compared with control rats. Thus, overall, alcohol-exposed rats expressed higher mRNA levels of endocannabinoid synthetic enzymes (N-acyl-phosphatidylethanolamine phospholipase D and diacylglycerol lipases) in the medial-prefrontal cortex (mPFC) but lower mRNA levels in the amygdala. Furthermore, we observed lower mRNA levels of receptors CB1 CB2 and peroxisome proliferator-activated receptor-α in the striatum. Regarding neuropeptide signaling, alcohol-exposed rats displayed lower mRNA levels of the neuropeptide Y signaling, particularly NPY receptor-2, in the amygdala and hippocampus and higher mRNA levels of corticotropin-releasing factor in the hippocampus. Additionally, we observed changes of several neuroinflammation-related factors. Whereas, the mRNA levels of toll-like receptor-4, tumor necrosis factor-α, cyclooxygenase-2 and glial fibrillary acidic protein were significantly increased in the mPFC, the mRNA levels of cyclooxygenase-2 and glial fibrillary acidic protein were decreased in the striatum and hippocampus. However, nuclear factor-κβ mRNA levels were lower in the mPFC and striatum and allograft inflammatory factor-1 levels were differentially expressed in the amygdala and hippocampus. In conclusion, rats exposed to adolescent intermittent alcohol displayed anxiety-like behavior and cognitive deficits in adulthood and these alterations were accompanied by brain region-dependent changes in the gene expression of the ECS and other signals associated with neuroinflammation and behavior. An intermittent adolescent alcohol exposure has behavioral and molecular consequences in the adult brain, which might be linked to higher vulnerability to addictive behaviors and psychopathologies. PMID:28223925

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

PubMed Central

2013-01-01

Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants. PMID:24053212

Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle

PubMed Central

Féasson, L; Stockholm, D; Freyssenet, D; Richard, I; Duguez, S; Beckmann, J S; Denis, C

2002-01-01

The molecular events by which eccentric muscle contractions induce muscle damage and remodelling remain largely unknown. We assessed whether eccentric exercise modulates the expression of proteinases (calpains 1, 2 and 3, proteasome, cathepsin B+L), muscle structural proteins (α-sarcoglycan and desmin), and the expression of the heat shock proteins Hsp27 and αB-crystallin. Vastus lateralis muscle biopsies from twelve healthy male volunteers were obtained before, immediately after, and 1 and 14 days after a 30 min downhill treadmill running exercise. Eccentric exercise induced muscle damage as evidenced by the analysis of muscle pain and weakness, creatine kinase serum activity, myoglobinaemia and ultrastructural analysis of muscle biopsies. The calpain 3 mRNA level was decreased immediately after exercise whereas calpain 2 mRNA level was increased at day 1. Both mRNA levels returned to control values by day 14. By contrast, cathepsin B+L and proteasome enzyme activities were increased at day 14. The α-sarcoglycan protein level was decreased immediately after exercise and at day 1, whereas the desmin level peaked at day 14. αB-crystallin and Hsp27 protein levels were increased at days 1 and 14. Our results suggest that the differential expression of calpain 2 and 3 mRNA levels may be important in the process of exercise-induced muscle damage, whereas expression of α-sarcoglycan, desmin, αB-crystallin and Hsp27 may be essentially involved in the subsequent remodelling of myofibrillar structure. This remodelling response may limit the extent of muscle damage upon a subsequent mechanical stress. PMID:12181300

Nucleus-specific expression in the multinuclear mushroom-forming fungus Agaricus bisporus reveals different nuclear regulatory programs.

PubMed

Gehrmann, Thies; Pelkmans, Jordi F; Ohm, Robin A; Vos, Aurin M; Sonnenberg, Anton S M; Baars, Johan J P; Wösten, Han A B; Reinders, Marcel J T; Abeel, Thomas

2018-04-24

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation. Copyright © 2018 the Author(s). Published by PNAS.

The IRE1/bZIP60 pathway and Bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants

USDA-ARS?s Scientific Manuscript database

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor and when activated it splices the bZIP60 mRNA producing a truncated transcription factor that upregulates expression of genes involved in the unfolded protein response (UPR). Bax inhibitor 1 (BI-1) is another ER stre...

Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase.

PubMed

Taniyama, Y; Shibata, S; Kita, S; Horikoshi, K; Fuse, H; Shirafuji, H; Sumino, Y; Fujino, M

1999-04-02

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in the esterification of plasma cholesterol and in the reverse cholesterol transport on high-density lipoprotein (HDL). We have found a novel LCAT-related gene among differentially expressed cDNA fragments between two types of foam cells derived from THP-1 cells, which are different in cholesterol efflux ability, using a subtractive PCR technique. The deduced 412-amino-acid sequence has 49% amino acid sequence similarity with human LCAT. In contrast to the liver-specific expression of LCAT, mRNA expression of the gene was observed mainly in peripheral tissues including kidney, placenta, pancreas, testis, spleen, heart, and skeletal muscle. The protein exists in human plasma and is probably associated with HDL. Moreover, we discovered that the recombinant protein hydrolyzed lysophosphatidylcholine (lysoPC), a proatherogenic lipid, to glycerophosphorylcholine and a free fatty acid. We have therefore named this novel enzyme LCAT-like lysophospholipase (LLPL), through which a new catabolic pathway for lysoPC on lipoproteins could be elucidated. Copyright 1999 Academic Press.

Effects of glutamine supplementation on oxidative stress-related gene expression and antioxidant properties in rats with streptozotocin-induced type 2 diabetes.

PubMed

Tsai, Pei-Hsuan; Liu, Jun-Jen; Yeh, Chui-Li; Chiu, Wan-Chun; Yeh, Sung-Ling

2012-04-01

There are close links among hyperglycaemia, oxidative stress and diabetic complications. Glutamine (GLN) is an amino acid with immunomodulatory properties. The present study investigated the effect of dietary GLN on oxidative stress-relative gene expressions and tissue oxidative damage in diabetes. There were one normal control (NC) and two diabetic groups in the present study. Diabetes was induced by an intraperitoneal injection of nicotinamide followed by streptozotocin (STZ). Rats in the NC group were fed a regular chow diet. In the two diabetic groups, one group (diabetes mellitus, DM) was fed a common semi-purified diet while the other group received a diet in which part of the casein was replaced by GLN (DM-GLN). GLN provided 25% of total amino acid N. The experimental groups were fed the respective diets for 8 weeks, and then the rats were killed for further analysis. The results showed that blood thioredoxin-interacting protein (Txnip) mRNA expression in the diabetic groups was higher than that in the NC group. Compared with the DM group, the DM-GLN group had lower glutamine fructose-6-phosphate transaminase 1, a receptor of advanced glycation end products, and Txnip gene expressions in blood mononuclear cells. The total antioxidant capacity was lower and antioxidant enzyme activities were altered by the diabetic condition. GLN supplementation increased antioxidant capacity and normalised antioxidant enzyme activities. Also, the renal nitrotyrosine level and Txnip mRNA expression were lower when GLN was administered. These results suggest that dietary GLN supplementation decreases oxidative stress-related gene expression, increases the antioxidant potential and may consequently attenuate renal oxidative damage in rats with STZ-induced diabetes.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

PubMed

Tuan, Pham Anh; Park, Sang Un

2013-01-01

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress. Copyright © 2012 Elsevier GmbH. All rights reserved.

Bovine ovarian follicular growth and development correlate with lysophosphatidic acid expression.

PubMed

Sinderewicz, Emilia; Grycmacher, Katarzyna; Boruszewska, Dorota; Kowalczyk-Zięba, Ilona; Staszkiewicz, Joanna; Ślężak, Tomasz; Woclawek-Potocka, Izabela

2018-01-15

The basis of successful reproduction is proper ovarian follicular growth and development. In addition to prostaglandins and vascular endothelial growth factor, a number of novel factors are suggested as important regulators of follicular growth and development: PGES, TFG, CD36, RABGAP1, DBI and BTC. This study focuses on examining the expression of these factors in granulosa and thecal cells that originate from different ovarian follicle types and their link with the expression of lysophosphatidic acid (LPA), known local regulator of reproductive functions in the cow. Ovarian follicles were divided into healthy, transitional, and atretic categories. The mRNA expression levels for PGES, TFG, CD36, RABGAP1, DBI and BTC in granulosa and thecal cells in different follicle types were measured by real-time PCR. The correlations among expression of enzymes synthesizing LPA (autotaxin, phospholipase A2), receptors for LPA and examined factors were measured. Immunolocalization of PGES, TFG, CD36, RABGAP1, DBI and BTC was examined by immunohistochemistry. We investigated follicle-type dependent mRNA expression of factors potentially involved in ovarian follicular growth and development, both in granulosa and thecal cells of bovine ovarian follicles. Strong correlations among receptors for LPA, enzymes synthesizing LPA, and the examined factors in healthy and transitional follicles were observed, with its strongest interconnection with TFG, DBI and RABGAP1 in granulosa cells, and TFG in thecal cells; whereas no correlations in atretic follicles were detected. A greater number of correlations were found in thecal cells than in granulosa cells as well as in healthy follicles than in transitional follicles. These data indicate the role of LPA in the growth, development and physiology of the bovine ovarian follicle. Copyright © 2017 Elsevier Inc. All rights reserved.

Studies on meat color, myoglobin content, enzyme activities, and genes associated with oxidative potential of pigs slaughtered at different growth stages

PubMed Central

Yu, Qin Ping; Feng, Ding Yuan; Xiao, Juan; Wu, Fan; He, Xiao Jun; Xia, Min Hao; Dong, Tao; Liu, Yi Hua; Tan, Hui Ze; Zou, Shi Geng; Zheng, Tao; Ou, Xian Hua; Zuo, Jian Jun

2017-01-01

Objective This experiment investigated meat color, myoglobin content, enzyme activities, and expression of genes associated with oxidative potential of pigs slaughtered at different growth stages. Methods Sixty 4-week-old Duroc×Landrace×Yorkshire pigs were assigned to 6 replicate groups, each containing 10 pigs. One pig from each group was sacrificed at day 35, 63, 98, and 161 to isolate longissimus dorsi and triceps muscles. Results Meat color scores were higher in pigs at 35 d than those at 63 d and 98 d (p<0.05), and those at 98 d were lower than those at 161 d (p<0.05). The total myoglobin was higher on 161 d compared with those at 63 d and 98 d (p<0.05). Increase in the proportions of metmyoglobin and deoxymyoglobin and a decrease in oxymyoglobin were observed between days 35 and 161 (p<0.05). Meat color scores were correlated to the proportion of oxymyoglobin (r = 0.59, p<0.01), and negatively correlated with deoxymyoglobin and metmyoglobin content (r = −0.48 and −0.62, p<0.05). Malate dehydrogenase (MDH) activity at 35 d and 98 d was higher than that at 161 d (p<0.05). The highest lactate dehydrogenase/MDH ratio was achieved at 161 d (p<0.05). Calcineurin mRNA expression decreased at 35 d compared to that at 63 d and 98 d (p<0.05). Myocyte enhancer factor 2 mRNA results indicated a higher expression at 161 d than that at 63 d and 98 d (p<0.05). Conclusion Porcine meat color, myoglobin content, enzyme activities, and genes associated with oxidative potential varied at different stages. PMID:28728400

Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia

PubMed Central

Altavilla, D; Minutoli, L; Polito, F; Irrera, N; Arena, S; Magno, C; Rinaldi, M; Burnett, BP; Squadrito, F; Bitto, A

2012-01-01

BACKGROUND AND PURPOSE Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5-lipoxygenase (5-LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid–based ‘dual inhibitor’ of the COX and 5-LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH. EXPERIMENTAL APPROACH Rats were treated daily with testosterone propionate (3 mg·kg−1 s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg−1, i.p.) or flavocoxid (20 mg·kg−1, i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells. KEY RESULTS Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE2 and leukotriene B4 (LTB4), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth. CONCLUSION AND IMPLICATIONS Our results show that a ‘dual inhibitor’ of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism. PMID:22471974

Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia.

PubMed

Altavilla, D; Minutoli, L; Polito, F; Irrera, N; Arena, S; Magno, C; Rinaldi, M; Burnett, B P; Squadrito, F; Bitto, A

2012-09-01

Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5-lipoxygenase (5-LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid-based 'dual inhibitor' of the COX and 5-LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH. Rats were treated daily with testosterone propionate (3 mg·kg(-1)  s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg(-1) , i.p.) or flavocoxid (20 mg·kg(-1) , i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells. Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE(2) and leukotriene B(4) (LTB(4) ), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth. Our results show that a 'dual inhibitor' of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Differential regulation of gene expression of neurotensin and prohormone convertases PC1 and PC2 in the bovine ocular ciliary epithelium: possible implications on neurotensin processing.

PubMed

Ortego, Javier; Wollmann, Guido; Coca-Prados, Miguel

2002-11-15

Prohormone convertases PC1 and PC2 are enzymes involved in the intracellular processing of pro-neurotensin/neuromedin N (pro-NT/NN) through the regulated secretory pathway. In this study, we present evidence of the differential gene expression of pro-NT/NN, pro-PC1 and pro-PC2 in two cell lines established from the neuroendocrine ocular ciliary epithelium. Dexamethasone and forskolin were found to synergistically up-regulate NT/NN mRNA expression in both cell types. The pigmented cells released NT, and this release was enhanced by agents that induced its biosynthesis. In contrast, nonpigmented cells exhibited a significantly reduced neurotensin secretion in response to inducers, leading to an accumulation of the peptide. PC1 and PC2 mRNA expression was induced in a cell-specific manner by the same agents that enhanced pro-NT/NN biosynthesis. These results demonstrate cell-specific processing of pro-NT/NN by the ciliary epithelium. Copyright 2002 Elsevier Science Ireland Ltd.

Quantifying Intrinsic and Extrinsic Variability in Stochastic Gene Expression Models

PubMed Central

Singh, Abhyudai; Soltani, Mohammad

2013-01-01

Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters. PMID:24391934

Quantifying intrinsic and extrinsic variability in stochastic gene expression models.

PubMed

Singh, Abhyudai; Soltani, Mohammad

2013-01-01

Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters.

Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae.

PubMed

Rhee, Jae-Sung; Kim, Bo-Mi; Kim, Ryeo-Ok; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

2013-09-15

To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4Gy of radiation, and biochemical and molecular damage became substantial from 8Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae. Copyright © 2013 Elsevier B.V. All rights reserved.

Up-regulated expression of cartilage intermediate-layer protein and ANK in articular hyaline cartilage from patients with calcium pyrophosphate dihydrate crystal deposition disease.

PubMed

Hirose, Jun; Ryan, Lawrence M; Masuda, Ikuko

2002-12-01

Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals. Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor beta1 (TGFbeta1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase-polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage. Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes or cartilage extracts. Both CILP and ANK mRNA expression and ePPi elaboration were stimulated by TGFbeta1 and inhibited by IGF-1 in chondrocytes from all sources. CILP and ANK mRNA expression correlates with chondrocyte ePPi accumulation around CPPD and OA chondrocytes, and all respond similarly to growth factor stimulation. These findings suggest that up-regulated CILP and ANK expression contributes to higher ePPi accumulation from CPPD crystal-forming cartilage.

Characterization of the cDNA coding for rat brain cysteine sulfinate decarboxylase: brain and liver enzymes are identical proteins encoded by two distinct mRNAs.

PubMed

Tappaz, M; Bitoun, M; Reymond, I; Sergeant, A

1999-09-01

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.

Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes.

PubMed Central

Han, Qian; Fang, Jianmin; Ding, Haizhen; Johnson, Jody K; Christensen, Bruce M; Li, Jianyong

2002-01-01

This study describes the identification of Drosophila yellow-f and yellow-f2 as dopachrome-conversion enzymes responsible for catalysing the conversion of dopachrome into 5,6-dihydroxyindole in the melanization pathway. Drosophila yellow -y gene and yellow -b, -c, -f and -f2 genes were expressed in an insect cell/baculovirus expression system and their corresponding recombinant proteins were screened for dopachrome-conversion enzyme activity. Among the yellow and yellow -related genes, the yellow -f and yellow -f2 genes were identified as the genes coding for Drosophila dopachrome-conversion enzyme based on the high activity of their recombinant proteins in catalysing the production of 5,6-dihydroxyindole from dopachrome. Both yellow-f and yellow-f2 are capable of mediating a decarboxylative structural rearrangement of dopachrome, as well as an isomerization/tautomerization of dopamine chrome and dopa methyl ester chrome. Northern hybridization revealed the transcription of yellow -f in larvae and pupae, but a high abundance of mRNA was observed in later larval and early pupal stages. In contrast, yellow-f2 transcripts were present at all stages, but high abundance of its mRNA was observed in later-stage pupae and adults. These data indicate that yellow-f and yellow-f2 complement each other during Drosophila development and that the yellow-f is involved in larval and pupal melanization, and yellow-f2 plays a major role in melanization reactions in Drosophila during later pupal and adult development. Results from this study provide the groundwork towards a better understanding of the physiological roles of the Drosophila yellow gene family. PMID:12164780

Loss of luteotropic prostaglandin E plays an important role in the regulation of luteolysis in women.

PubMed

Nio-Kobayashi, Junko; Kudo, Masataka; Sakuragi, Noriaki; Iwanaga, Toshihiko; Duncan, W Colin

2017-05-01

Do intraluteal prostaglandins (PG) contribute to luteal regulation in women? Prostaglandin E (PGE), which is produced in human granulosa-lutein cells stimulated with luteotropic hCG, exerts similar luteotropic effects to hCG, and the expression of PG synthetic and metabolic enzymes in the human CL is driven toward less PGE but more prostaglandin F (PGF) during luteolysis. Uterine PGF is a major luteolysin in many non-primate species but not in women. Increases in the PGF synthase, aldo-ketoreductase family one member C3 (AKR1C3), have been observed in the CL of marmoset monkeys during luteolysis. PGE prevents spontaneous or induced luteolysis in domestic animals. Human CL tissues staged as the early-luteal (n = 6), mid-luteal (n = 6), late-luteal (n = 5) and menstrual (n = 3) phases were obtained at the time of hysterectomy for benign gynecological conditions. Luteinized granulosa cells (LGCs) were purified from follicular fluids obtained from patients undergoing assisted conception. Upon collection, one half of the CL was snap-frozen and the other was fixed with formalin and processed for immunohistochemical analysis of a PGE synthase (PTGES). Quantitative RT-PCR was employed to examine changes in the mRNA abundance of PG synthetic and metabolic enzymes, steroidogenic enzymes, and luteolytic molecules in the staged human CL and in human LGCs in vitro treated with hCG, PGE and PGF. A PGE withdrawal experiment was also conducted in order to reveal the effects of the loss of PGE in LGCs. Progesterone concentrations in the culture medium were measured. The key enzyme for PGE synthesis, PTGES mRNA was abundant in the functional CL during the mid-luteal phase (P < 0.01), while mRNA abundance for genes involved in PGF synthesis (AKR1B1 and AKR1C1-3) increased in the CL during the late-luteal phase and menstruation (P < 0.05-0.001). PTGES mRNA expression positively correlated with that of 3β-hydroxysteroid dehydrogenase (HSD3B1; r = 0.7836, P < 0.001), while AKR1C3 expression inversely correlated with that of HSD3B1 (r = -0.7514, P = 0.0012) and PTGES (r = -0.6923, P = 0.0042). PGE exerted similar effects to hCG-promoting genes, such as steroidogenic acute regulatory protein (STAR) and HSD3B1, to produce progesterone and luteotropic PGE, suppress PGF synthetic enzymes and down-regulate luteolytic molecules such as βA- and βB-inhibin subunits (INHBA and INHBB) and bone morphogenetic proteins (BMP2, BMP4 and BMP6). PGE withdrawal resulted in reductions in the enzymes that produce progesterone (STAR; P < 0.001) and PGE (PTGES; P < 0.001), and the capacity to produce PGE decreased, while the capacity to produce PGF increased during the culture. The addition of PGF did not recapitulate the luteolytic effects of PGE withdrawal. None. Changes in mRNA expression of PG synthetic and metabolic enzymes may not represent actual increases in PGF during luteolysis in the CL. The effects of PGF on luteal cells currently remain unclear and the mechanisms responsible for decreases in the synthesis of PGE in vitro and at luteolysis have not been elucidated in detail. The results obtained strongly support a luteotropic function of PGE in regulation of the human CL. They suggest that the main PG produced in human luteal tissue changes from PGE to PGF during the maturation and regression of the CL, and the loss of PGE is more important than the effects of PGF during luteolysis in women. This may be accompanied by reduced effects of LH/hCG in luteal cells, particularly decreased activation of cAMP/protein kinase A; however, the underlying mechanisms remain unknown. This study was supported by the Cunningham Trust to WCD, a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science and the Suntory Foundation for Life Sciences to J.N.-K.; W.C.D. is supported by an MRC Centre Grant G1002033 and a Scottish Senior Clinical Fellowship. The authors have nothing to disclose. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Upregulation of genes encoding digestive enzymes and nutrient transporters in the digestive system of broiler chickens by dietary supplementation of fiber and inclusion of coarse particle size corn.

PubMed

Kheravii, Sarbast K; Swick, Robert A; Choct, Mingan; Wu, Shu-Biao

2018-03-20

Measures to improve bird performance have been sought due to the imminent phase out of in-feed antibiotics in poultry and continued demand for higher poultry feeding efficiency. Increasing grain particle size and dietary fibre may improve gizzard function, digestive efficiency and nutrient absorption. This study was conducted to evaluate the effect increased particle size of corn and inclusion of sugarcane bagasse (SB) on mRNA expression of genes encoding digestive enzymes and nutrient transporters in broilers. A total of 336 day-old Ross 308 males were assigned in a 2 × 2 factorial arrangement of treatments with corn particle size - coarse 3576 μm or fine 1113 μm geometric mean diameter, and SB - 0 or 2% inclusion. Feed conversion ratio (FCR), weight gain and feed intake were measured from d 0-10 and d 10-24. The relative gizzard weight and mRNA expression of genes encoding digestive enzymes and intestinal nutrient transporters were measured on d 24. During d 10-24, a particle size × SB interaction was observed for FCR (P < 0.01), where birds fed coarsely ground corn (CC) with 2% SB had lower FCR than those fed CC without SB. A particle size × SB interaction was observed for both expression of pepsinogen A and C (P < 0.01) which were negatively correlated with FCR on d 24. Addition of 2% SB upregulated pepsinogen A and C only in CC fed birds. Further, 2% SB also upregulated pancreatic amylase (AMY2A) and intestinal cationic amino acid transporter-1 (CAT1). Inclusion of dietary CC upregulated duodenal amino peptidase N (APN), jejunal alanine, serine, cysteine and threonine transporter-1 (ASCT1), and ileal peptide transporter-2 (PepT2). These results suggest that both SB and coarse particle size modulate expression of genes encoding important digestive enzymes and nutrient transporters and thus are directly related to bird performance. These findings provide insights into the combination effects of dietary fiber and particle size in the future management of broiler feeding.

Effects of Carbenoxolone on the Canine Pituitary-Adrenal Axis.

PubMed

Teshima, Takahiro; Matsumoto, Hirotaka; Okusa, Tomoko; Nakamura, Yumi; Koyama, Hidekazu

2015-01-01

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to suppressive negative feedback by glucocorticoids. The abnormal expression of 11beta-hydroxysteroid dehydrogenase (11HSD), which is a cortisol metabolic enzyme, is found in human and murine corticotroph adenomas. Our recent studies demonstrated that canine corticotroph adenomas also have abnormal expression of 11HSD. 11HSD has two isoforms in dogs, 11HSD type1 (HSD11B1), which converts cortisone into active cortisol, and 11HSD type2 (HSD11B2), which converts cortisol into inactive cortisone. It has been suggested that glucocorticoid resistance in corticotroph tumors is related to the overexpression of HSD11B2. Therefore it was our aim to investigate the effects of carbenoxolone (CBX), an 11HSD inhibitor, on the healthy dog's pituitary-adrenal axis. Dogs were administered 50 mg/kg of CBX twice each day for 15 days. During CBX administration, no adverse effects were observed in any dogs. The plasma adrenocorticotropic hormone (ACTH), and serum cortisol and cortisone concentrations were significantly lower at day 7 and 15 following corticotropin releasing hormone stimulation. After completion of CBX administration, the HSD11B1 mRNA expression was higher, and HSD11B2 mRNA expression was significantly lower in the pituitaries. Moreover, proopiomelanocortin mRNA expression was lower, and the ratio of ACTH-positive cells in the anterior pituitary was also significantly lower after CBX treatment. In adrenal glands treated with CBX, HSD11B1 and HSD11B2 mRNA expression were both lower compared to normal canine adrenal glands. The results of this study suggested that CBX inhibits ACTH secretion from pituitary due to altered 11HSD expressions, and is potentially useful for the treatment of canine Cushing's disease.

7-Ketocholesterol inhibits isocitrate dehydrogenase 2 expression and impairs endothelial function via microRNA-144.

PubMed

Fu, Xiaodong; Huang, Xiuwei; Li, Ping; Chen, Weiyu; Xia, Min

2014-06-01

Oxysterol is associated with the induction of endothelial oxidative stress and impaired endothelial function. Mitochondria play a central role in oxidative energy metabolism and the maintenance of proper redox status. The purpose of this study was to determine the effects and mechanisms of 7-ketocholesterol (7-KC) on isocitrate dehydrogenase 2 (IDH2) and its impact on endothelial function in both human aortic endothelial cells (HAECs) and C57BL/6J mice. HAECs treated with 7-KC showed significant reductions of IDH2 mRNA and protein levels and enzyme activity, leading to decreased NADPH concentration and an increased ratio of reduced-to-oxidized glutathione in the mitochondria. 7-KC induced the expression of a specific microRNA, miR-144, which in turn targets and downregulates IDH2. In silico analysis predicted that miR-144 could bind to the 3'-untranslated region of IDH2 mRNA. Overexpression of miR-144 decreased the expression of IDH2 and the levels of NADPH. A complementary finding is that a miR-144 inhibitor increased the mRNA and protein expression levels of IDH2. Furthermore, miR-144 level was elevated in HAECs in response to 7-KC. Anti-Ago1/2 immunoprecipitation coupled with a real-time polymerase chain reaction assay revealed that 7-KC increased the functional targeting of miR-144/IDH2 mRNA in HAECs. Infusion of 7-KC in vivo decreased vascular IDH2 expression and impaired vascular reactivity via miR-144. 7-KC controls miR-144 expression, which in turn decreases IDH2 expression and attenuates NO bioavailability to impair endothelial homeostasis. The newly identified 7-KC-miR-144-IDH2 pathway may contribute to atherosclerosis progression and provides new insight into 7-KC function and microRNA biology in cardiovascular disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Anti-proliferative and apoptotic effects of the novel taspine derivative tas41 in the Caco-2 cell line.

PubMed

Zhang, Yanmin; Zhang, Jie; Dai, Bingling; Wang, Nan; He, Langchong

2011-05-01

Taspine was screened and isolated for the first time from Radix et Rhizoma Leonticis. Tas41 is a novel taspine derivative. We investigated the effects of tas41 on proliferation of the Caco-2 cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a fluorescence-activated cell sorter (FACS), enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (WB). Changes in the cell cycle, apoptosis, activation of caspase-3, caspase-8 and caspase-9, and expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) were investigated after Caco-2 cells were treated with tas41. At the same time, expressions of apoptosis protein bcl-2 and bax were determined. Tas41 was found to induce apoptosis in a concentration-dependent manner as confirmed by DNA fragmentation analysis, TUNEL assay and flow cytometry. Protein and mRNA expressions of EGF, VEGF, CDK2, bcl-2 and bax were evaluated by ELISA, WB and RT-PCR. Tas41 had a better anti-proliferative effect than taspine on Caco-2 cells. A DNA ladder and apoptosis was observed, and the increased apoptotic activity by tas41 was accompanied by a decrease in the expression of VEGF protein and mRNA. The activities of caspase-3, caspase-8 and caspase-9 were significantly increased in cells treated with tas41 compared with those in the control group. In addition, protein and mRNA expressions of bcl-2 were decreased, and protein and mRNA expressions of bax were increased. These findings demonstrate that tas41 can inhibit the proliferation of, and induce apoptosis in, Caco-2 cells by activating caspase-3, caspase-8 and caspase-9, downregulating the expressions of VEGF, upregulating the ratio of bax/bcl-2. Copyright © 2011 Elsevier B.V. All rights reserved.

γ-Amino-butyric acid (GABA) receptor subunit and transporter expression in the gonad and liver of the fathead minnow (Pimephales promelas).

PubMed

Biggs, Katie; Seidel, Jason S; Wilson, Alex; Martyniuk, Christopher J

2013-09-01

γ-Amino-butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate central nervous system. GABA receptors and synthesizing enzymes have also been localized to peripheral tissues including the liver, oviduct, uterus and ovary of mammals but the distribution and role of GABA in peripheral tissues of fish has not been fully investigated. The objectives of this study were to (1) determine if mRNA encoding GABA synthesizing enzymes (glutamic acid decarboxylase 65 and 67; gad65 and gad67), GABA transporters, and GABAA receptor subunits are localized to liver and gonad of fathead minnow (Pimephales promelas) (FHM) (2) investigate the effects of GABA on ovarian 17β-estradiol (E2) production, and (3) measure transcript responses in the ovary after in vitro incubation to GABA. Real-time PCR assays were developed for gad65, gad67, vesicular GABA transporter (vgat) and GABA transporter 1 (gat1), and select GABAA receptor subunits (gabra1, gabra5, gabrb1, gabrb2, gabrg1, gabrg2). All transcripts were localized to the brain as expected; however transcripts were also detected in the liver, ovary, and testis of FHMs. In the female liver, gad65 mRNA was significantly higher in expression compared to the male liver. Transcripts for gad67 were the highest in the brain>gonad>liver and in the gonads, gad67 was significantly higher in expression than gad65 mRNA. In the liver and gonad, the relative abundance of the subunits followed a general trend of gabrb1>gabrb2=gabrg1=gabrg2>gabra1=gabra5. To explore the effects of GABA in the ovary, tissue explants from reproductive female FHMs were treated with GABA (10(-10), 10(-8) and 10(-6)M) for 12h. GABA had no significant effect on 17β-estradiol production or on mRNA abundance for genes involved in ovarian steroidogenesis (e.g., 11βhsd, cyp17, cyp19a). There was a significant decrease in estrogen receptor 2a (esr2a) mRNA with 10(-10)M GABA. This study begins to investigate the GABA system in non-neural tissues of teleost fish and addresses the broader topic regarding the peripheral roles of neurotransmitters. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of Remote Ischemic Preconditioning on Heme Oxygenase-1 Expression and Cutaneous Wound Repair

PubMed Central

Cremers, Niels A. J.; Wever, Kimberley E.; Wong, Ronald J.; van Rheden, René E. M.; Vermeij, Eline A.; van Dam, Gooitzen M.; Carels, Carine E.; Lundvig, Ditte M. S.; Wagener, Frank A. D. T. G.

2017-01-01

Skin wounds may lead to scar formation and impaired functionality. Remote ischemic preconditioning (RIPC) can induce the anti-inflammatory enzyme heme oxygenase-1 (HO-1) and protect against tissue injury. We aim to improve cutaneous wound repair by RIPC treatment via induction of HO-1. RIPC was applied to HO-1-luc transgenic mice and HO-1 promoter activity and mRNA expression in skin and several other organs were determined in real-time. In parallel, RIPC was applied directly or 24h prior to excisional wounding in mice to investigate the early and late protective effects of RIPC on cutaneous wound repair, respectively. HO-1 promoter activity was significantly induced on the dorsal side and locally in the kidneys following RIPC treatment. Next, we investigated the origin of this RIPC-induced HO-1 promoter activity and demonstrated increased mRNA in the ligated muscle, heart and kidneys, but not in the skin. RIPC did not change HO-1 mRNA and protein levels in the wound 7 days after cutaneous injury. Both early and late RIPC did not accelerate wound closure nor affect collagen deposition. RIPC induces HO-1 expression in several organs, but not the skin, and did not improve excisional wound repair, suggesting that the skin is insensitive to RIPC-mediated protection. PMID:28218659

Expression and Function of Chemokines CXCL9-11 in Micturition Pathways in Cyclophosphamide (CYP)-Induced Cystitis and Somatic Sensitivity in Mice

PubMed Central

Guo, Michael; Chang, Phat; Hauke, Eric; Girard, Beatrice M.; Tooke, Katharine; Ojala, Jacqueline; Malley, Susan M.; Hsiang, Harrison; Vizzard, Margaret A.

2018-01-01

Changes in urinary bladder function and somatic sensation may be mediated, in part, by inflammatory changes in the urinary bladder including the expression of chemokines. Male and female C57BL/6 mice were treated with cyclophosphamide (CYP; 75 mg/kg, 200 mg/kg, i.p.) to induce bladder inflammation (4 h, 48 h, chronic). We characterized the expression of CXC chemokines (CXCL9, CXCL10 and CXCL11) in the urinary bladder and determined the effects of blockade of their common receptor, CXCR3, at the level urinary bladder on bladder function and somatic (hindpaw and pelvic) sensation. qRT-PCR and Enzyme-Linked Immunoassays (ELISAs) were used to determine mRNA and protein expression of CXCL9, CXCL10 and CXCL11 in urothelium and detrusor. In urothelium of female mice treated with CYP, CXCL9 and CXCL10 mRNA significantly (p ≤ 0.01) increased with CYP treatment whereas CXC mRNA expression in the detrusor exhibited both increases and decreases in expression with CYP treatment. CXC mRNA expression urothelium and detrusor of male mice was more variable with both significant (p ≤ 0.01) increases and decreases in expression depending on the specific CXC chemokine and CYP treatment. CXCL9 and CXCL10 protein expression was significantly (p ≤ 0.01) increased in the urinary bladder with 4 h CYP treatment in female mice whereas CXC protein expression in the urinary bladder of male mice did not exhibit an overall change in expression. CXCR3 blockade with intravesical instillation of AMG487 (5 mg/kg) significantly (p ≤ 0.01) increased bladder capacity, reduced voiding frequency and reduced non-voiding contractions in female mice treated with CYP (4 h, 48 h). CXCR3 blockade also reduced (p ≤ 0.01) hindpaw and pelvic sensitivity in female mice treated with CYP (4 h, 48 h). CXC chemokines may be novel targets for treating urinary bladder dysfunction and somatic sensitization resulting from urinary bladder inflammation. PMID:29681802

From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes

PubMed Central

Corcoran, Callan C.; Grady, Cameron R.; Pisitkun, Trairak; Parulekar, Jaya

2017-01-01

The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. PMID:27974320

Shikonin, an ingredient of Lithospermum erythrorhizon, down-regulates the expression of steroid sulfatase genes in breast cancer cells.

PubMed

Zhang, Yi; Qian, Rui-Qin; Li, Ping-Ping

2009-10-18

Steroid sulfatase (STS) has an important role in regulating the biosynthesis of estrogen within breast tumors. We aimed to investigate whether shikonin, an ingredient of Lithospermum erythrorhizon, could modulate STS expression in breast cancer cells. By MTT assay, shikonin inhibited the cell proliferation of breast cancer cells MCF-7 and SK-BR-3. Moreover, by semi-quantitative/quantitative reverse transcription polymerase chain reaction and dual-luciferase reporter based bioluminescent measurements, the mRNA and enzymatic activity levels of STS were decreased after shikonin treatment. Concluding, shikonin could act as a selective estrogen enzyme modulator by down-regulating the STS expression.

Expression of Ras-related C3 botulinum toxin substrate 1 (RAC1) in human cholesteatoma.

PubMed

Lee, No Hee; Chang, Ji-Won; Choi, June; Jung, Hak Hyun; Im, Gi Jung

2013-02-01

Ras-related C3 botulinum toxin substrate 1 (RAC1) is a 21-kDa signaling G protein that functions as a pleiotropic regulator of many cellular processes including epithelial differentiation. RAC1 activates the nicotinamide adenine dinucleotide phosphate oxidase complex which promotes formation of reactive oxygen species and degradation enzymes. RAC1 has been associated with rapid epithelial differentiation and invasive properties in human cholesteatoma. This study aimed to identify the presence of RAC1 in human cholesteatoma and analyze its functional role as a regulator of proteolysis and overgrowth. Tissue samples from human cholesteatoma and normal postaural skin were obtained from patients during otologic surgery for cholesteatoma. The expression of RAC1 mRNA was quantified by real-time RT-PCR, and localization of RAC1 expression was confirmed using immunohistochemical staining. Expression of RAC1 mRNA in the epithelium of cholesteatoma was significantly elevated 2.94 fold on average, compared with normal control skin. RAC1 expression in the suprabasal and basal layer of cholesteatoma epithelium was stronger than normal control skin. Our results suggest that RAC1 can be associated with rapid epithelial differentiation and invasive properties of human cholesteatoma.

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

PubMed

Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

2016-06-12

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants.

PubMed

Yang, Qingjie; Yuan, Dawei; Shi, Lianxuan; Capell, Teresa; Bai, Chao; Wen, Nuan; Lu, Xiaodan; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2012-10-01

The accumulation of carotenoids in plants depends critically on the spatiotemporal expression profiles of the genes encoding enzymes in the carotenogenic pathway. We cloned and characterized the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter to determine its role in the regulation of carotenogenesis, because the native gene is expressed at high levels in petals, which contain abundant chromoplasts. We transformed tomato (Solanum lycopersicum cv. Micro-Tom) plants with the gusA gene encoding the reporter enzyme β-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated the reporter expression profile at the mRNA and protein levels. We detected high levels of gusA expression and GUS activity in chromoplast-containing flowers and fruits, but minimal levels in immature fruits containing green chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictly associated with fruit development and chromoplast differentiation, suggesting an evolutionarily-conserved link between ZEP and the differentiation of organelles that store carotenoid pigments. The impact of our results on current models for the regulation of carotenogenesis in plants is discussed.

Effects of triclosan (TCS) on hormonal balance and genes of hypothalamus-pituitary- gonad axis of juvenile male Yellow River carp (Cyprinus carpio).

PubMed

Wang, Fan; Liu, Fei; Chen, Wanguang; Xu, Ruijie; Wang, Wei

2018-02-01

Triclosan (TCS) is a broad spectrum antimicrobial agent which has been widely dispersed and determinated in the aquatic environment. However, the effects of TCS on reproductive endocrine in male fish are poorly understood. In this study, male Yellow River carp (Cyprinus carpio) were exposed to 0, 1/5, 1/10 and 1/20 LC 50 (96 h LC 50 of TCS to carp) TCS under semi-static conditions for 42 d. Vitellogenin (Vtg), 17β-estradiol (E 2 ), testosterone(T), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-β, GnRH, estrogen receptor (Er), and androgen receptor (Ar) by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). TCS induced Vtg levels of hepatopancreas, E 2 levels of serum, and inhibited Ar and Er mRNA levels, suggesting that the induction of Vtg production by TCS was indirectly caused by non-Er pathways. TCS-induced Vtg levels by interfering with the reproductive axis at plenty of latent loci of male carps: (a) TCS exposure increased the aromatase mRNA expression of hypothalamus and gonad aromatase, consequently increasing serum concentrations of E 2 to induce Vtg in hepatopancreas; (b) TCS treatment changed GtH-β and GnRH mRNA expression and secretion, causing the disturbance of reproductive endocrine; (c) TCS exposure decreased Ar mRNA levels, indicating potential Ar-mediated antiandrogen action. These mechanisms showed that TCS may induce Vtg production in male carp by non-Er-mediated pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protective effect of [6]-gingerol on the ethanol-induced teratogenesis of cultured mouse embryos.

PubMed

Yon, Jung-Min; Baek, In-Jeoung; Lee, Se-Ra; Kim, Mi-Ra; Hong, Jin Tae; Yong, Hwanyul; Lee, Beom Jun; Yun, Young Won; Nam, Sang-Yoon

2012-01-01

Excessive ethanol consumption during pregnancy causes fetal alcohol syndrome. We investigated the effect of [6]-gingerol on ethanol-induced embryotoxicity using a whole embryo culture system. The morphological changes of embryos and the gene expression patterns of the antioxidant enzymes cytosolic glutathione peroxidase (cGPx), cytoplasmic Cu/Zn superoxide dismutase (SOD1), and Mn-SOD (SOD2), and SOD activity were examined in the cultured mouse embryos exposed to ethanol (5 μL/3 mL) and/or [6]-gingerol (1×10(-8) or 1×10(-7) μg/mL) for 2 days. In ethanol-exposed embryos, the standard morphological score of embryos was significantly decreased compared with those of the control (vehicle) group. However, cotreatment of embryos with [6]-gingerol and ethanol significantly improved all of the developmental parameters except crownrump length and head length, compared with those of the ethanol alone group. The mRNA expression levels of cGPx and SOD2, not SOD1, were decreased consistently, SOD activity were significantly decreased compared with the control group. However, the decreases in mRNA levels of antioxidant enzymes and SOD activity were significantly restored to the control levels by [6]-gingerol supplement. These results indicate that [6]-gingerol has a protective effect against ethanol-induced teratogenicity during mouse embryogenesis.

DD-RT-PCR identifies 7-dehydrocholesterol reductase as a key marker of early Leydig cell steroidogenesis.

PubMed

Anbalagan, M; Yashwanth, R; Jagannadha Rao, A

2004-04-30

Postnatal Leydig cell development in rat involves an initial phase of proliferation of progenitor Leydig cells (PLCs) and subsequent differentiation of these cells into immature Leydig cells (ILCs) and adult Leydig cells (ALCs). With an objective to identify the molecular changes associated with Leydig cell differentiation, the mRNA population in PLCs and ILCs were analyzed by the technique of differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed differential expression of several transcripts in PLCs and ILCs. Of the several differentially expressed transcripts, the expression of transcripts corresponding to collagen IV alpha6 (Col IV alpha6) and ribosomal protein L 41 (RpL41) decreased during the differentiation of PLC to ILC. Also there was an increase in the expression of transcripts encoding enzymes such as microsomal glutathione-S-transferase (mGST 1) and 7-dehydrocholesterol reductase (7-DHCR) during this process. While Col IV alpha6 and RpL41 are known to be involved in cellular proliferation, mGST 1 and 7-DHCR are essential for normal Leydig cell steroidogenesis. A detailed study on 7-DHCR expression in Leydig cells revealed that this enzyme plays a crucial role in steroidogenesis. Interestingly expression of this enzyme is not under acute regulation by Luteinizing hormone (LH). Copyright 2004 Elsevier Ireland Ltd.

Bisphenol S induces obesogenic effects through deregulating lipid metabolism in zebrafish (Danio rerio) larvae.

PubMed

Wang, Weiwei; Zhang, Xiaona; Wang, Zihao; Qin, Jingyu; Wang, Wei; Tian, Hua; Ru, Shaoguo

2018-05-01

It has been suggested that dramatic increase in obesity may be caused by growing exposure to environmental chemicals. In vitro data has suggested bisphenol S (BPS), a compound widely used in polycarbonate plastic production, can induce lipid accumulation in preadipocytes. However, the mechanisms responsible for BPS-induced obesity in vivo remain unclear. In this study, we used translucent zebrafish (Danio rerio) larvae as a model to investigate the effect of environmentally relevant BPS exposure (1, 10, and 100 μg/L from 2 h to 15 d post fertilization) on lipid accumulation, triacylglycerol (TAG) and lipoproteins content, and mRNA expression of genes involved in the regulation of lipid synthesis, transport, degradation, and storage. We also analyzed activities of two enzymes critical to TAG metabolism: lipoprotein lipase and diglyceride acyltransferase. Overfed, obese larvae were used as positive control. The results indicated that BPS-treated and overfed larvae had much higher TAG levels and visceral fat accumulation compared with control. BPS exhibited obesogenic effects by interfering with lipid metabolism as evidenced by (a) upregulation of the mRNA expression of fasn, acc1, and agpat4 genes encoding enzymes involved in the de novo synthesis of TAG in the liver, (b) downregulation of apolipoprotein expression, which should reduce TAG transport from the liver, and (c) increase in rxrα expression, which should promote visceral fat accumulation. Our study is the first to demonstrate that the obesogenic effects of BPS in zebrafish are related to the disruption of TAG metabolism. Copyright © 2018 Elsevier Ltd. All rights reserved.

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

PubMed

Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

2016-01-01

Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

The role of fructose‑1,6‑bisphosphatase 1 in abnormal development of ovarian follicles caused by high testosterone concentration.

PubMed

Liu, Tao; Zhao, Han; Wang, Jianfeng; Shu, Xin; Gao, Yuan; Mu, Xiaoli; Gao, Fei; Liu, Hongbin

2017-11-01

The present study aimed to identify the molecular mechanisms underlying the effects of the fructose‑1,6‑bisphosphatase 1 (FBP1) signaling pathway within normal follicle development and in hyperandrogenism‑induced abnormal follicle growth. To achieve this, murine primary follicles, granulosa cells (GCs) and theca‑interstitial cells (TICs) were isolated, cultured in vitro and treated with a high concentration of androgens. A concentration of 1x10‑5 mol/l testosterone was considerable to induce hyperandrogenism by MTT assay. All cells were divided into four groups, as follows: Control group, testosterone group, androgen receptor antagonist‑flutamide group and flutamide + testosterone group. Flutamide was used in the present study as it blocks the effects of the androgen receptor. The mRNA expression levels of FBP1 were detected using reverse transcription‑quantitative polymerase chain reaction. The expression levels and localization of FBP1 were analyzed by western blot analysis and immunofluorescence staining. The experimental results demonstrated that androgen presence stimulated follicle development, whereas excessive testosterone inhibited development. FBP1 was identified as being mainly expressed in follicles; FBP1 protein was significantly expressed in GCs of the 14‑day‑cultured follicle, as well as in the cytoplasm and nuclei of GCs and TICs in vitro. Testosterone increased FBP1 expression during a specific range of testosterone concentrations. Testosterone increased the expression of FBP1 within GCs. Furthermore, FBP1 and phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression was increased in GCs treated with testosterone, whereas forkhead box protein O1 (FOXO1) and peroxisome proliferator‑activated receptor γ coactivator‑1α mRNA expression was significantly decreased in the testosterone group. In TICs, testosterone and flutamide inhibited the mRNA expression levels of FOXO1 and glucose‑6‑phosphatase enzyme, and promoted the expression of PCK1. These results suggested that the FBP1 signaling pathway may serve an important role in normal follicle growth and hyperandrogenism‑induced abnormal development, which may be associated with abnormal glucose metabolism induced by high concentrations of testosterone.

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

PubMed

Mishina, Kei; Shinkai, Masaharu; Shimokawaji, Tadasuke; Nagashima, Akimichi; Hashimoto, Yusuke; Inoue, Yoriko; Inayama, Yoshiaki; Rubin, Bruce K; Ishigatsubo, Yoshiaki; Kaneko, Takeshi

2015-12-01

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 μM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.