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Sample records for abca1 mrna levels

  1. An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations

    SciTech Connect

    Sorrenson, Brie; Suetani, Rachel J.; Bickley, Vivienne M.; George, Peter M.; Williams, Michael J.A.; Scott, Russell S.; McCormick, Sally P.A.

    2011-06-10

    Highlights: {yields} Characterisation of an ABCA1 truncation mutant, C978fsX988, in a pedigree with three ABCA1 mutations. {yields} Functional analysis of C978fsX988 in patient fibroblasts and HEK 293 cells shows no cholesterol efflux function. {yields} Allele-specific quantification shows C978fsX988 not expressed at mRNA level in fibroblasts. {yields} Unlike other ABCA1 truncations, C978fsX988 mutant shows no dominant negative effect at mRNA or protein level. -- Abstract: The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.

  2. Differential Regulation of ABCA1 and Macrophage Cholesterol Efflux By Elaidic and Oleic Acids

    PubMed Central

    Shao, Fei; Ford, David A.

    2013-01-01

    Trans fatty acid consumption is associated with an increased risk of coronary heart disease. This increased risk has been attributed to decreased levels of HDL cholesterol and increased levels of LDL cholesterol. However, the mechanism by which trans fatty acid modulates cholesterol transit remains poorly defined. ATP-binding cassette transporter A1 (ABCA1)-mediated macrophage cholesterol efflux is the rate-limiting step initiating apolipoprotein A-I lipidation. In this study, elaidic acid, the most abundant trans fatty acid in partially hydrogenated vegetable oil, was shown to stabilize macrophage ABCA1 protein levels in comparison to that of its cis fatty acid isomer, oleic acid. The mechanism responsible for the disparate effects of oleic and elaidic acid on ABCA1 levels was through accelerated ABCA1 protein degradation in cells treated with oleic acid. In contrast, no apparent differences were observed in ABCA1 mRNA levels, and only minor changes were observed in Liver X receptor/Retinoic X receptor promoter activity in cells treated with elaidic and oleic acid. Efflux of both tracers and cholesterol mass revealed that elaidic acid slightly increased ABCA1-mediated cholesterol efflux, while oleic acid led to decreased ABCA1-mediated efflux. In conclusion, these studies sho that cis and trans structural differences in eighteen carbon n-9 monoenoic fatty acids variably impact cholesterol efflux through disparate effects on ABCA1 protein degradation. PMID:23800855

  3. Dietary High Cholesterol and Trace Metals in the Drinking Water Increase Levels of ABCA1 in the Rabbit Hippocampus and Temporal Cortex

    PubMed Central

    Schreurs, Bernard G.; Sparks, D. Larry

    2015-01-01

    Background Cholesterol-fed rabbits have been documented to show increased amyloid-β (Aβ) deposits in the brain that can be exacerbated by the quality of drinking water especially if rabbits drink tap water or distilled water containing copper. One mechanism of cholesterol and Aβ clearance may be through the ATP-binding cassette transporter A1 (ABCA1). Objective and Methods Using an ABCA1 antibody, we determined the number of ABCA1-immunopositive neurons in three areas of rabbit brain as a function of feeding 2% cholesterol and providing tap water, distilled water, or distilled water to which aluminum, copper, or zinc was added. Results The number of neurons with ABCA1 immunoreactivity was increased significantly as a result of dietary cholesterol in the rabbit hippocampus and inferior and superior temporal cortex. The number of neurons with ABCA1 immunoreactivity was further increased in all three areas as a result of cholesterol-fed rabbits drinking tap water or distilled water with copper. Finally, cholesterol-fed rabbits that drank distilled water with aluminum also showed an increased number of ABCA1-immunopositive neurons in inferior and superior temporal cortex. Conclusions These data suggest that ABCA1 levels increase in parallel with previously documented increases in Aβ levels as a result of high dietary cholesterol and copper in the drinking water. Addition of aluminum to distilled water may have a similar effect in the temporal cortex. ABCA1 has been proposed as a means of clearing Aβ from the brain and manipulations that increase Aβ also result in an increase of clearance machinery. PMID:26444796

  4. Adaptations of placental and cord blood ABCA1 DNA methylation profile to maternal metabolic status.

    PubMed

    Houde, Andrée-Anne; Guay, Simon-Pierre; Desgagné, Véronique; Hivert, Marie-France; Baillargeon, Jean-Patrice; St-Pierre, Julie; Perron, Patrice; Gaudet, Daniel; Brisson, Diane; Bouchard, Luigi

    2013-12-01

    In utero environmental perturbations have been associated with epigenetic changes in the offspring and a lifelong susceptibility to cardiovascular diseases (CVD). DNA methylation at the ATP-binding cassette transporter A1 (ABCA1) gene was previously associated with CVD, but whether these epigenetic marks respond to changes in the maternal environment is unknown. This study was undertaken to assess the associations between the maternal metabolic profile and ABCA1 DNA methylation levels in placenta and cord blood. Placenta and cord blood samples were obtained at delivery from 100 women including 26 with impaired glucose tolerance (IGT) diagnosed following a 75 g-oral glucose tolerance test (OGTT) between week 24 and 28 of gestation. ABCA1 DNA methylation and mRNA levels were measured using bisulfite pyrosequencing and quantitative real-time PCR, respectively. We report that ABCA1 DNA methylation levels on the maternal side of the placenta are correlated with maternal high density lipoprotein cholesterol (HDL-C) levels (r<-0.21; P<0.04) and glucose levels 2 h post-OGTT (r = 0.25; P = 0.02). On the fetal side of the placenta, ABCA1 DNA methylation levels are associated with cord blood triglyceride levels (r = -0.28; P = 0.01). ABCA1 DNA methylation variability on both sides of the placenta are also associated with ABCA1 mRNA levels (r<-0.35; P = 0.05). As opposed to placenta, cord blood DNA methylation levels are negatively correlated with maternal glucose 2 h post-OGTT (r = -0.26; P = 0.02). In conclusion, the epivariations observed in placenta and cord blood likely contribute to an optimal materno-fetal cholesterol transfer. These in utero epigenetics adaptations may also potentially trigger the long-term susceptibility of the newborn to dyslipidemia and CVD. PMID:24113149

  5. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.

    PubMed

    Goedeke, Leigh; Rotllan, Noemi; Canfrán-Duque, Alberto; Aranda, Juan F; Ramírez, Cristina M; Araldi, Elisa; Lin, Chin-Sheng; Anderson, Norma N; Wagschal, Alexandre; de Cabo, Rafael; Horton, Jay D; Lasunción, Miguel A; Näär, Anders M; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-11-01

    The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease. PMID:26437365

  6. Ligand, receptor, and cell type-dependent regulation of ABCA1 and ABCG1 mRNA in prostate cancer epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent evidence suggests that the liver X receptor (LXR) is a potential anti-cancer target in prostate carcinoma. There is little characterization, however, of how the two major isoforms LXRa or LXRß regulate the LXR-responsive genes ATP-binding cassette sub-family A 1 (ABCA1) and sub-family member ...

  7. Methyl protodioscin increases ABCA1 expression and cholesterol efflux while inhibiting gene expressions for synthesis of cholesterol and triglycerides by suppressing SREBP transcription and microRNA 33a/b levels.

    PubMed

    Ma, Weilie; Ding, Hang; Gong, Xiaohua; Liu, Zhen; Lin, Yalin; Zhang, Zhizhen; Lin, Guorong

    2015-04-01

    Sterol regulatory element-binding proteins (SREBPs) regulate homeostasis of LDL, HDL and triglycerides. This study was aimed to determine if inhibition of SREBPs by methyl protodioscin (MPD) regulates downstream gene and protein expressions of lipid metabolisms. In THP-1 macrophages, MPD increases levels of ABCA1 mRNA and protein in dose- and time-dependent manners, and apoA-1-mediated cholesterol efflux. The underlying mechanisms for the effects is that MPD inhibits the transcription of SREBP1c and SREBP2, and decreases levels of microRNA 33a/b hosted in the introns of SREBPs, which leads to reciprocally increase ABCA1 levels. In HepG2 cells, MPD shows the same effects as these observed in THP-1 macrophages. MPD also decreases the gene expressions of HMGCR, FAS and ACC for cholesterol and fatty acid synthesis. MPD further promotes LDL receptor through reducing the PCSK9 level. Collectively, the study demonstrates that MPD potentially increase HDL cholesterol while reducing LDL cholesterol and triglycerides. PMID:25733328

  8. Down-regulation of lipoprotein lipase increases ABCA1-mediated cholesterol efflux in THP-1 macrophages.

    PubMed

    Kawashima, Ryoko L; Medh, Jheem D

    2014-08-01

    The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of excess cholesterol from foam cells to lipid-poor apolipoprotein A-I, in a process called reverse cholesterol transport. Lipoprotein lipase (LPL) is a lipolytic enzyme expressed by macrophages within atherosclerotic lesions. Lentivirus-mediated RNA interference was used to genetically knock-down (KD) the expression of LPL in THP-1 macrophages. Silencing of the LPL gene was confirmed by end-point PCR, real time PCR, and protein analysis. Suppression of LPL expression correlated with a 1.6-fold up-regulation of ABCA1 mRNA levels, and resulted in a 4.5-fold increase in ABCA1-dependent cholesterol efflux. Replenishing LPL by addition of purified bovine LPL to the cell culture media resulted in down-regulation of ABCA1-mediated cholesterol efflux in both wild-type and LPL knockdown cells. These findings suggest an inverse correlation between macrophage LPL levels and ABCA1 cholesterol transport activity. PMID:25017912

  9. Adiponectin upregulates ABCA1 expression through liver X receptor alpha signaling pathway in RAW 264.7 macrophages

    PubMed Central

    Liang, Bin; Wang, Xin; Guo, Xiaohong; Yang, Zhiming; Bai, Rui; Liu, Ming; Xiao, Chuanshi; Bian, Yunfei

    2015-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and anti-atherosclerosis. Liver X receptor alpha (LXRα) can stimulate cholesterol efflux through ABCA1. It has been well known that adiponectin has cardiovascular protection. In this study, we attempted to clarify the effect of adiponectin on expression of ABCA1, and explored the role of LXRα in the regulation of ABCA1 in RAW 264.7 macrophages. Our results showed that adiponectin increased ABCA1 expression at both the mRNA and protein levels in a dose-dependent and time-dependent manner. Consequently, adiponectin promoted cholesterol efflux and decreased cholesterol content in RAW 264.7 macrophages. Moreover, adiponectin up-regulated the expression of LXRα in a dose-dependent and time-dependent manner in RAW 264.7 macrophages. LXRα small interfering RNA completely abolished the promotion effects of adiponectin. In summary, adiponectin up-regulates ABCA1 expression via the LXRα pathway in RAW 264.7 macrophages. This novel insight could prove useful for developing new treatment strategies for cardiovascular diseases. PMID:25755733

  10. Inhibition of ABCA1 Protein Expression and Cholesterol Efflux by TNF α in MLO-Y4 Osteocytes.

    PubMed

    Wehmeier, Kent R; Kurban, William; Chandrasekharan, Chandrikha; Onstead-Haas, Luisa; Mooradian, Arshag D; Haas, Michael J

    2016-06-01

    Hip fracture and myocardial infarction cause significant morbidity and mortality. In vivo studies raising serum cholesterol levels as well as pro-inflammatory cytokines such as TNF α manifest bone loss and atherosclerotic vascular disease, suggesting that abnormalities of cholesterol transport may contribute to osteoporosis. We used the mouse osteocyte cell line (MLO-Y4) to investigate the effects of TNF α on the expression of cholesterol acceptor proteins such as apolipoprotein A-I (apo A-I) and apolipoprotein E (apo E), as well as on the cholesterol transporters ATP-binding cassette-1 (ABCA1), scavenger receptor class B type 1 (SRB1), and cluster of differentiation 36 (CD36). MLO-Y4 cells do not express apo A-I or apo E; however, they do express all three cholesterol transporters (ABCA1, SRB1, and CD36). Treatment of MLO-Y4 cells with TNF α had no effect on SRB1, CD36, and osteocalcin levels; however, TNF α reduced ABCA1 protein levels in a dose-dependent manner and cholesterol efflux to apo A-I. Interestingly, TNF α treatment increased ABCA1 promoter activity and ABCA1 mRNA levels, and increased liver X receptor α protein expression, but had no effect on retinoid X receptor α and retinoic acid receptor α levels. Pharmacological inhibition of p38 mitogen-activated protein (MAP) kinase, but not c-jun-N-terminal kinase 1 or mitogen-activated protein kinase (MEK), restored ABCA1 protein levels in TNF α-treated cells. These results suggest that pro-inflammatory cytokines regulate cholesterol metabolism in osteocytes in part by suppressing ABCA1 levels post-translationally in a p38 MAP kinase-dependent manner. PMID:26759003

  11. The effect of ABCG5/G8 polymorphisms on plasma HDL cholesterol levels depends on the ABCA1 gene variation in the Boston Puerto Rican Health Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: ATP-binding cassette transporters G5/G8 have shown an association with HDL-C. One of the most likely mechanisms to explain those associations is through ABCA1. Objective: To assess whether the effect of ABCG5/G8 polymorphisms on HDL-C is dependent on ABCA1, we studied potential interacti...

  12. Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues

    PubMed Central

    Van Eck, Miranda; Bos, I. Sophie T.; Kaminski, Wolfgang E.; Orsó, Evelyn; Rothe, Gregor; Twisk, Jaap; Böttcher, Alfred; Van Amersfoort, Edwin S.; Christiansen-Weber, Trudy A.; Fung-Leung, Wai-Ping; Van Berkel, Theo J. C.; Schmitz, Gerd

    2002-01-01

    The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr−/−) mice that are selectively deficient in leukocyte ABCA1 (ABCA1−/−) by using bone marrow transfer (ABCA1−/− → LDLr−/−). Here we demonstrate that ABCA1−/− → LDLr−/− chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr−/− mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1−/− → LDLr−/− chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells. PMID:11972062

  13. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans.

    PubMed

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-07-15

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Americans and 863 individuals from other ethnic groups to investigate genetic evidence of positive selection, to assess its functional effect in vitro and to explore associations with HDL-C levels and other metabolic traits. The C230 allele was found in 29 of 36 Native American groups, but not in European, Asian or African individuals. C230 was observed on a single haplotype, and C230-bearing chromosomes showed longer relative haplotype extension compared with other haplotypes in the Americas. Additionally, single-nucleotide polymorphism data from the Human Genome Diversity Panel Native American populations were enriched in significant integrated haplotype score values in the region upstream of the ABCA1 gene. Cells expressing the C230 allele showed a 27% cholesterol efflux reduction (P< 0.001), confirming this variant has a functional effect in vitro. Moreover, the C230 allele was associated with lower HDL-C levels (P = 1.77 x 10(-11)) and with higher body mass index (P = 0.0001) in the combined analysis of Native American populations. This is the first report of a common functional variant exclusive to Native American and descent populations, which is a major determinant of HDL-C levels and may have contributed to the adaptive evolution of Native American populations. PMID:20418488

  14. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    PubMed Central

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K.; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D.; de Ángeles Granados-Silvestre, Ma; Montufar-Robles, Isela; Tito-Alvarez, Ana M.; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P.; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L.; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A.; Lisker, Ruben; Moises, Regina S.; Menjivar, Marta; Salzano, Francisco M.; Knowler, William C.; Bortolini, M. Cátira; Hayden, Michael R.; Baier, Leslie J.; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Americans and 863 individuals from other ethnic groups to investigate genetic evidence of positive selection, to assess its functional effect in vitro and to explore associations with HDL-C levels and other metabolic traits. The C230 allele was found in 29 of 36 Native American groups, but not in European, Asian or African individuals. C230 was observed on a single haplotype, and C230-bearing chromosomes showed longer relative haplotype extension compared with other haplotypes in the Americas. Additionally, single-nucleotide polymorphism data from the Human Genome Diversity Panel Native American populations were enriched in significant integrated haplotype score values in the region upstream of the ABCA1 gene. Cells expressing the C230 allele showed a 27% cholesterol efflux reduction (P< 0.001), confirming this variant has a functional effect in vitro. Moreover, the C230 allele was associated with lower HDL-C levels (P = 1.77 × 10−11) and with higher body mass index (P = 0.0001) in the combined analysis of Native American populations. This is the first report of a common functional variant exclusive to Native American and descent populations, which is a major determinant of HDL-C levels and may have contributed to the adaptive evolution of Native American populations. PMID:20418488

  15. Oxysterols trigger ABCA1-mediated basolateral surfactant efflux.

    PubMed

    Agassandian, Marianna; Mathur, Satya N; Zhou, Jiming; Field, F Jeffrey; Mallampalli, Rama K

    2004-08-01

    Surfactant is an apically-secreted surface-active material containing primarily disaturated phosphatidylcholine (DSPtdCho) that is released from alveolar epithelia into the alveolus. Surfactant deficiency is an important aspect of inflammatory lung disease and may result from extravasation of serum lipoproteins into the alveolus. We investigated whether one bioactive component of modified lipoproteins, oxysterols, might reduce surfactant PtdCho availability by altering its trafficking. The oxysterol, 22-hydroxycholesterol (22HC), in combination with its obligate partner, 9 cis-retinoic acid (RA), decreased surfactant PtdCho levels, in part, by stimulating basolateral phospholipid export in murine lung epithelia. 22HC/RA stimulated basolateral PtdCho efflux in cells via transcriptional activation of the ATP-binding cassette transporter 1 (ABCA1) gene. This effect was mediated by a DR-4 locus within the ABCA1 promoter. ABCA1 knockdown studies using ABCA1 siRNA or the ABCA1 inhibitor, glyburide, selectively attenuated 22HC/RA-driven basolateral PtdCho efflux. 22HC/RA significantly increased export of PtdCho molecular species containing saturated (16:0) fatty-acyl species typical of DSPtdCho. Overexpression of ABCA1 mimicked 22HC/RA effects by increasing cellular PtdCho efflux, whereas mutagenesis of ABCA1 at Trp590 attenuated PtdCho release. The results indicate the existence of an oxysterol-activated basolateral exit pathway for surfactant that might impact the availability of phospholipid destined for apical secretion. PMID:15039140

  16. Difference in expression patterns of placental cholesterol transporters, ABCA1 and SR-BI, in Meishan and Yorkshire pigs with different placental efficiency

    PubMed Central

    Hong, Linjun; Xu, Xiangdong; Huang, Ji; Lei, Minggang; Xu, Dequan; Zhao, Shuhong; Yu, Mei

    2016-01-01

    Cholesterol is a key cell membrane component and precursor of steroid hormones. The maternal cholesterol is an important exogenous cholesterol source for the developing embryos and its transportation is mediated by ABCA1 and SR-BI. Here we reported that during the peri-implantation period in pigs, ABCA1 was expressed by uterine luminal epithelium (LE) and interestingly, its expression was more abundantly in LE on mesometrial side of uterus. However, SR-BI was expressed primarily by LE, glandular epithelial cells (GE) and trophoblast cells (Tr). During the placentation period, the expression levels of ABCA1 and SR-BI proteins at epithelial bilayer and placental areolae were significantly higher in Chinese Meishan pigs compared to Yorkshire pigs. Consisitently, mRNA levels of HMGCR, the rate-limiting enzyme for cholesterol synthesis, were significantly higher in Meishan placentas than in Yorkshire placentas. Our findings revealed the routes of transplacental cholesterol transport mediated by ABCA1 and SR-BI in pigs and indicated that ABCA1 related pathway may participate in anchoring the conceptus to the mesometrial side of uterus. Additionally, an ABCA1 dependent compensatory mechanism related to the placental efficiency in response to the smaller placenta size in Meishan pigs was suggested. PMID:26852751

  17. Difference in expression patterns of placental cholesterol transporters, ABCA1 and SR-BI, in Meishan and Yorkshire pigs with different placental efficiency.

    PubMed

    Hong, Linjun; Xu, Xiangdong; Huang, Ji; Lei, Minggang; Xu, Dequan; Zhao, Shuhong; Yu, Mei

    2016-01-01

    Cholesterol is a key cell membrane component and precursor of steroid hormones. The maternal cholesterol is an important exogenous cholesterol source for the developing embryos and its transportation is mediated by ABCA1 and SR-BI. Here we reported that during the peri-implantation period in pigs, ABCA1 was expressed by uterine luminal epithelium (LE) and interestingly, its expression was more abundantly in LE on mesometrial side of uterus. However, SR-BI was expressed primarily by LE, glandular epithelial cells (GE) and trophoblast cells (Tr). During the placentation period, the expression levels of ABCA1 and SR-BI proteins at epithelial bilayer and placental areolae were significantly higher in Chinese Meishan pigs compared to Yorkshire pigs. Consisitently, mRNA levels of HMGCR, the rate-limiting enzyme for cholesterol synthesis, were significantly higher in Meishan placentas than in Yorkshire placentas. Our findings revealed the routes of transplacental cholesterol transport mediated by ABCA1 and SR-BI in pigs and indicated that ABCA1 related pathway may participate in anchoring the conceptus to the mesometrial side of uterus. Additionally, an ABCA1 dependent compensatory mechanism related to the placental efficiency in response to the smaller placenta size in Meishan pigs was suggested. PMID:26852751

  18. Hepatobiliary cholesterol transport is not impaired in Abca1-null mice lacking HDL

    PubMed Central

    Groen, Albert K.; Bloks, Vincent W.; Bandsma, Robert H.J.; Ottenhoff, Roelof; Chimini, Giovanna; Kuipers, Folkert

    2001-01-01

    The ABC transporter ABCA1 regulates HDL levels and is considered to control the first step of reverse cholesterol transport from the periphery to the liver. To test this concept, we studied the effect of ABCA1 deficiency on hepatic metabolism and hepatobiliary flux of cholesterol in mice. Hepatic lipid contents and biliary secretion rates were determined in Abca1–/–, Abca1+/–, and Abca1+/+ mice with a DBA background that were fed either standard chow or a high-fat, high-cholesterol diet. Hepatic cholesterol and phospholipid contents in Abca1–/– mice were indistinguishable from those in Abca1+/– and Abca1+/+ mice on both diets. In spite of the absence of HDL, biliary secretion rates of cholesterol, bile salts, and phospholipid were unimpaired in Abca1–/– mice. Neither the hepatic expression levels of genes controlling key steps in cholesterol metabolism nor the contribution of de novo synthesis to biliary cholesterol and bile salts were affected by Abca genotype. Finally, fecal excretion of neutral and acidic sterols was similar in all groups. We conclude that plasma HDL levels and ABCA1 activity do not control net cholesterol transport from the periphery via the liver into the bile, indicating that the importance of HDL in reverse cholesterol transport requires re-evaluation. PMID:11560953

  19. Anti-cancer activity of the cholesterol exporter ABCA1 gene

    PubMed Central

    Smith, Bradley; Land, Hartmut

    2012-01-01

    Summary The ABCA1 protein mediates the transfer of cellular cholesterol across the plasma membrane to apolipoprotein A-I. Loss-of-function mutations in the ABCA1 gene induce Tangier disease and familial hypoalphalipoproteinemia, both cardio-vascular conditions characterized by abnormally low levels of serum cholesterol, increased cholesterol in macrophages and subsequent formation of vascular plaque. Increased intra-cellular cholesterol levels are also frequently found in cancer cells. Here we demonstrate anti-cancer activity of ABCA1 efflux function, which is compromised following inhibition of ABCA1 gene expression by oncogenic mutations or cancer-specific ABCA1 loss-of-function mutations. In concert with elevated cholesterol synthesis found in cancer cells, ABCA1 deficiency allows for increased mitochondrial cholesterol, inhibits release of mitochondrial cell death-promoting molecules and thus facilitates cancer cell survival, overall suggesting that elevated mitochondrial cholesterol is essential to the cancer phenotype. PMID:22981231

  20. ABCA1 and nascent HDL biogenesis.

    PubMed

    Wang, Shuhui; Smith, Jonathan D

    2014-01-01

    ABCA1 mediates the secretion of cellular free cholesterol and phospholipids to an extracellular acceptor, apolipoprotein AI, to form nascent high-density lipoprotein (HDL). Thus, ABCA1 is a key molecule in cholesterol homeostasis. Functional studies of certain Tangier disease mutations demonstrate that ABCA1 has multiple activities, including plasma membrane remodeling and apoAI binding to cell surface, which participate in nascent HDL biogenesis. Recent advances in our understanding of ABCA1 have demonstrated that ABCA1also mediates unfolding the N terminus of apoAI on the cell surface, followed by lipidation of apoAI and release of nascent HDL. Although ABCA1-mediated cholesterol efflux to apoAI can occur on the plasma membrane, the role of apoAI retroendocytosis during cholesterol efflux may play a role in macrophage foam cells that store cholesterol esters in cytoplasmic lipid droplets. PMID:25359426

  1. Novel effect of paeonol on the formation of foam cells: promotion of LXRα-ABCA1-dependent cholesterol efflux in macrophages.

    PubMed

    Zhao, Jin-Feng; Jim Leu, Shr-Jeng; Shyue, Song-Kun; Su, Kuo-Hui; Wei, Jeng; Lee, Tzong-Shyuan

    2013-01-01

    Paeonol, a phenolic component purified from Paeonia suffruticosa (Cortex Moutan), is used in traditional Chinese medicine to treat inflammatory diseases. However, little is known about the effect of paeonol on cholesterol metabolism. We investigated the efficacy of paeonol on cholesterol metabolism and the underlying mechanism in macrophages and apolipoprotein E deficient (apoE(-/-)) mice. Treatment with paeonol markedly attenuated cholesterol accumulation induced by oxidized LDL in macrophages, which was due to increased cholesterol efflux. Additionally, paeonol enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Inhibition of ABCA1 activity with a pharmacological inhibitor, neutralizing antibody or small interfering RNA (siRNA), negated the effects of paeonol on cholesterol efflux and cholesterol accumulation. Furthermore, paeonol induced the nuclear translocation of liver X receptor α (LXRα) by increasing its activity. siRNA knockdown of LXRα abolished the paeonol-induced upregulation of ABCA1, promotion of cholesterol efflux and suppression of cholesterol accumulation. Moreover, atherosclerotic lesions, hyperlipidemia and systemic inflammation were reduced and the protein expression of ABCA1 was increased in aortas of paeonol-treated apoE(-/-) mice. Paeonol may alleviate the formation of foam cells by enhancing LXRα-ABCA1-dependent cholesterol efflux. PMID:24117070

  2. Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis

    PubMed Central

    Wan, Wei; Yao, Feng; He, Ping-Ping; Xie, Wei; Mo, Zhong-Cheng; Shi, Jin-Feng; Wu, Jian-Feng; Peng, Juan; Liu, Dan; Cayabyab, Francisco S.; Zheng, Xi-Long; Tang, Xiang-Yang; Ouyang, Xin-Ping; Tang, Chao-Ke

    2016-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter. PMID:27295295

  3. Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis.

    PubMed

    Lv, Yun-Cheng; Tang, Yan-Yan; Zhang, Ping; Wan, Wei; Yao, Feng; He, Ping-Ping; Xie, Wei; Mo, Zhong-Cheng; Shi, Jin-Feng; Wu, Jian-Feng; Peng, Juan; Liu, Dan; Cayabyab, Francisco S; Zheng, Xi-Long; Tang, Xiang-Yang; Ouyang, Xin-Ping; Tang, Chao-Ke

    2016-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter. PMID:27295295

  4. LXR driven induction of HDL-cholesterol is independent of intestinal cholesterol absorption and ABCA1 protein expression.

    PubMed

    Kannisto, Kristina; Gåfvels, Mats; Jiang, Zhao-Yan; Slätis, Katharina; Hu, Xiaoli; Jorns, Carl; Steffensen, Knut R; Eggertsen, Gösta

    2014-01-01

    We investigated whether: (1) liver X receptor (LXR)-driven induction of high-density lipoprotein cholesterol (HDL-C) and other LXR-mediated effects on cholesterol metabolism depend on intestinal cholesterol absorption; and (2) combined treatment with the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe results in synergistic effects on cholesterol metabolism that could be beneficial for treatment of atherosclerosis. Mice were fed 0.2 % cholesterol and treated with GW3965+ezetimibe, GW3965 or ezetimibe. GW3965+ezetimibe treatment elevated serum HDL-C and Apolipoprotein (Apo) AI, effectively reduced the intestinal cholesterol absorption and increased the excretion of faecal neutral sterols. No changes in intestinal ATP-binding cassette (ABC) A1 or ABCG5 protein expression were observed, despite increased mRNA expression, while hepatic ABCA1 was slightly reduced. The combined treatment caused a pronounced down-regulation of intestinal Niemann-Pick C1-like 1 (NPC1L1) and reduced hepatic and intestinal cholesterol levels. GW3965 did not affect the intestinal cholesterol absorption, but increased serum HDL-C and ApoAI levels. GW3965 also increased Apoa1 mRNA levels in primary mouse hepatocytes and HEPA1-6 cells. Ezetimibe reduced the intestinal cholesterol absorption, ABCA1 and ABCG5, but did not affect the serum HDL-C or ApoAI levels. Thus, the LXR-driven induction of HDL-C and ApoAI was independent of the intestinal cholesterol absorption and increased expression of intestinal or hepatic ABCA1 was not required. Inhibited influx of cholesterol via NPC1L1 and/or low levels of intracellular cholesterol prevented post-transcriptional expression of intestinal ABCA1 and ABCG5, despite increased mRNA levels. Combined LXR activation and blocked intestinal cholesterol absorption induced effective faecal elimination of cholesterol. PMID:24163219

  5. In type 2 diabetes mellitus glycated albumin alters macrophage gene expression impairing ABCA1-mediated cholesterol efflux.

    PubMed

    Machado-Lima, Adriana; Iborra, Rodrigo T; Pinto, Raphael S; Castilho, Gabriela; Sartori, Camila H; Oliveira, Erika R; Okuda, Ligia S; Nakandakare, Edna R; Giannella-Neto, Daniel; Machado, Ubiratan F; Corrêa-Giannella, Maria Lucia C; Traldi, Pietro; Porcu, Simona; Roverso, Marco; Lapolla, Annunziata; Passarelli, Marisa

    2015-06-01

    Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2. PMID:25413254

  6. ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice

    PubMed Central

    Vaisman, Boris L.; Lambert, Gilles; Amar, Marcelo; Joyce, Charles; Ito, Toshimitsu; Shamburek, Robert D.; Cain, William J.; Fruchart-Najib, Jamila; Neufeld, Edward D.; Remaley, Alan T.; Brewer, H. Bryan; Santamarina-Fojo, Silvia

    2001-01-01

    The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans. PMID:11457883

  7. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1.

    PubMed

    Fond, Aaron M; Lee, Chang Sup; Schulman, Ira G; Kiss, Robert S; Ravichandran, Kodi S

    2015-07-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane-initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell-initiated pathway functioned independently of the liver X receptor (LXR) sterol-sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  8. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1

    PubMed Central

    Fond, Aaron M.; Lee, Chang Sup; Schulman, Ira G.; Kiss, Robert S.; Ravichandran, Kodi S.

    2015-01-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane–initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell–initiated pathway functioned independently of the liver X receptor (LXR) sterol–sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  9. Salvianolic acid B accelerated ABCA1-dependent cholesterol efflux by targeting PPAR-γ and LXRα

    SciTech Connect

    Yue, Jianmei; Li, Bo; Jing, Qingping; Guan, Qingbo

    2015-07-03

    Objectives: Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. Methods: After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [{sup 3}H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL{sub 2} or HDL{sub 3}. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. Results: The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. Conclusion: These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway. - Highlights: • Sal B promotes the expression of ABCA1. • Sal B promotes cholesterol efflux in macrophages. • Sal B promotes the expression of ABCA1 and cholesterol efflux through PPAR-γ/LXRα signaling pathway.

  10. Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism.

    PubMed

    Zhou, Xiaoye; He, Wei; Huang, Zhiping; Gotto, Antonio M; Hajjar, David P; Han, Jihong

    2008-01-25

    Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions

  11. Inhibition of ABCA1 protein degradation promotes HDL cholesterol efflux capacity and RCT and reduces atherosclerosis in mice.

    PubMed

    Huang, LinZhang; Fan, BaoYan; Ma, Ang; Shaul, Philip W; Zhu, HaiBo

    2015-05-01

    ABCA1 plays a key role in the initial lipidation of apoA-I, which generates circulating HDL cholesterol. Whereas it is known that the transcriptional upregulation of ABCA1 promotes HDL formation and reverse cholesterol transport (RCT), it is not known how the inhibition of ABCA1 protein degradation impacts HDL function. Employing the small molecule triacetyl-3-hydroxyphenyladenosine (IMM-H007), we determined how the attenuation of ABCA1 protein degradation affects HDL cholesterol efflux capacity, RCT, and atherosclerotic lesion formation. Pulse-chase analysis revealed that IMM-H007 inhibits ABCA1 degradation and facilitates its cell-surface localization in macrophages, and additional studies in macrophages showed that IMM-H007 thereby promotes cholesterol efflux. IMM-H007 treatment of Paigen diet-fed mice caused an increase in circulating HDL level, it increased the cholesterol efflux capacity of HDL, and it enhanced in vivo RCT from macrophages to the plasma, liver, and feces. Furthermore, ABCA1 degradation suppression by IMM-H007 reduced atherosclerotic plaque formation in apoE(-/-) mice. Thus, via effects on both ABCA1-expressing cells and circulating HDL function, the inhibition of ABCA1 protein degradation by IMM-H007 promotes HDL cholesterol efflux capacity and RCT and attenuates atherogenesis. IMM-H007 potentially represents a lead compound for the development of agents to augment HDL function. PMID:25761370

  12. Dysregulation of cholesterol homeostasis in human prostate cancer through loss of ABCA1

    PubMed Central

    Lee, Byron H.; Taylor, Margaret G.; Robinet, Peggy; Smith, Jonathan D.; Schweitzer, Jessica; Sehayek, Ephraim; Falzarano, Sara M.; Magi-Galluzzi, Cristina; Klein, Eric A.; Ting, Angela H.

    2012-01-01

    Recent epidemiologic data show that low serum cholesterol level as well as statin use is associated with a decreased risk of developing aggressive or advanced prostate cancer, suggesting a role for cholesterol in aggressive prostate cancer development. Intracellular cholesterol promotes prostate cancer progression as a substrate for de novo androgen synthesis and through regulation of AKT signaling. By performing next-generation sequencing-based DNA methylome analysis, we have discovered marked hypermethylation at the promoter of the major cellular cholesterol efflux transporter, ABCA1, in LNCaP prostate cancer cells. ABCA1 promoter hypermethylation renders the promoter unresponsive to trans-activation and leads to elevated cholesterol levels in LNCaP. ABCA1 promoter hypermethylation is enriched in intermediate to high grade prostate cancers and not detectable in benign prostate. Remarkably, ABCA1 down-regulation is evident in all prostate cancers examined, and expression levels are inversely correlated with Gleason grade. Our results suggest cancer-specific ABCA1 hypermethylation and loss of protein expression direct high intracellular cholesterol levels and hence contribute to an environment conducive to tumor progression. PMID:23233737

  13. Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages.

    PubMed

    Hong, Yi-Fan; Kim, Hangeun; Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

    2016-01-01

    Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent. PMID:27120199

  14. Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages

    PubMed Central

    Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

    2016-01-01

    Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent. PMID:27120199

  15. Cystathionine γ-lyase(CSE)/hydrogen sulfide system is regulated by miR-216a and influences cholesterol efflux in macrophages via the PI3K/AKT/ABCA1 pathway.

    PubMed

    Gong, Duo; Cheng, Hai-peng; Xie, Wei; Zhang, Min; Liu, Dan; Lan, Gang; Huang, Chong; Zhao, Zhen-wang; Chen, Ling-yan; Yao, Feng; Tan, Yu-lin; Li, Liang; Xia, Xiao-dan; Zheng, Xi-long; Wang, Zong-bao; Tang, Chao-ke

    2016-01-29

    This study was designed to evaluate whether CSE/H2S system, which is regulated by miR-216a, regulated ABCA1-mediated cholesterol efflux and cholesterol contents in THP-1 macrophages-derived foam cells. Our qPCR and western blotting results showed that CSE/H2S significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via PI3K/AKT pathway in foam cells derived from human THP-1 macrophages. The miR-216a directly targeted 3' untranslated region of CSE. It significantly reduced CSE and ABCA1 expression, and also decreased the phosphorylation of PI3K and AKT. Additionally, cholesterol efflux decreased, and cholesterol levels increased in THP-1 macrophage-derived foam cells in response to treatment with miR-216a. Our study demonstrates that CSE/H2S system is regulated by miR-216a, and regulates ABCA1-mediated cholesterol efflux and cholesterol levels through the PI3K/AKT pathway. PMID:26772887

  16. A novel role for ABCA1-generated large pre-β migrating nascent HDL in the regulation of hepatic VLDL triglyceride secretion[S

    PubMed Central

    Chung, Soonkyu; Gebre, Abraham K.; Seo, Jeongmin; Shelness, Gregory S.; Parks, John S.

    2010-01-01

    In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function. PMID:20215580

  17. Urotensin II increases foam cell formation by repressing ABCA1 expression through the ERK/NF-κB pathway in THP-1 macrophages

    SciTech Connect

    Wang, Yan; Wu, Jian-Feng; Tang, Yan-Yan; Zhang, Min; Li, Yuan; Chen, Kong; Zeng, Meng-Ya; Yao, Feng; Xie, Wei; Zheng, Xi-Long; Zeng, Gao-Feng; Tang, Chao-Ke

    2014-10-03

    Highlights: • U II reduces cholesterol efflux in THP-1 macrophages. • U II decreases the expression of ABCA1. • Inhibition of the ERK/NF-κB pathway reduces U II effects on ABCA1 expression and cholesterol efflux. - Abstract: Objective: Foam cell formation in the arterial wall plays a key role in the development of atherosclerosis. Recent studies showed that Urotensin II (U II) is involved in the pathogenesis of atherosclerosis. Here we examined the effects of human U II on ATP-binding cassette transporter A1 (ABCA1) expression and the underlying mechanism in THP-1 macrophages. Methods and results: Cultured THP-1 macrophages were treated with U II, followed by measuring the intracellular lipid contents, cholesterol efflux and ABCA1 levels. The results showed that U II dramatically decreased ABCA1 levels and impaired cholesterol efflux. However, the effects of U II on ABCA1 protein expression and cellular cholesterol efflux were partially reversed by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa B (NF-κB) activity, suggesting the potential roles of ERK1/2 and NF-κB in ABCA1 expression, respectively. Conclusion: Our current data indicate that U II may have promoting effects on the progression of atherosclerosis, likely through suppressing ABCA1 expression via activation of the ERK/NF-κB pathway and reducing cholesterol efflux to promote macrophage foam cell formation.

  18. LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2.

    PubMed

    Hussein, Maryem A; Shrestha, Elina; Ouimet, Mireille; Barrett, Tessa J; Leone, Sarah; Moore, Kathryn J; Hérault, Yann; Fisher, Edward A; Garabedian, Michael J

    2015-01-01

    High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients. PMID:26288135

  19. Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages

    PubMed Central

    Wang, Limei; Rotter, Susanne; Ladurner, Angela; Heiss, Elke H.; Oberlies, Nicholas H.; Dirsch, Verena M.; Atanasov, Atanas G.

    2016-01-01

    Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease. PMID:26729088

  20. Quercetin increases macrophage cholesterol efflux to inhibit foam cell formation through activating PPARγ-ABCA1 pathway

    PubMed Central

    Sun, Liqiang; Li, En; Wang, Feng; Wang, Tao; Qin, Zhiping; Niu, Shaohui; Qiu, Chunguang

    2015-01-01

    The accumulation of cholesterol in macrophages could induce the formation of foam cells and increase the risk of developing atherosclerosis. We wonder if quercetin, one of flavonoids with anti-inflammation functions in different cell types, could elevate the development of foam cells formation in atherosclerosis. We treated foam cells derived from oxLDL induced THP-1 cells with quercetin, and evaluated the foam cells formation, cholesterol content and apoptosis of the cells. We found that quercetin induced the expression of ABCA1 in differentiated THP-1 cells, and increased the cholesterol efflux from THP-1 cell derived foam cells. Eventually, cholesterol level and the formation of foam cell derived from THP-1 cells decreased after quercetin treatment. In addition, quercetin activated PPARγ-LXRα pathway to upregulate ABCA1 expression through increasing protein level of PPARγ and its transcriptional activity. Inhibition of PPARγ activity by siRNA knockdown or the addition of chemical inhibitor, GW9662, abolished quercetin induced ABCA1 expression and cholesterol efflux in THP-1 derived macrophages. Our data demonstrated that quercetin increased cholesterol efflux from macrophages through upregulating the expressions of PPARγ and ABCA1. Taken together, increasing uptake of quercetin or quercetin-rich foods would be an effective way to lower the risk of atherosclerosis. PMID:26617799

  1. Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR{gamma}-LXR{alpha}-ABCA1 pathway

    SciTech Connect

    Xu, Yanni; Lai, Fangfang; Xu, Yang; Wu, Yexiang; Liu, Qi; Li, Ni; Wei, Yuzhen; Feng, Tingting; Zheng, Zhihui; Jiang, Wei; Yu, Liyan; Hong, Bin; Si, Shuyi

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. Black-Right-Pointing-Pointer MPA induced ABCA1 and LXR{alpha} protein expression in HepG2 cells. Black-Right-Pointing-Pointer PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. Black-Right-Pointing-Pointer The effect of MPA upregulating ABCA1 was due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 pathway. -- Abstract: ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 {mu}M). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}; EC50 = 5.2-9.3 {mu}M). Liver X receptor {alpha} (LXR{alpha}) is a target gene of PPAR{gamma} and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXR{alpha} protein expression in HepG2 cells. Addition of PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPAR{gamma}. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

  2. Glucagon-like peptide 1 (GLP-1)-based therapy upregulates LXR-ABCA1/ABCG1 cascade in adipocytes.

    PubMed

    Mostafa, Ahmed M; Hamdy, Nadia M; El-Mesallamy, Hala O; Abdel-Rahman, Sherif Z

    2015-12-25

    A promising treatment for obesity involves the use of therapeutic agents that increase the level of the glucagon-like peptide (GLP-1) which reduces appetite and food intake. Native GLP-1 is rapidly metabolized by the dipeptidyl peptidase-4 (DPP-4) enzyme and, as such, GLP-1 mimetics or DPP-4 inhibitors represent promising treatment approaches. Interestingly, obese patient receiving such medications showed improved lipid profiles and cholesterol homeostasis, however the mechanism(s) involved are not known. Members of the ATP-binding cassette (ABC) transporters, including ABCA1 and ABCG1, play essential roles in reverse cholesterol transport and in high density lipoprotein (HDL) formation. These transporters are under the transcriptional regulation of liver X receptor alpha (LXR-α). We hypothesize that GLP-1 mimetics and/or DPP-4 inhibitors modulate ABCA1/ABCG1 expression in adipocytes through an LXR-α mediated process and thus affecting cholesterol homeostasis. 3T3-L1 adipocytes were treated with the DPP-4 inhibitor vildagliptin (2 nM) or the GLP-1 mimetic exendin-4 (5 nM). Gene and protein expression of ABCA1, ABCG1 and LXR-α were determined and correlated with cholesterol efflux. Expression levels of interleukin-6 (IL-6), leptin and the glucose transporter-4 (GLUT-4) were also determined. Treatment with both medications significantly increased the expression of ABCA1, ABCG1, LXR-α and GLUT-4, decreased IL-6 and leptin, and improved cholesterol efflux from adipocytes (P < 0.05). Our data suggest that GLP-1-based therapy modulate ABCA1/ABCG1 expression in adipocytes potentially through an LXR-α mediated process. PMID:26603933

  3. Interactions of Six SNPs in ABCA1gene and Obesity in Low HDL-C Disease in Kazakh of China

    PubMed Central

    Yao, Ming-hong; Guo, Heng; He, Jia; Yan, Yi-zhong; Ma, Ru-lin; Ding, Yu-song; Zhang, Jing-yu; Liu, Jia-ming; Zhang, Mei; Li, Shu-gang; Xu, Shang-zhi; Niu, Qiang; Ma, Jiao-long; Guo, Shu-xia

    2016-01-01

    Objective: To detect the interactions between six functional polymorphisms in ABCA1 and obesity in Kazakhs with low HDL-C levels. Methods: A total of 204 patients with low HDL-C and 207 health control subjects, which were randomly selected from among 5692 adult Kazakhs, were matched for age and sex. We genotyped ABCA1 single nucleotide polymorphisms of rs2515602, rs3890182, rs2275542, rs2230806, rs1800976, and rs4149313. Results: (1) The genotypic and allelic frequencies of rs2515602, rs2230806 and rs4149313 were different between normal HDL-C and low HDL-C subjects, the genotypic frequency of rs2275542 was also different between normal HDL-C and low HDL-C subjects (p < 0.05); (2) the level of HDL-C (rs2515602 and rs2275542) in normal HDL-C subjects were different among the genotypes (p < 0.05); the levels of TC, LDL-C (rs2515602, rs4149313); TG (rs2515602, rs1800976, rs4149313) in low HDL-C patients were different among the genotypes (p < 0.05); (3) interactions between the rs3890182, rs2275542, rs180096, and rs4149313 polymorphisms in ABCA1 gene and obesity may be associated with low HDL-C disease; (4) the C-C-C-A-A-G, T-C-C-A-A-A, T-C-C-A-A-G, C-C-C-A-A-A, C-T-G-G-A-A, and T-T-C-G-A-A haplotypes were significant between the subjects with normal HDL-C and low HDL-C level (p < 0.05). Conclusions: The differences in serum lipid levels between normal HDL-C and low HDL-C subjects among Kazakhs might partly result from ABCA1 gene polymorphisms; ABCA1 gene polymorphisms may be associated with low HDL-C disease; the low HDL-C disease might partly result from interactions between ABCA1 gene polymorphisms and obesity; the C-C-C-A-A-G, T-C-C-A-A-A, and T-C-C-A-A-G haplotypes may serve as risk factors of low HDL-C disease among Kazakhs, the C-C-C-A-A-A, C-T-G-G-A-A, and T-T-C-G-A-A haplotypes may serve as protective factor of low HDL-C disease among Kazakhs. PMID:26828509

  4. Cyclosporine A and PSC833 inhibit ABCA1 function via direct binding.

    PubMed

    Nagao, Kohjiro; Maeda, Minami; Mañucat, Noralyn B; Ueda, Kazumitsu

    2013-02-01

    ATP-binding cassette protein A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL). However, the detailed mechanism of HDL formation remains unclear; in order to reveal it, chemicals that specifically block each step of HDL formation would be useful. Cyclosporine A inhibits ABCA1-mediated cholesterol efflux, but it is not clear whether this is mediated via inhibition of calcineurin. We analyzed the effects of cyclosporine A and related compounds on ABCA1 function in BHK/ABCA1 cells. Cyclosporine A, FK506, and pimecrolimus inhibited ABCA1-mediated cholesterol efflux in a concentration-dependent manner, with IC(50) of 7.6, 13.6, and 7.0μM, respectively. An mTOR inhibitor, rapamycin also inhibited ABCA1, with IC(50) of 18.8μM. The primary targets for these drugs were inhibited at much lower concentrations in BHK/ABCA1 cells, suggesting that they were not involved. Binding of [(3)H] cyclosporine A to purified ABCA1 could be clearly detected. Furthermore, a non-immunosuppressive cyclosporine, PSC833, inhibited ABCA1-mediated cholesterol efflux with IC(50) of 1.9μM, and efficiently competed with [(3)H] cyclosporine A binding to ABCA1. These results indicate that cyclosporine A and PSC833 inhibit ABCA1 via direct binding, and that the ABCA1 inhibitor PSC833 is an excellent candidate for further investigations of the detailed mechanisms underlying formation of HDL. PMID:23153588

  5. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    PubMed

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters. PMID:25302608

  6. 22(R)-hydroxycholesterol and pioglitazone synergistically decrease cholesterol ester via the PPARγ–LXRα–ABCA1 pathway in cholesterosis of the gallbladder

    SciTech Connect

    Wang, Jing-Min Wang, Dong Tan, Yu-Yan Zhao, Gang Ji, Zhen-Ling

    2014-04-25

    Highlights: • Cholesterosis is a metabolic disease characterized by excessive lipid droplets. • Lipid droplet efflux is mediated by the ABCA1 transporter. • 22(R)-hydroxycholesterol can activate LXRα and up-regulate ABCA1. • Pioglitazone up-regulates ABCA1 in a PPARγ–LXRα–ABCA1-dependent manner. • 22(R)-hydroxycholesterol and pioglitazone synergistically decrease lipid droplets. - Abstract: Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets

  7. Impact of HDL oxidation by the myeloperoxidase system on sterol efflux by the ABCA1 pathway.

    PubMed

    Shao, Baohai; Heinecke, Jay W

    2011-10-19

    Protein oxidation by phagocytic white blood cells is implicated in tissue injury during inflammation. One important target might be high-density lipoprotein (HDL), which protects against atherosclerosis by removing excess cholesterol from artery wall macrophages. In the human artery wall, cholesterol-laden macrophages are a rich source of myeloperoxidase (MPO), which uses hydrogen peroxide for oxidative reactions in the extracellular milieu. Levels of two characteristic products of MPO-chlorotyrosine and nitrotyrosine-are markedly elevated in HDL from human atherosclerotic lesions. Here, we describe how MPO-dependent chlorination impairs the ability of apolipoprotein A-I (apoA-I), HDL's major protein, to transport cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. Faulty interactions between apoA-I and ABCA1 are involved. Tandem mass spectrometry and investigations of mutated forms of apoA-I demonstrate that tyrosine residues in apoA-I are chlorinated in a site-specific manner by chloramine intermediates on suitably juxtaposed lysine residues. Plasma HDL isolated from subjects with coronary artery disease (CAD) also contains higher levels of chlorinated and nitrated tyrosine residues than HDL from healthy subjects. Thus, the presence of chlorinated HDL might serve as a marker of CAD risk. Because HDL damaged by MPO in vitro becomes dysfunctional, inhibiting MPO in vivo might be cardioprotective. PMID:21501700

  8. Cholesterol transport via ABCA1: new insights from solid-phase binding assay.

    PubMed

    Reboul, Emmanuelle; Dyka, Frank M; Quazi, Faraz; Molday, Robert S

    2013-04-01

    It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s). PMID:23201557

  9. miR-146a-5p Antagonized AGEs- and P.g-LPS-Induced ABCA1 and ABCG1 Dysregulation in Macrophages via IRAK-1 Downregulation.

    PubMed

    Li, Xia; Ji, Zheng; Li, Si; Sun, Ya-Nan; Liu, Jia; Liu, Ying; Tian, Wei; Zhou, Yun-Tao; Shang, Xiao-Ming

    2015-10-01

    The miR-146-mediated IL-1 receptor-associated kinase 1 (IRAK-1) feedback circuit has been shown to inhibit inflammatory response in macrophages against lipopolysaccharide (LPS). The aim of this study is to compare the antagonized effects of miR-146a-5p on Porphyromonas gingivalis (P.g) lipolysaccharide (LPS)- and advanced glycation end products (AGEs)-triggered ABCA1 and ABCG1 dysregulation and explore the underlying mechanism. THP-1-derived macrophages transfected with miRNA mimics or not were treated with P.g LPS or AGE-BSA, respectively. The mechanism of endotoxin tolerance was mimicked. miR-146a-5p levels and protein levels of IRAK-1, LXRα/β, ABCA1, and ABCG1 were detected by stem-loop reverse transcription followed by TaqMan PCR analysis and Western blotting. Our results showed that miR-146a-5p levels were significantly increased in macrophages after 24 h of stimulation with high dose of P.g LPS or AGE-BSA. Macrophages transfected with miR-146a-5p mimics attenuated the dysregulation of ABCA1/G1 induced by P.g LPS and AGEs through IRAK-1 downregulation. In low-dose LPS-tolerized cells, elevated miR-146a-5p antagonized the increase of ABCA1, ABCG1, and IRAK-1. However, low-dose AGE-BSA did not increase miR-146a-5p levels. In conclusion, the model of endotoxin tolerance is suitable for the antagonistic effects on the dysregulation of ABCA1/G1 induced by high dose of P.g LPS. Conversely, low-dose AGEs did not induce the model of P.g LPS-mediated tolerance. PMID:25805648

  10. Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

    PubMed

    Fournier, Natalie; Tardivel, Sylviane; Benoist, Jean-François; Vedie, Benoît; Rousseau-Ralliard, Delphine; Nowak, Maxime; Allaoui, Fatima; Paul, Jean-Louis

    2016-04-01

    A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages. PMID:26776055

  11. Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

    PubMed Central

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; Genest, Jacques

    2015-01-01

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  12. Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of preβ-1 HDL in vitro

    DOE PAGESBeta

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; Genest, Jacques; Zhu, Xuewei

    2015-07-24

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from themore » carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  13. Paraoxonase 1-treated oxLDL promotes cholesterol efflux from macrophages by stimulating the PPARγ-LXRα-ABCA1 pathway.

    PubMed

    Ikhlef, Souade; Berrougui, Hicham; Kamtchueng Simo, Olivier; Khalil, Abdelouahed

    2016-06-01

    Here, we investigate the mechanism through which paraoxonase 1 (PON1) may regulate cholesterol efflux. Pretreatment of oxLDL with PON1 (oxLDL-PON1) contributed to the formation of LysoPC. In J774 macrophages, oxLDL-PON1 increased cholesterol efflux by more than 47% compared to oxLDL alone. oxLDL-PON1 significantly increased mRNA and protein expression of ABCA1 and ABCG1, as well as of PPARγ and LXRα compared to oxLDL alone. Intraperitoneal injection of oxLDL-PON1- or LysoPC-treated J774 macrophages significantly increased the fecal elimination of macrophage-derived cholesterol in these mice. Our results suggest that PON1 stimulates cholesterol efflux via a mechanism that involves oxidized phospholipid hydrolysis. PMID:27148853

  14. ATP-Binding Membrane Cassette Transporter A1 (ABCA1): A Possible Link between Inflammation and Reverse Cholesterol Transport

    PubMed Central

    Yin, Kai; Liao, Duan-fang; Tang, Chao-ke

    2010-01-01

    Atherosclerosis is characterized by a chronic inflammatory condition that involves numerous cellular and molecular inflammatory components. A wide array of inflammatory mediators, such as cytokines and proteins produced by macrophages and other cells, play a critical role in the development and progression of the disease. ATP-binding membrane cassette transporter A1 (ABCA1) is crucial for cellular cholesterol efflux and reverse cholesterol transport (RCT) and is also identified as an important target in antiatherosclerosis treatment. Evidence from several recent studies indicates that inflammation, along with other atherogenic-related mediators, plays distinct regulating roles in ABCA1 expression. Proatherogenic cytokines such as interferon (IFN)-γ and interleukin (IL)-1β have been shown to inhibit the expression of ABCA1, while antiatherogenic cytokines, including IL-10 and transforming growth factor (TGF)-β1, have been shown to promote the expression of ABCA1. Moreover, some cytokines such as tumor necrosis factor (TNF)-α seem to regulate ABCA1 expression in species-specific and dose-dependent manners. Inflammatory proteins such as C-reactive protein (CRP) and cyclooxygenase (COX)-2 are likely to inhibit ABCA1 expression during inflammation, and inflammation induced by lipopolysaccharide (LPS) was also found to block the expression of ABCA1. Interestingly, recent experiments revealed ABCA1 can function as an antiinflammatory receptor to suppress the expression of inflammatory factors, suggesting that ABCA1 may be the molecular basis for the interaction between inflammation and RCT. This review aims to summarize recent findings on the role of inflammatory cytokines, inflammatory proteins, inflammatory lipids, and the endotoxin-mediated inflammatory process in expression of ABCA1. Also covered is the current understanding of the function of ABCA1 in modulating the immune response and inflammation through its direct and indirect antiinflammatory mechanisms

  15. ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT[S

    PubMed Central

    Duka, Adelina; Fotakis, Panagiotis; Georgiadou, Dimitra; Kateifides, Andreas; Tzavlaki, Kalliopi; von Eckardstein, Leonard; Stratikos, Efstratios; Kardassis, Dimitris; Zannis, Vassilis I.

    2013-01-01

    The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I−/− mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-β-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I−/− × apoE−/− mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-β-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1−/− or LCAT−/− mice. Coexpression of apoA-IV and LCAT in apoA-I−/− mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV. PMID:23132909

  16. Impairment of the ABCA1 and SR-BI-mediated cholesterol efflux pathways and HDL anti-inflammatory activity in Alzheimer's disease.

    PubMed

    Khalil, Abdelouahed; Berrougui, Hicham; Pawelec, Graham; Fulop, Tamas

    2012-01-01

    The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation. PMID:22178419

  17. ABCA1 promotes the efflux of bacterial LPS from macrophages and accelerates recovery from LPS-induced tolerance[S

    PubMed Central

    Thompson, Patricia A.; Gauthier, Karine C.; Varley, Alan W.; Kitchens, Richard L.

    2010-01-01

    Macrophages play important roles in both lipid metabolism and innate immunity. We show here that macrophage ATP-binding cassette transporter A1 (ABCA1), a transporter known for its ability to promote apolipoprotein-dependent cholesterol efflux, also participates in the removal of an immunostimulatory bacterial lipid, lipopolysaccharide (LPS). Whereas monocytes require an exogenous lipoprotein acceptor to remove cell-associated LPS, macrophages released LPS in the absence of an exogenous acceptor by a mechanism that was driven, in part, by endogenous apolipoprotein E (apoE). Agents that increased ABCA1 expression increased LPS efflux from wild-type but not ABCA1-deficient macrophages. Preexposure of peritoneal macrophages to LPS for 24 h increased the expression of ABCA1 and increased LPS efflux with a requirement for exogenous apolipoproteins due to suppression of endogenous apoE production. In contrast, LPS preconditioning of ABCA1-deficient macrophages significantly decreased LPS efflux and led to prolonged retention of cell-surface LPS. Although the initial response to LPS was similar in wild-type and ABCA1-deficient macrophages, LPS-induced tolerance was greater and more prolonged in macrophages that lacked ABCA1. Our results define a new role for macrophage ABCA1 in removing cell-associated LPS and restoring normal macrophage responsiveness. PMID:20472936

  18. HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation

    PubMed Central

    Tardy, Claudine; Goffinet, Marine; Boubekeur, Nadia; Cholez, Guy; Ackermann, Rose; Sy, Gavin; Keyserling, Constance; Lalwani, Narendra; Paolini, John F.; Dasseux, Jean-Louis; Barbaras, Ronald; Baron, Rudi

    2015-01-01

    Objective CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3). Methods and Results CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL3 compared to non-lipidated apoA-I. In vivo, in an apoE-/- mouse “flow cessation model,” in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively. Conclusions These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that

  19. Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

    SciTech Connect

    Ohara, Kazuaki; Wakabayashi, Hideyuki; Taniguchi, Yoshimasa; Shindo, Kazutoshi; Yajima, Hiroaki; Yoshida, Aruto

    2013-11-29

    Highlights: •The major circulating quercetin metabolite (Q3GA) activated LXRα. •Q3GA induced ABCA1 via LXRα activation in macrophages. •Nelumbo nucifera leaf extracts contained quercetin glycosides. •N. nucifera leaf extract feeding elevated HDLC in mice. -- Abstract: Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

  20. ABCA1 gene variants regulate posprandial lipid metabolism in healthy men

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Genetic variants of ABCA1, a member of a large family of conserved transmembrane proteins, have been linked to altered atherosclerosis progression and fasting lipid concentration, mainly HDL and Apolipoprotein A, but results from different studies have been inconsistent. Methods and res...

  1. Hepatic ACAT2 Knock Down Increases ABCA1 and Modifies HDL Metabolism in Mice

    PubMed Central

    Degirolamo, Chiara; Gomaraschi, Monica; Graham, Mark; Ossoli, Alice; Larsson, Lilian; Calabresi, Laura; Gustafsson, Jan-Åke; Steffensen, Knut R.; Eriksson, Mats; Parini, Paolo

    2014-01-01

    Objectives ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism. Design WT and LXRα/β double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet. Results ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE. Conclusions The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1. PMID:24695360

  2. Direct intracerebral delivery of a miR-33 antisense oligonucleotide into mouse brain increases brain ABCA1 expression. [Corrected].

    PubMed

    Jan, Asad; Karasinska, Joanna M; Kang, Martin H; de Haan, Willeke; Ruddle, Piers; Kaur, Achint; Connolly, Colum; Leavitt, Blair R; Sorensen, Poul H; Hayden, Michael R

    2015-06-26

    The ATP-binding cassette transporter A1 (ABCA1) is a membrane bound protein that serves to efflux cholesterol and phospholipids onto lipid poor apolipoproteins during HDL biogenesis. Increasing the expression and activity of ABCA1 have beneficial effects in experimental models of various neurologic and cardiovascular diseases including Alzheimer's disease. Despite the beneficial effects of liver X receptor (LXR) agonists--compounds that increase ABCA1 expression--in preclinical studies, their therapeutic utility is limited by systemic adverse effects on lipid metabolism. Interestingly, microRNA-33 (miR-33) inhibition increases ABCA1 expression and activity in rodents and non-human primates without severe metabolic adverse effects. Herein, we demonstrate that treatment of cultured mouse neurons, astrocytes and microglia with an antisense oligonucleotide (ASO) targeting miR-33 increased ABCA1 expression, which was accompanied by increased cholesterol efflux and apoE secretion in astrocytic cultures. We also show that intracerebral delivery of an ASO targeting miR-33 leads to increased ABCA1 expression in cerebral cortex or subcortical structures such as hippocampus. These findings highlight an effective strategy for increasing brain ABCA1 expression/activity for relevant mechanistic studies. [Corrected] PMID:25957561

  3. Both STAT3 activation and cholesterol efflux contribute to the anti-inflammatory effect of apoA-I/ABCA1 interaction in macrophages.

    PubMed

    Tang, Chongren; Houston, Barbara A; Storey, Carl; LeBoeuf, Renee C

    2016-05-01

    ABCA1 exports excess cholesterol from cells to apoA-I and is essential for HDL synthesis. Genetic studies have shown that ABCA1 protects against cardiovascular disease. We have previously shown that the interaction of apoA-I with ABCA1 activates signaling molecule Janus kinase 2 (JAK2), which optimizes the cholesterol efflux activity of ABCA1. ABCA1-mediated activation of JAK2 also activates signal transducer and activator of transcription 3 (STAT3), which significantly attenuates proinflammatory cytokine expression in macrophages. To determine the mechanisms of the anti-inflammatory effects of apoA-I/ABCA1 interaction, we identified two special ABCA1 mutants, one with normal STAT3-activating capacity but lacking cholesterol efflux ability and the other with normal cholesterol efflux ability but lacking STAT3-activating capacity. We showed that activation of STAT3 by the interaction of apoA-I/ABCA1 without cholesterol efflux could significantly decrease proinflammatory cytokine expression in macrophages. Mechanistic studies showed that the anti-inflammatory effect of the apoA-I/ABCA1/STAT3 pathway is suppressor of cytokine signaling 3 dependent. Moreover, we showed that apoA-I/ABCA1-mediated cholesterol efflux without STAT3 activation can also reduce proinflammatory cytokine expression in macrophages. These findings suggest that the interaction of apoA-I/ABCA1 activates cholesterol efflux and STAT3 branch pathways to synergistically suppress inflammation in macrophages. PMID:26989082

  4. Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of preβ-1 HDL in vitro

    SciTech Connect

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; Genest, Jacques; Zhu, Xuewei

    2015-07-24

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These

  5. Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.

    PubMed

    Timmins, Jenelle M; Lee, Ji-Young; Boudyguina, Elena; Kluckman, Kimberly D; Brunham, Liam R; Mulya, Anny; Gebre, Abraham K; Coutinho, Jonathan M; Colvin, Perry L; Smith, Thomas L; Hayden, Michael R; Maeda, Nobuyo; Parks, John S

    2005-05-01

    Patients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1(-L/-L)). Abca1(-L/-L) mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1(-L/-L) mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time. PMID:15841208

  6. Cholesterol Transporters ABCA1 and ABCG1 Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Metabolic Syndrome

    PubMed Central

    Tavoosi, Zahra; Moradi-Sardareh, Hemen; Saidijam, Massoud; Yadegarazari, Reza; Borzuei, Shiva; Soltanian, Alireza; Goodarzi, Mohammad Taghi

    2015-01-01

    ABCA1 and ABCG1 genes encode the cholesterol transporter proteins that play a key role in cholesterol and phospholipids homeostasis. This study was aimed at evaluating and comparing ABCA1 and ABCG1 genes expression in metabolic syndrome patients and healthy individuals. This case-control study was performed on 36 patients with metabolic syndrome and the same number of healthy individuals in Hamadan (west of Iran) during 2013-2014. Total RNA was extracted from mononuclear cells and purified using RNeasy Mini Kit column. The expression of ABCA1 and ABCG1 genes was performed by qRT-PCR. Lipid profile and fasting blood glucose were measured using colorimetric procedures. ABCG1 expression in metabolic syndrome patients was significantly lower (about 75%) compared to that of control group, while for ABCA1 expression, there was no significant difference between the two studied groups. Comparison of other parameters such as HDL-C, FBS, BMI, waist circumference, and systolic and diastolic blood pressure between metabolic syndrome patients and healthy individuals showed significant differences (P < 0.05). Decrease in ABCG1 expression in metabolic syndrome patients compared to healthy individuals suggests that hyperglycemia, related metabolites, and hyperlipidemia over the transporter capacity resulted in decreased expression of ABCG1. Absence of a significant change in ABCA1 gene expression between two groups can indicate a different regulation mechanism for ABCA1 expression. PMID:26788366

  7. Association between Polymorphisms and Haplotype in the ABCA1 Gene and Overweight/Obesity Patients in the Uyghur Population of China

    PubMed Central

    Yao, Ming-Hong; He, Jia; Ma, Ru-Lin; Ding, Yu-Song; Guo, Heng; Yan, Yi-Zhong; Zhang, Jing-Yu; Liu, Jia-Ming; Zhang, Mei; Rui, Dong-Shen; Niu, Qiang; Guo, Shu-Xia

    2016-01-01

    Objective: This study aimed to detect the association between polymorphisms and haplotype in the ATP-binding cassette transporter A1 (ABCA1) gene and overweight/obese Uyghur patients in China. Methods: A total of 259 overweight/obese patients and 276 normal weight subjects, which were randomly selected from among 3049 adult Uyghurs, were matched for age. We genotyped ABCA1 single nucleotide polymorphisms of rs2515602, rs3890182, rs2275542, rs2230806, rs1800976, and rs4149313. Results: (1) The genotypic and allelic frequencies of rs2515602 and rs4149313 differed between the control group and case group. The genotypic frequency of rs2275542 also differed between the control group and case group (p < 0.05); (2) rs2515602, rs2230806, and rs4149313 polymorphisms were significantly related to risk of overweight/obese; (3) a significant linkage disequilibrium (LD) was observed between the ABCA1 gene rs2275542 with rs3890182 and rs2515602 with rs4149313. (4) the C-C-C-A-G-G, T-C-G-A-G-G, and T-T-G-G-G-A haplotypes were significant in normal weight and overweight/obese subjects (p < 0.05); (5) the levels of HDL-C (rs2515602, rs2275542, rs4149313) in normal weight subjects were different among the genotypes (p < 0.05); the levels of TC, LDL-C and TG (rs1800976) in overweight/obese subjects were different among the genotypes (p < 0.05). Conclusions: The rs2515602, rs4149313, and rs2275542 polymorphisms were associated with overweight/obese conditions among Uyghurs. Strong LD was noted between rs2275542 with rs3890182 and rs2515602 with rs4149313. The C-C-C-A-G-G and T-C-G-A-G-G haplotypes may serve as risk factors of overweight/obesity among Uyghurs. The T-T-G-G-G-A haplotype may serve as a protective factor of overweight/obesity among Uyghurs. Rs2515602, rs2275542, rs4149313, and rs1800976 polymorphisms in the ABCA1 gene may influence lipid profiles. PMID:26891315

  8. Liver X receptor reduces proliferation of human oral cancer cells by promoting cholesterol efflux via up-regulation of ABCA1 expression

    PubMed Central

    Kashiwagi, Korehito; Yaginuma, Nanae; Ohkoshi, Chihiro; Tanaka, Mizuko; Sugino, Takashi; Imura, Tetsuya; Hasegawa, Hiroshi; Chiba, Hideki

    2015-01-01

    Liver X receptors (LXRs) contribute not only to maintain cholesterol homeostasis but also to control cell growth. However, the molecular mechanisms behind the LXR-mediated anti-proliferative effects are largely unknown. Here we show, by immunohistochemistry, that LXRα and LXRβ are differentially distributed in oral stratified squamous epithelia. By immunohistochemical and Western blot analyses, we also reveal that LXRα is abundantly expressed in human oral squamous cell carcinoma (HOSCC) tissues and cell lines. Cell counting, BrdU labeling and cell cycle assay indicated that LXR stimulation led to significant reduction of proliferation in HOSCC cells. Importantly, our study highlights, by using RNA interference, that the ATP-binding cassette transporter A1 (ABCA1)-accelerated cholesterol efflux is critical for the growth inhibitory action of LXRs in HOSCC cells. Moreover, we demonstrate that LXR activation reduces the growth of xenograft tumour of HOSCC cells in mice accompanied by the upregulation of ABCA1 expression and the decline of cholesterol levels in the tumour. These findings strongly suggested that targeting the LXR-regulated cholesterol transport, yielding in lowering intracellular cholesterol levels, could be a promising therapeutic option for certain types of cancers. PMID:26452260

  9. BIOMARKERS OF ENDOCRINE DISRUPTION AT THE MRNA LEVEL

    EPA Science Inventory

    Denslow, Nancy D., Christopher J. Bowman, Gillian Robinson, H. Stephen Lee, Ronald J. Ferguson, Michael J. Hemmer and Leroy C. Folmar. 1999. Biomarkers of Endocrine Disruption at the mRNA Level. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for ...

  10. Cooperation between Engulfment Receptors: The Case of ABCA1 and MEGF10

    PubMed Central

    Hamon, Yannick; Trompier, Doriane; Ma, Zhong; Venegas, Victor; Pophillat, Matthieu; Mignotte, Vincent; Zhou, Zheng; Chimini, Giovanna

    2006-01-01

    The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway. PMID:17205124

  11. LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling.

    PubMed

    Ito, Ayaka; Hong, Cynthia; Rong, Xin; Zhu, Xuewei; Tarling, Elizabeth J; Hedde, Per Niklas; Gratton, Enrico; Parks, John; Tontonoz, Peter

    2015-01-01

    The liver X receptors (LXRs) are transcriptional regulators of lipid homeostasis that also have potent anti-inflammatory effects. The molecular basis for their anti-inflammatory effects is incompletely understood, but has been proposed to involve the indirect tethering of LXRs to inflammatory gene promoters. Here we demonstrate that the ability of LXRs to repress inflammatory gene expression in cells and mice derives primarily from their ability to regulate lipid metabolism through transcriptional activation and can occur in the absence of SUMOylation. Moreover, we identify the putative lipid transporter Abca1 as a critical mediator of LXR's anti-inflammatory effects. Activation of LXR inhibits signaling from TLRs 2, 4 and 9 to their downstream NF-κB and MAPK effectors through Abca1-dependent changes in membrane lipid organization that disrupt the recruitment of MyD88 and TRAF6. These data suggest that a common mechanism-direct transcriptional activation-underlies the dual biological functions of LXRs in metabolism and inflammation. PMID:26173179

  12. Plasma Membrane Profiling Reveals Upregulation of ABCA1 by Infected Macrophages Leading to Restriction of Mycobacterial Growth

    PubMed Central

    Long, Jing; Basu Roy, Robindra; Zhang, Yanjia J.; Antrobus, Robin; Du, Yuxian; Smith, Duncan L.; Weekes, Michael P.; Javid, Babak

    2016-01-01

    The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection. We applied plasma membrane profiling, a technique that combines quantitative mass spectrometry with selective cell surface aminooxy-biotinylation, to Bacille Calmette–Guérin (BCG)-infected THP-1 macrophages. We quantified 559 PM proteins in BCG-infected THP-1 cells. One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1. We showed that ABCA1 was upregulated on the macrophage cell-surface following infection with pathogenic mycobacteria and knockdown of ABCA1 resulted in increased mycobacterial survival within macrophages, suggesting that it may be a novel mycobacterial host-restriction factor. PMID:27462310

  13. Subfraction analysis of circulating lipoproteins in a patient with Tangier disease due to a novel ABCA1 mutation.

    PubMed

    Murano, Takeyoshi; Yamaguchi, Takashi; Tatsuno, Ichiro; Suzuki, Masayo; Noike, Hirofumi; Takanami, Tarou; Yoshida, Tomoe; Suzuki, Mitsuya; Hashimoto, Ryuya; Maeno, Takatoshi; Terai, Kensuke; Tokuyama, Wataru; Hiruta, Nobuyuki; Schneider, Wolfgang J; Bujo, Hideaki

    2016-01-15

    Tangier disease, characterized by low or absent high-density lipoprotein (HDL), is a rare hereditary lipid storage disorder associated with frequent, but not obligatory, severe premature atherosclerosis due to disturbed reverse cholesterol transport from tissues. The reasons for the heterogeneity in atherogenicity in certain dyslipidemias have not been fully elucidated. Here, using high-performance liquid chromatography with a gel filtration column (HPLC-GFC), we have studied the lipoprotein profile of a 17-year old male patient with Tangier disease who to date has not developed manifest coronary atherosclerosis. The patient was shown to be homozygous for a novel mutation (Leu1097Pro) in the central cytoplasmic region of ATP-binding cassette transporter A1 (ABCA1). Serum total and HDL-cholesterol levels were 59mg/dl and 2mg/dl, respectively. Lipoprotein electrophoretic analyses on agarose and polyacrylamide gels showed the presence of massively abnormal lipoproteins. Further analysis by HPLC-GFC identified significant amounts of lipoproteins in low-density lipoprotein (LDL) subfractions. The lipoprotein particles found in the peak subfraction were smaller than normal LDL, were rich in triglycerides, but poor in cholesterol and phospholipids. These findings in an adolescent Tangier patient suggest that patients in whom these triglyceride-rich, cholesterol- and phospholipid-poor LDL-type particles accumulate over time, would experience an increased propensity for developing atherosclerosis. PMID:26616730

  14. An ABCA1-independent pathway for recycling a poorly lipidated 8.1 nm apolipoprotein E particle from glia

    PubMed Central

    Fan, Jianjia; Stukas, Sophie; Wong, Charmaine; Chan, Jennifer; May, Sharon; DeValle, Nicole; Hirsch-Reinshagen, Veronica; Wilkinson, Anna; Oda, Michael N.; Wellington, Cheryl L.

    2011-01-01

    Lipid transport in the brain is coordinated by glial-derived lipoproteins that contain apolipoprotein E (apoE) as their primary protein. Here we show that apoE is secreted from wild-type (WT) primary murine mixed glia as nascent lipoprotein subspecies ranging from 7.5 to 17 nm in diameter. Negative-staining electron microscropy (EM) revealed rouleaux, suggesting a discoidal structure. Potassium bromide (KBr) density gradient ultracentrifugation showed that all subspecies, except an 8.1 nm particle, were lipidated. Glia lacking the cholesterol transporter ABCA1 secreted only 8.1 nm particles, which were poorly lipidated and nondiscoidal but could accept lipids to form the full repertoire of WT apoE particles. Receptor-associated-protein (RAP)-mediated inhibition of apoE receptor function blocked appearance of the 8.1 nm species, suggesting that this particle may arise through apoE recycling. Selective deletion of the LDL receptor (LDLR) reduced the level of 8.1 nm particle production by approximately 90%, suggesting that apoE is preferentially recycled through the LDLR. Finally, apoA-I stimulated secretion of 8.1 nm particles in a dose-dependent manner. These results suggest that nascent glial apoE lipoproteins are secreted through multiple pathways and that a greater understanding of these mechanisms may be relevant to several neurological disorders. PMID:21705806

  15. Hfq affects mRNA levels independently of degradation

    PubMed Central

    2010-01-01

    Background The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. Results The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain. Conclusions The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript would help to overcome

  16. Heart ABCA1 and PPAR- α Genes Expression Responses in Male rats: Effects of High Intensity Treadmill Running Training and Aqueous Extraction of Black Crataegus-Pentaegyna

    PubMed Central

    Ghanbari-Niaki, Abbass; Ghanbari-Abarghooi, Safieyh; Rahbarizadeh, Fatemeh; Zare-Kookandeh, Navabeh; Gholizadeh, Monireh; Roudbari, Fatemeh; Zare-Kookandeh, Asghar

    2013-01-01

    Introduction: Heart as a high metabolic and aerobic tissue is consuming lipid as a fuel for its energy provision at rest during light and moderate exercise, except when lactate level is higher in blood circulation. It has been shown that any type of regular exercise and crataegus species would improve cardiovascular function and minimizes several risk factors via stimulating lipid metabolism by acting on enzymes and genes expression such as ABCA1 and PPAR α which are involving in this process. Materials and Methods: Twenty Wistar male rats (4-6 weeks old, 140-173 g weight) were used. Animals were randomly classified into training (n = 10) and control (n = 10) groups and then divided into saline-control (SC), saline-training (ST), Crataegus-Pentaegyna -control (CPC), and Crataegus-Pentaegyna -training (CPT) groups. Training groups have performed a high-intensity running program (at 34 m/min (0% grade), 60 min/day, 5 days/week) on a motor-driven treadmill for eight weeks. Animals were orally fed with Crataegus-Pentaegyna extraction (500mg/kg) and saline solution for six weeks. Seventy- two hours after the last training session, rats were sacrificed, hearts were excised, cleaned and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Plasma also was collected for plasma variable measurements. Statistical analysis was performed using a two way analysis of variance, and significance was accepted at P < 0.05. Results: A non-significant (P < 0.4, P < 0.79, respectively) increase in ABCA1 and PPAR α genes expression was accompanied by a significant (P < 0.01, P < 0.04, P < 0.04, respectively) reduction in TC, TG, and VLDL-C levels in Crataegus-Pentaegyna groups. Conclusions: Our findings show that a high intensity treadmill running was able to express ABCA1 and PPAR α in rat heart. Data also possibly indicate that the Crataeguse-Pentaegyna supplementation solely could mimic training effect on the mentioned genes and lipid profiles via

  17. Effects of DHA Supplementation on Vascular Function, Telomerase Activity in PBMC, Expression of Inflammatory Cytokines, and PPARγ-LXRα-ABCA1 Pathway in Patients With Type 2 Diabetes Mellitus: Study Protocol for Randomized Controlled Clinical Trial.

    PubMed

    Toupchian, Omid; Sotoudeh, Gity; Mansoori, Anahita; Djalali, Mahmoud; Keshavarz, Seyyed Ali; Nasli-Esfahani, Ensieh; Alvandi, Ehsan; Koohdani, Fariba

    2016-07-01

    Docosahexaenoic acid (DHA), as an omega-3 fatty acid, in a natural ligand of peroxisome proliferator-activated receptors (PPARs). Regarding the combinative effects of Nutrigenomics and Nutrigenetics and due to the lack of in vivo studies conducted using natural ligands of PPARs, we aimed to evaluate the effects of DHA supplementation on vascular function, telomerase activity, and PPARγ-LXRα-ABCA1 pathway, in patients with type 2 diabetes mellitus (T2DM), based on the Pro12Ala polymorphism in PPARγ encoding gene. 72 T2DM patients (36 dominant and 36 recessive allele carriers), aged 30-70, with body mass index of 18.5 to 35 kg/m2, will be participated in this double blind randomized controlled trial. In each group, stratification will be performed based on sex and age and participants will be randomly assigned to receive 2.4 g/day DHA or placebo (paraffin) for 8 weeks. PPARγ genotyping will be carried out using PCR-RFLP method; Telomerase activity will be estimated by PCR-ELISA TRAP assay; mRNA expression levels of target genes will be assessed using real time PCR. Serum levels of ADMA, sCD163 and adiponectin, will be measured using ELISA commercial kits. The present study is designed in order to help T2DM patients to modify their health conditions based on their genetic backgrounds, and to recommend the proper food ingredients as the natural agonists for PPARs in order to prevent and treat metabolic abnormalities of the disease. PMID:27424010

  18. A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis

    PubMed Central

    Hu, Yan-Wei; Yang, Jun-Yao; Ma, Xin; Chen, Zhi-Ping; Hu, Ya-Rong; Zhao, Jia-Yi; Li, Shu-Fen; Qiu, Yu-Rong; Lu, Jing-Bo; Wang, Yan-Chao; Gao, Ji-Juan; Sha, Yan-Hua; Zheng, Lei; Wang, Qian

    2014-01-01

    Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE−/− mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE−/− mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1β, IL-6, and TNF-α levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE−/− mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE−/− mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent. PMID:24493833

  19. Influence of four polymorphisms in ABCA1 and PTGS2 genes on risk of Alzheimer's disease: a meta-analysis.

    PubMed

    Chen, Qicong; Liang, Biyu; Wang, Ziyou; Cheng, Xiaoguang; Huang, Yifeng; Liu, Yong; Huang, Zunnan

    2016-08-01

    We preformed this meta-analysis to investigate the influence of ABCA1 (ATP-binding cassette sub-family A member 1) rs2422493 (C-477T), rs1800977 (C-14T), rs2066718 (V771M), and PTGS2 (Prostaglandin-endoperoxide synthase 2) rs20417 (G-765C) polymorphisms on the risk of Alzheimer's disease (AD). Seventeen eligible case-control studies were acquired from PubMed, Embase, Alzgene, Chinese National Knowledge Infrastructure and Wanfang databases. The pooled odds ratios (ORs) with 95 % confidence intervals (95 % CI) were calculated to evaluate the association under five genetic models. Combined data indicated that ABCA1 rs2422493 polymorphism was statistically significant associated with increasing AD risk in three genetic models (allelic T vs C: OR = 1.12, 95 % CI: 1.01-1.24; homozygous TT vs CC: OR = 1.26, 95 % CI: 1.03-1.55; and recessive TT vs TC + CC: OR = 1.33, 95 % CI: 1.12-1.58) while no association was found between two other ABCA1 polymorphisms and AD susceptibility. Nevertheless, a further risk-stratification analysis showed that ApoE-ε4 carriers with any ABCA1 polymorphism suffered a much higher probability to be AD patients. Meanwhile, PTGS2 rs20417 polymorphism was linked to decreasing AD risk with a P < 0.0001 in five genetic models (e.g., allelic C vs G: OR = 0.59, 95 % CI: 0.50-0.70; homozygous CC vs GG: OR = 0.31, 95 % CI: 0.18-0.52; and heterozygous CG vs GG: OR = 0.64, 95 % CI: 0.52-0.78). In summary, our meta-analysis results showed that ABCA1 rs2422493 polymorphism was a risk factor for AD while PTGS2 rs20417 variant showed a protective effect on AD risk. In addition, ABCA1 rs2066718 and rs1800977 polymorphisms might not contribute to AD susceptibility in general population, but they should play a role on AD development when interacted with ApoE-ε4. PMID:27215623

  20. Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages.

    PubMed

    Shrestha, Elina; Hussein, Maryem A; Savas, Jeffery N; Ouimet, Mireille; Barrett, Tessa J; Leone, Sarah; Yates, John R; Moore, Kathryn J; Fisher, Edward A; Garabedian, Michael J

    2016-05-20

    Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis. PMID:27026705

  1. The Novel Anticancer Agent JNJ-26854165 Induces Cell Death through Inhibition of Cholesterol Transport and Degradation of ABCA1

    PubMed Central

    Gu, Dongmin; Bjorklund, Chad C.; Kuiatse, Isere; Remaley, Alan T.; Bashir, Tarig; Vreys, Veronique; Orlowski, Robert Z.

    2013-01-01

    JNJ-26854165 (serdemetan) has previously been reported to inhibit the function of the E3 ligase human double minute 2, and we initially sought to characterize its activity in models of mantle cell lymphoma (MCL) and multiple myeloma (MM). Serdemetan induced a dose-dependent inhibition of proliferation in both wild-type (wt) and mutant (mut) p53 cell lines, with IC50 values from 0.25 to 3 μM/l, in association with an S phase cell cycle arrest. Caspase-3 activation was primarily seen in wtp53-bearing cells but also occurred in mutp53-bearing cells, albeit to a lesser extent. 293T cells treated with JNJ-26854165 and serdemetan-resistant fibroblasts displayed accumulation of cholesterol within endosomes, a phenotype reminiscent of that seen in the ATP-binding cassette subfamily A member-1 (ABCA1) cholesterol transport disorder, Tangiers disease. MM and MCL cells had decreased cholesterol efflux and electron microscopy demonstrated the accumulation of lipid whorls, confirming the lysosomal storage disease phenotype. JNJ-26854165 induced induction of cholesterol regulatory genes, sterol regulatory element-binding transcription factor-1 and -2, liver X receptors α and β, along with increased expression of Niemann-Pick disease type-C1 and -C2. However, JNJ-26854165 induced enhanced ABCA1 turnover despite enhancing transcription. Finally, ABCA1 depletion resulted in enhanced sensitivity to JNJ-26854165. Overall, these findings support the hypothesis that serdemetan functions in part by inhibiting cholesterol transport and that this pathway is a potential new target for the treatment of MCL and MM. PMID:23820125

  2. Lipoxin A4 promotes ABCA1 expression and cholesterol efflux through the LXRα signaling pathway in THP-1 macrophage-derived foam cells

    PubMed Central

    Sha, Yan-Hua; Hu, Yan-Wei; Gao, Ji-Juan; Wang, Yan-Chao; Ma, Xin; Qiu, Yu-Rong; Li, Shu-Fen; Zhao, Jia-Yi; Huang, Chuan; Zhao, Jing-Jing; Lu, Jing-Bo; Kang, Chun-Min; Zheng, Lei; Wang, Qian

    2015-01-01

    Adenosine triphosphate-binding cassette transporter A1 (ABCA1) is a crucial cholesterol transporter and plays a central role in the high density lipoproteins (HDL) cholesterol metabolism and lipid clearance from the foam cell. Lipoxin A4 (LXA4) is an endogenous lipid mediator that requires cell-cell interaction or cell-platelet interaction for its synthesis. The roles of LXA4 on inflammatory responses are well described, while its effects on mediating ABCA1 and underlying mechanisms remain unclear. In this study, we showed that LXA4 significantly increases expression of ABCA1 and LXRα in a dose-dependent manner in THP-1 macrophage-derived foam cells. Cellular cholesterol content was decreased while cholesterol efflux was increased by LXA4 treatment. However, after short interfering RNA of LXRα, the effects of LXA4 on ABCA1 expression and cholesterol metabolism were significantly abolished. These results provide evidence that LXA4 increases ABCA1 expression and promotes cholesterol efflux through LXRα pathway in THP-1 macrophage-derived foam cells. PMID:26261553

  3. HDLs in apoA-I transgenic Abca1 knockout mice are remodelednormally in plasma but are hypercatabolized by the kidney.

    SciTech Connect

    Lee, Ji-Young; Timmins, Jenelle M.; Mulya, Anny; Smith, ThomasL.; Zhu, Yiwen; Rubin, Edward M.; Chisholm, Jeffrey W.; Colvin, Perry L.; Parks, John S.

    2005-07-05

    Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with 125I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-ITg) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P<0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-ITg mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo.

  4. Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway[S

    PubMed Central

    Pamir, Nathalie; Hutchins, Patrick; Ronsein, Graziella; Vaisar, Tomas; Reardon, Catherine A.; Getz, Godfrey S.; Lusis, Aldons J.; Heinecke, Jay W.

    2016-01-01

    Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages. PMID:26673204

  5. Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

    PubMed Central

    Fellous, A; Ginzburg, I; Littauer, U Z

    1982-01-01

    Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6964957

  6. Chromosome 9p21 and ABCA1 Genetic Variants and Their Interactions on Coronary Heart Disease and Ischemic Stroke in a Chinese Han Population.

    PubMed

    Cao, Xiao-Li; Yin, Rui-Xing; Huang, Feng; Wu, Jin-Zhen; Chen, Wu-Xian

    2016-01-01

    The single nucleotide polymorphisms (SNPs) related to both coronary heart disease (CHD) and ischemic stroke (IS) in Chinese individuals have not been identified definitely. This study was developed to evaluate the genetic susceptibility to CHD and IS on the chromosome 9p21 and the adenosine triphosphate (ATP)-binding cassette transporter A1 genes (ABCA1) in a Chinese Han population. Genotypes of the rs1333040, rs1333042, rs4977574, rs2066715 and rs2740483 SNPs were determined in 1134 unrelated patients (CHD, 565 and IS, 569) and 541 controls. The frequencies of the rs4977574 genotypes and alleles between CHD and control groups, and the rs2740483 genotypes and alleles between IS and control groups were different (p = 0.006-0.001). The subjects with rs1333042GG genotype and the carriers of the rs4977574G allele were associated with increased risk of CHD. The carriers of the rs4977574G allele were associated with increased risk of IS. However, the carriers of the rs2740483C allele had lower risk of IS than the non-carriers of the rs2740483C allele after controlling for potential confounders. The rs4977574GG-age (>60 year) interaction increased the risk of CHD (p = 0.022), whereas the rs2740483CG/CC-body mass index (>24 kg/m²) interaction decreased the risk of IS (p = 0.035). The interactions of rs1333040-rs1333042 on the risk of CHD and IS were relatively strong, whereas the interactions of rs1333040-rs1333042-rs2066715 and rs1333040-rs1333042-rs2066715-rs2740483 on the risk of CHD, and rs1333040-rs1333042-rs4977574 and rs1333040-rs1333042-rs4977574-rs2740483 on the risk of IS were relatively weak. These findings suggest that some common variants on the chromosome 9p21 and ABCA1 and their interactions may significantly modify the risk of CHD and IS independent of effects on serum lipid levels. PMID:27096864

  7. Chromosome 9p21 and ABCA1 Genetic Variants and Their Interactions on Coronary Heart Disease and Ischemic Stroke in a Chinese Han Population

    PubMed Central

    Cao, Xiao-Li; Yin, Rui-Xing; Huang, Feng; Wu, Jin-Zhen; Chen, Wu-Xian

    2016-01-01

    The single nucleotide polymorphisms (SNPs) related to both coronary heart disease (CHD) and ischemic stroke (IS) in Chinese individuals have not been identified definitely. This study was developed to evaluate the genetic susceptibility to CHD and IS on the chromosome 9p21 and the adenosine triphosphate (ATP)-binding cassette transporter A1 genes (ABCA1) in a Chinese Han population. Genotypes of the rs1333040, rs1333042, rs4977574, rs2066715 and rs2740483 SNPs were determined in 1134 unrelated patients (CHD, 565 and IS, 569) and 541 controls. The frequencies of the rs4977574 genotypes and alleles between CHD and control groups, and the rs2740483 genotypes and alleles between IS and control groups were different (p = 0.006–0.001). The subjects with rs1333042GG genotype and the carriers of the rs4977574G allele were associated with increased risk of CHD. The carriers of the rs4977574G allele were associated with increased risk of IS. However, the carriers of the rs2740483C allele had lower risk of IS than the non-carriers of the rs2740483C allele after controlling for potential confounders. The rs4977574GG-age (>60 year) interaction increased the risk of CHD (p = 0.022), whereas the rs2740483CG/CC-body mass index (>24 kg/m2) interaction decreased the risk of IS (p = 0.035). The interactions of rs1333040-rs1333042 on the risk of CHD and IS were relatively strong, whereas the interactions of rs1333040-rs1333042-rs2066715 and rs1333040-rs1333042-rs2066715-rs2740483 on the risk of CHD, and rs1333040-rs1333042-rs4977574 and rs1333040-rs1333042-rs4977574-rs2740483 on the risk of IS were relatively weak. These findings suggest that some common variants on the chromosome 9p21 and ABCA1 and their interactions may significantly modify the risk of CHD and IS independent of effects on serum lipid levels. PMID:27096864

  8. Common variants near ABCA1, AFAP1 and GMDS confer risk of primary open-angle glaucoma

    PubMed Central

    Fogarty, Rhys; Sharma, Shiwani; Hewitt, Alex W.; Martin, Sarah; Law, Matthew H.; Cremin, Katie; Bailey, Jessica N. Cooke; Loomis, Stephanie J.; Pasquale, Louis R.; Haines, Jonathan L.; Hauser, Michael A.; Viswanathan, Ananth C.; McGuffin, Peter; Topouzis, Fotis; Foster, Paul J.; Graham, Stuart L; Casson, Robert J; Chehade, Mark; White, Andrew J; Zhou, Tiger; Souzeau, Emmanuelle; Landers, John; Fitzgerald, Jude T; Klebe, Sonja; Ruddle, Jonathan B; Goldberg, Ivan; Healey, Paul R; Mills, Richard A.; Wang, Jie Jin; Montgomery, Grant W.; Martin, Nicholas G.; Radford-Smith, Graham; Whiteman, David C.; Brown, Matthew A.; Wiggs, Janey L.; Mackey, David A; Mitchell, Paul; MacGregor, Stuart; Craig, Jamie E.

    2014-01-01

    Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness worldwide. We performed a genome-wide association study in an Australian discovery cohort comprising 1,155 advanced POAG cases and 1,992 controls. Association of the top SNPs from the discovery stage was investigated in two Australian replication cohorts (total 932 cases, 6,862 controls) and two US replication cohorts (total 2,616 cases, 2,634 controls). Meta-analysis of all cohorts revealed three novel loci associated with development of POAG. These loci are located upstream of ABCA1 (rs2472493 [G] OR=1.31, P= 2.1 × 10−19), within AFAP1 (rs4619890 [G] OR=1.20, P= 7.0 × 10−10) and within GMDS (rs11969985 [G] OR=1.31, and P= 7.7 × 10−10). Using RT-PCR and immunolabelling, we also showed that these genes are expressed within human retina, optic nerve and trabecular meshwork and that ABCA1 and AFAP1 are also expressed in retinal ganglion cells. PMID:25173105

  9. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  10. PROLONGED FASTING AND CORTISOL REDUCE MYOSTATIN MRNA LEVELS IN TILAPIA LARVAE, SHORT-TERM FASTING ELEVATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indices in adults, skeletal muscle myostatin mRNA...

  11. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    SciTech Connect

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  12. Impact of STAT/SOCS mRNA Expression Levels after Major Injury

    PubMed Central

    Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

    2014-01-01

    Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

  13. The effects of ABCG5/G8 polymorphisms on HDL-cholesterol concentrations depend on ABCA1 genetic variants in the Boston Puerto Rican health study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background and aims: ATP-binding cassette transporters G5/G8 (ABCG5/G8) are associated with HDL-C concentrations. To assess whether the effect of ABCG5/G8 genetic variants on HDL-C concentrations is dependent on ATP-binding cassette transporters A1 (ABCA1), we studied potential interactions between ...

  14. Differential Control of Interleukin-6 mRNA Levels by Cellular Distribution of YB-1

    PubMed Central

    Kang, Sujin; Lee, Taeyun A.; Ra, Eun A.; Lee, Eunhye; Choi, Hyun jin; Lee, Sungwook; Park, Boyoun

    2014-01-01

    Cytokine production is essential for innate and adaptive immunity against microbial invaders and must be tightly controlled. Cytokine messenger RNA (mRNA) is in constant flux between the nucleus and the cytoplasm and in transcription, splicing, or decay; such processes must be tightly controlled. Here, we report a novel function of Y-box-binding protein 1 (YB-1) in modulating interleukin-6 (IL-6) mRNA levels in a cell type-specific manner. In lipopolysaccharide (LPS)-stimulated macrophages, YB-1 interacts with IL-6 mRNA and actively transports it to the extracellular space by YB-1-enriched vesicles, resulting in the proper maintenance of intracellular IL-6 mRNA levels. YB-1 secretion occurs in a cell type-specific manner. Whereas macrophages actively secret YB-1, dendritic cells maintain it predominantly in the cytoplasm even in response to LPS. Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells. Moreover, because LPS differentially regulates the expression of histone deacetylase 6 (HDAC6) in macrophages and dendritic cells, this stimulus might control YB-1 acetylation differentially in both cell types. Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1. PMID:25398005

  15. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus

    SciTech Connect

    Koller, K.J.; Wolff, R.S.; Warden, M.K.; Zoeller, R.T.

    1987-10-01

    Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone, replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a /sup 35/S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatments with exogenous L-triiodothyronine (T/sub 3/) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T/sub 3/ replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-..beta.. mRNA levels were elevated after thyroidectomy and reduced by T/sub 3/ treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

  16. Myeloid Cell-Specific ABCA1 Deletion Has Minimal Impact on Atherogenesis in Atherogenic Diet-Fed LDL Receptor Knockout Mice

    PubMed Central

    Bi, Xin; Zhu, Xuewei; Gao, Chuan; Shewale, Swapnil; Cao, Qiang; Liu, Mingxia; Boudyguina, Elena; Gebre, Abraham K.; Wilson, Martha D.; Brown, Amanda L.; Parks, John S.

    2014-01-01

    Objective Transplantation studies suggest that bone marrow (BM) cell ABCA1 protects against atherosclerosis development. However, the in vivo impact of macrophage ABCA1 expression on atherogenesis is not fully understood because BM contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ABCA1 knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question. Approach and Results Chow-fed MSKO/LDLrKO (DKO) vs. LDLrKO (SKO) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma VLDL/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO vs. SKO mice had significantly higher cholesterol content, but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10–16 wks of AD, but increased modestly in DKO mice by 24 wks of AD. Lesional macrophage content was similar, likely due to higher monocyte flux through aortic root lesions in DKO vs. SKO mice. After transplantation of DKO or SKO BM into SKO mice and 16 wk of AD feeding, atherosclerosis extent was similar and plasma apoB lipoproteins was reduced in mice receiving DKO BM. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 wks, DKO mice had modest, but significantly more, atherosclerosis compared to SKO mice. Conclusions Myeloid cell ABCA1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDLrKO mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in minimal increase in atherosclerosis. PMID:24833800

  17. Corticotropin-Releasing Hormone (CRH) Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1).

    PubMed

    Cho, Wonkyoung; Kang, Jihee Lee; Park, Young Mi

    2015-01-01

    Atherosclerosis, the major pathology of cardiovascular disease, is caused by multiple factors involving psychological stress. Corticotropin-releasing hormone (CRH), which is released by neurosecretory cells in the hypothalamus, peripheral nerve terminals and epithelial cells, regulates various stress-related responses. Our current study aimed to verify the role of CRH in macrophage foam cell formation, the initial critical stage of atherosclerosis. Our quantitative real-time reverse transcriptase PCR (qRT-PCR), semi-quantitative reverse transcriptase PCR, and Western blot results indicate that CRH down-regulates ATP-binding cassette transporter-1 (ABCA1) and liver X receptor (LXR)-α, a transcription factor for ABCA1, in murine peritoneal macrophages and human monocyte-derived macrophages. Oil-red O (ORO) staining and intracellular cholesterol measurement of macrophages treated with or without oxidized LDL (oxLDL) and with or without CRH (10 nM) in the presence of apolipoprotein A1 (apoA1) revealed that CRH treatment promotes macrophage foam cell formation. The boron-dipyrromethene (BODIPY)-conjugated cholesterol efflux assay showed that CRH treatment reduces macrophage cholesterol efflux. Western blot analysis showed that CRH-induced down-regulation of ABCA1 is dependent on phosphorylation of Akt (Ser473) induced by interaction between CRH and CRH receptor 1(CRHR1). We conclude that activation of this pathway by CRH accelerates macrophage foam cell formation and may promote stress-related atherosclerosis. PMID:26110874

  18. Transcriptional Bursting Explains the Noise–Versus–Mean Relationship in mRNA and Protein Levels

    PubMed Central

    Dar, Roy D.; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean. PMID:27467384

  19. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    DOE PAGESBeta

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  20. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGESBeta

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  1. Ammonium Chloride Ingestion Attenuates Exercise-Induced mRNA Levels in Human Muscle

    PubMed Central

    Mündel, Toby; Pilegaard, Henriette; Hawke, Emma; Leikis, Murray; Lopez-Villalobos, Nicolas; Oliveira, Rodrigo S. F.; Bishop, David J.

    2015-01-01

    Minimizing the decrease in intracellular pH during high-intensity exercise training promotes greater improvements in mitochondrial respiration. This raises the intriguing hypothesis that pH may affect the exercise-induced transcription of genes that regulate mitochondrial biogenesis. Eight males performed 10x2-min cycle intervals at 80% V˙O2peak intensity on two occasions separated by ~2 weeks. Participants ingested either ammonium chloride (ACID) or calcium carbonate (PLA) the day before and on the day of the exercise trial in a randomized, counterbalanced order, using a crossover design. Biopsies were taken from the vastus lateralis muscle before and after exercise. The mRNA level of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α), citrate synthase, cytochome c and FOXO1 was elevated at rest following ACID (P<0.05). During the PLA condition, the mRNA content of mitochondrial- and glucose-regulating proteins was elevated immediately following exercise (P<0.05). In the early phase (0–2 h) of post-exercise recovery during ACID, PGC-1α, citrate synthase, cytochome C, FOXO1, GLUT4, and HKII mRNA levels were not different from resting levels (P>0.05); the difference in PGC-1α mRNA content 2 h post-exercise between ACID and PLA was not significant (P = 0.08). Thus, metabolic acidosis abolished the early post-exercise increase of PGC-1α mRNA and the mRNA of downstream mitochondrial and glucose-regulating proteins. These findings indicate that metabolic acidosis may affect mitochondrial biogenesis, with divergent responses in resting and post-exercise skeletal muscle. PMID:26656911

  2. Ascorbate free radical reductase mRNA levels are induced by wounding.

    PubMed Central

    Grantz, A A; Brummell, D A; Bennett, A B

    1995-01-01

    A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. PMID:7784511

  3. Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

    NASA Astrophysics Data System (ADS)

    Luo, Wen; Zhao, Yunlong; Zhou, Zhongliang; An, Chuanguang; Ma, Qiang

    2008-02-01

    The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR (FQ—PCR) methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

  4. Osmoregulation of Na(+)-inositol cotransporter activity and mRNA levels in brain glial cells.

    PubMed

    Paredes, A; McManus, M; Kwon, H M; Strange, K

    1992-12-01

    During plasma hypertonicity brain volume is regulated acutely by electrolyte uptake and chronically by accumulation of organic solutes such as inositol. Cultured rat C6 glioma cells, an astrocyte-like cell line, show a similar pattern of volume control. Volume regulatory accumulation of inositol requires external inositol, indicating that membrane transport plays a central role in this process. The inositol uptake pathway is Na+ dependent and exhibits Michaelis-Menten kinetics. Chronic hypertonic acclimation results in a twofold increase in the maximum velocity of the transporter without changing the Km. Hypertonic stress also results in a 17-fold increase in transporter mRNA. Elevation of mRNA levels precedes activation of the transporter by 4-6 h, suggesting that increased inositol uptake is mediated by synthesis and membrane insertion of new transport proteins. Reacclimation of hypertonic cells to isotonicity causes a rapid reduction of transporter mRNA levels to control levels within 4 h. In contrast, downregulation of transport activity does not begin until between 10 and 24 h after reexposure to isotonicity. PMID:1476169

  5. Comparison of IgE expression at the mRNA and protein levels in vitro.

    PubMed Central

    Turner, K J; Creany, J; Coelen, R J; Cameron, K J; Holt, B J; Beilharz, M W

    1991-01-01

    The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay. Images Figure 1 PMID:1783428

  6. Smooth Muscle Cell Foam Cell Formation, Apolipoproteins, and ABCA1 in Intracranial Aneurysms: Implications for Lipid Accumulation as a Promoter of Aneurysm Wall Rupture.

    PubMed

    Ollikainen, Eliisa; Tulamo, Riikka; Lehti, Satu; Lee-Rueckert, Miriam; Hernesniemi, Juha; Niemelä, Mika; Ylä-Herttuala, Seppo; Kovanen, Petri T; Frösen, Juhana

    2016-07-01

    Saccular intracranial aneurysm (sIA) aneurysm causes intracranial hemorrhages that are associated with high mortality. Lipid accumulation and chronic inflammation occur in the sIA wall. A major mechanism for lipid clearance from arteries is adenosine triphosphate-binding cassette A1 (ABCA1)-mediated lipid efflux from foam cells to apolipoprotein A-I (apoA-I). We investigated the association of wall degeneration, inflammation, and lipid-related parameters in tissue samples of 16 unruptured and 20 ruptured sIAs using histology and immunohistochemistry. Intracellular lipid accumulation was associated with wall remodeling (p = 0.005) and rupture (p = 0.020). Foam cell formation was observed in smooth muscle cells, in addition to CD68- and CD163-positive macrophages. Macrophage infiltration correlated with intracellular lipid accumulation and apolipoproteins, including apoA-I. ApoA-I correlated with markers of lipid accumulation and wall degeneration (p = 0.01). ApoA-I-positive staining colocalized with ABCA1-positive cells particularly in sIAs with high number of smooth muscle cells (p = 0.003); absence of such colocalization was associated with wall degeneration (p = 0.017). Known clinical risk factors for sIA rupture correlated inversely with apoA-I. We conclude that lipid accumulation associates with sIA wall degeneration and risk of rupture, possibly via formation of foam cells and subsequent loss of mural cells. Reduced removal of lipids from the sIA wall via ABCA1-apoA-I pathway may contribute to this process. PMID:27283327

  7. Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

    PubMed

    Schäfer-Somi, S; Palme, N

    2016-04-01

    The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings. PMID:26848092

  8. Codon influence on protein expression in E. coli correlates with mRNA levels.

    PubMed

    Boël, Grégory; Letso, Reka; Neely, Helen; Price, W Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D; Valecha, Mayank; Everett, John K; Acton, Thomas B; Xiao, Rong; Montelione, Gaetano T; Aalberts, Daniel P; Hunt, John F

    2016-01-21

    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

  9. Novel beta-spectrin mutations in hereditary spherocytosis associated with decreased levels of mRNA.

    PubMed

    Maciag, Monika; Płochocka, Danuta; Adamowicz-Salach, Anna; Burzyńska, Beata

    2009-08-01

    Hereditary spherocytosis (HS) is one of the most frequent and heterogeneous inherited haemolytic anaemias. It is associated with abnormalities of several erythrocyte membrane proteins. We investigated relative mRNA quantification of red blood cell membrane protein genes using real-time quantitative polymerase chain reaction (qPCR) in order to better characterize HS cases and to select genes to search for mutations in patients with spherocytosis. qPCR experiments indicated that the spectrin beta gene (SPTB) could be involved in anaemia pathogenesis. DNA analysis of SPTB in the HS subjects with decreased SPTB mRNA levels revealed the presence of five previously undescribed mutations: R1756X, 781delT and IVS22nt-4G>A, 1502insA and IVS20nt-2A>G. PMID:19538529

  10. Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates.

    PubMed

    Rodgers, Buel D; Weber, Gregory M; Kelley, Kevin M; Levine, Michael A

    2003-05-01

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indexes in adults, skeletal muscle myostatin mRNA levels were unaffected. By contrast, larval myostatin mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting. These effects were specific for myostatin, as mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase were unchanged. Cortisol levels were elevated in fasted larvae with reduced myostatin mRNA, whereas in addition immersion of larvae in 1 ppm (2.8 microM) cortisol reduced myostatin mRNA in a time-dependent fashion. These results suggest that larval myostatin mRNA levels may initially rise but ultimately fall during a prolonged fast. The reduction is likely mediated by fasting-induced hypercortisolemia, indicating divergent evolutionary mechanisms of glucocorticoid regulation of myostatin mRNA, since these steroids upregulate myostatin gene expression in mammals. PMID:12676749

  11. Analysis of xanthine dehydrogenase mRNA levels in mutants affecting the expression of the rosy locus.

    PubMed Central

    Covington, M; Fleenor, D; Devlin, R B

    1984-01-01

    Xanthine dehydrogenase (XDH) mRNA levels were measured in a number of mutants and natural variants affecting XDH gene expression. Two variants, ry+4 and ry+10, contain cis-acting elements which map to a region flanking the 5' end of the XDH gene. Ry+4, which has 2-3 times more XDH protein than a wild type strain, has 3.2 times more XDH mRNA. Ry+10 has 50% of the wild type XDH level and 54% of the wild type XDH mRNA level. Three rosy mutants which map within the structural gene were also examined. Two of these had little if any XDH mRNA, but the third mutant had 1.3 times more XDH mRNA than wild type flies. Another mutant, ry2 , which contains no XDH protein and has a 9KB transposable element inserted into the XDH gene, has normal levels of XDH mRNA transcripts which are also the same size as those found in the wild type strain. Changes in XDH mRNA levels were measured during Drosophila development and found to parallel changes in the amount of XDH protein. In addition, there were no large changes in the size of XDH mRNA during development. Images PMID:6588363

  12. Reduced mRNA expression levels of MBD2 and MBD3 in gastric carcinogenesis.

    PubMed

    Pontes, Thaís Brilhante; Chen, Elizabeth Suchi; Gigek, Carolina Oliveira; Calcagno, Danielle Queiroz; Wisnieski, Fernanda; Leal, Mariana Ferreira; Demachki, Samia; Assumpção, Paulo Pimentel; Artigiani, Ricardo; Lourenço, Laércio Gomes; Burbano, Rommel Rodriguez; Arruda Cardoso Smith, Marília

    2014-04-01

    Aberrant methylation has been reported in several neoplasias, including gastric cancer. The methyl-CpG-binding domain (MBD) family proteins have been implicated in the chromatin remodeling process, leading to the modulation of gene expression. To evaluate the role of MBD2 and MBD3 in gastric carcinogenesis and the possible association with clinicopathological characteristics, we assessed the mRNA levels and promoter methylation patterns in gastric tissues. In this study, MBD2 and MBD3 mRNA levels were determined by RT-qPCR in 28 neoplastic and adjacent nonneoplastic and 27 gastritis and non-gastritis samples. The promoter methylation status was determined by bisulfite sequencing, and we found reduced MBD2 and MBD3 levels in the neoplastic samples compared with the other groups. Moreover, a strong correlation between the MBD2 and MBD3 expression levels was observed in each set of paired samples. Our data also showed that the neoplastic tissues exhibited higher MBD2 promoter methylation than the other groups. Interestingly, the non-gastritis group was the only one with positive methylation in the MBD3 promoter region. Furthermore, a weak correlation between gene expression and methylation was observed. Therefore, our data suggest that DNA methylation plays a minor role in the regulation of MBD2 and MBD3 expression, and the presence of methylation at CpGs that interact with transcription factor complexes might also be involved in the modulation of these genes. Moreover, reduced mRNA expression of MBD2 and MBD3 is implicated in gastric carcinogenesis, and thus, further investigations about these genes should be conducted for a better understanding of the role of abnormal methylation involved in this neoplasia. PMID:24338710

  13. Hibiscus sabdariffa leaf polyphenolic extract inhibits LDL oxidation and foam cell formation involving up-regulation of LXRα/ABCA1 pathway.

    PubMed

    Chen, Jing-Hsien; Wang, Chau-Jong; Wang, Chi-Ping; Sheu, Jenn-Yuan; Lin, Chia-Liang; Lin, Hui-Hsuan

    2013-11-01

    The oxidative modification of low-density lipoprotein (LDL) is involved in the pathogenesis of atherosclerotic lesions through the formation of macrophage-derived foam cells. In the present study, we aimed to investigate the anti-atherosclerotic effect of Hibiscus sabdariffa leaf polyphenolic extract (HLP), which is rich in flavonoid. The inhibitory effect of HLP on oxidation and lipid peroxidation of LDL was defined in vitro. HLP showed potential in reducing foam cell formation and intracellular lipid accumulation in oxidised-LDL (ox-LDL)-induced macrophage J774A.1 cells under non-cytotoxic concentrations. Molecular data showed these influences of HLP might be mediated via liver-X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) pathway, as demonstrated by the transfection of LXRα siRNA. Our data implied that HLP up-regulated the LXRα/ABCA1 pathway, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that HLP potentially could be developed as an anti-atherosclerotic agent. PMID:23768373

  14. Acute intermittent morphine increases preprodynorphin and kappa opioid receptor mRNA levels in the rat brain.

    PubMed

    Wang, X M; Zhou, Y; Spangler, R; Ho, A; Han, J S; Kreek, M J

    1999-03-20

    We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain. PMID:10095091

  15. Thyroid sialyltransferase mRNA level and activity are increased in Graves' disease.

    PubMed

    Kiljański, Jacek; Ambroziak, Michał; Pachucki, Janusz; Jazdzewski, Krystian; Wiechno, Wieslaw; Stachlewska, Elzbieta; Górnicka, Barbara; Bogdańska, Magdalena; Nauman, Janusz; Bartoszewicz, Zbigniew

    2005-07-01

    Sialylation of cell components is an important immunomodulating mechanism affecting cell response to hormones and adhesion molecules. To study alterations in sialic acid metabolism in Graves' disease (GD) we measured the following parameters in various human thyroid tissues: lipid-bound sialic acid (LBSA) content, ganglioside profile, total sialyltransferase activity, and the two major sialyltransferase mRNAs for sialyltransferase-1 (ST6Gal I) and for sialyltransferase-4A (ST3Gal I). Fragments of toxic thyroid nodules (TN), nontoxic thyroid nodules (NN) and nontumorous tissue from patients with nodular goiter or thyroid cancer were used as a control (C). The LBSA content and sialyltransferase activity were the highest in the GD group (164 +/- 4.44 versus 120 +/- 2.00 nmoL/g, p = 0.005 and 1625 +/- 283.5 versus 324 +/- 54.2 cpm/mg of protein, p < 0.005 compared to control group C). Ganglioside profile in the GD group was similar to that in control tissues. Sialyltransferase- 1 mRNA and sialyltransferase-4A mRNA levels were significantly higher in the GD group than in the control group (12.52 +/- 6.90 versus 2.54 +/- 1.24 arbitrary units, p < 0.005 and 2,49 +/- 1.16 versus 1.23 +/- 0.46 arbitrary units, p < 0.05, respectively). There was a positive correlation between the increased sialyltransferase-1 mRNA level and the TSH-receptor antibody titer determined by the TRAK test. These results indicate that sialyltransferases expression and activity are increased in GD. Exact mechanism of this upregulation remains unknown, though one of possible explanations is the activation of the thyrotropin (TSH) receptor. PMID:16053379

  16. Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

    PubMed

    Hashikawa, Naoya; Ogawa, Takumi; Sakamoto, Yusuke; Ogawa, Mami; Matsuo, Yumi; Zamami, Yoshito; Hashikawa-Hobara, Narumi

    2015-08-01

    Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test. PMID:25820756

  17. Dietary copper can regulate the level of mRNA for dopamine B-hydroxylase in rat adrenal gland

    SciTech Connect

    Sabban, E.L.; Failla, M.L.; McMahon, A.; Seidel, K.E. Dept. of Agriculture, Beltsville, MD )

    1991-03-15

    Recent studies have shown that Cu deficiency markedly alters the levels of dopamine (DA) and norepinephrine (NE) in several peripheral tissues of rodents. Conversion of DA to NE is mediated by dopamine B-hydroxylase (DBM). Here the authors examined the effect of dietary Cu deficiency on the levels of DA, NE and DBM mRNA in rat adrenal gland. Severe Cu deficiency was induced by feeding low Cu diet to dams beginning at 17d gestation and weaning pups to the same diet. At 7 wks of age rats fed {minus}Cu diet were characterized by depressed growth, low tissue Cu, enlarged hearts and moderate anemia. Concentrations of DA were higher in adrenals and hearts of {minus}Cu rats compared to +Cu controls. While cardiac level of NE in {minus}Cu rats were reduced to 17% that of controls, adrenal NE was unchanged by Cu deficiency. To investigate possible mechanisms responsible for the response of adrenal gland to Cu deficiency, RNA was isolated and the levels of DBH mRNA and tyrosine hydroxylase (TH) mRNA were analyzed by Northern blots. Steady state levels of adrenal DBH mRNA was increased 2-3 fold in {minus}Cu rats, whereas TH mRNA were unchanged by dietary Cu status. Upon feeding the {minus}Cu rats the Cu adequate diet overnight, there was a further increase in DBH mRNA and a slight elevation of TH mRNA levels. The results indicate that dietary copper can markedly affect the level of DBH mRNA in rat adrenal gland.

  18. Melatonin receptor deficiency decreases and temporally shifts ecto-5'-nucleotidase mRNA levels in mouse prosencephalon.

    PubMed

    Homola, Moran; Pfeffer, Martina; Robson, Simon C; Fischer, Claudia; Zimmermann, Herbert; Korf, Horst-Werner

    2016-07-01

    Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse brain. Via the production of adenosine, eN participates in many physiological and pathological processes, such as wakefulness, inflammation, nociception and neuroprotection. The mechanisms regulating the expression of eN are therefore of considerable neurobiological and clinical interest. Having previously described a modulatory effect of melatonin in the regulation of eN mRNA levels, we decided to analyze the melatonin receptor subtype involved in the regulation of eN mRNA levels by comparing eN mRNA patterns in melatonin-proficient transgenic mice lacking either the melatonin receptor subtype 1 (MT1 KO) or both melatonin receptor subtypes (MT1 and MT2; MT1/2 KO) with the corresponding melatonin-proficient wild-type (WT) controls. By means of radioactive in situ hybridization, eN mRNA levels were found to be diminished in both MT1 and MT1/2 KO mice compared with WT controls suggesting stimulatory impacts of melatonin receptors on eN mRNA levels. Whereas eN mRNA levels increased during the day and peaked at night in WT and MT1 KO mice, eN mRNA levels at night were reduced and the peak was shifted toward day-time in double MT1/2 KO mice. These data suggest that the MT2 receptor subtype may play a role in the temporal regulation of eN mRNA availability. Notably, day-time locomotor activity was significantly higher in MT1/2 KO compared with WT mice. Our results suggest melatoninergic signaling as an interface between the purinergic system and the circadian system. PMID:26917036

  19. RIP140 triggers foam-cell formation by repressing ABCA1/G1 expression and cholesterol efflux via liver X receptor.

    PubMed

    He, Yanhong; Zhang, Luankun; Li, Zhuoming; Gao, Hui; Yue, Zhongbao; Liu, Zhiping; Liu, Xueping; Feng, Xiaojun; Liu, Peiqing

    2015-02-13

    Receptor-interacting protein 140 (RIP140) is a multifunctional coregulator of lipid metabolism and inflammation. However, the potential role of RIP140 in atherosclerosis remains unknown. The present study investigated the impact of RIP140 on foam cell formation, a critical step in pathogenesis of atherosclerosis. The expression of RIP140 was increased in foam cells. RIP140 overexpression resulted in decreased cholesterol efflux in macrophages and their concomitant differentiation into foam cells. Moreover, RIP140 negatively regulated the macrophage expression of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), by suppressing the expression and activity of liver X receptor (LXR). These findings shed light onto the contribution of RIP140 to the development and progression of atherosclerosis, and suggest a novel therapeutic target for the treatment of atherosclerosis. PMID:25616132

  20. Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis☆

    PubMed Central

    Chou, Chi-Wen; Chiang, Tsay-I; Chang, I-Chang; Huang, Chung-Hung; Cheng, Ya-Wen

    2016-01-01

    Background The up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. Methods In this study, we enrolled 107 women older than 45 years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. Results ERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (> 55 years), and low ERα mRNA levels in PBLs (≦ 250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. Conclusion We conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significance Our findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels. PMID:27051599

  1. mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

    PubMed

    Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

    2015-06-01

    The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme. PMID:25963717

  2. Reduced XPC DNA repair gene mRNA levels in clinically normal parents of xeroderma pigmentosum patients.

    PubMed

    Khan, Sikandar G; Oh, Kyu-Seon; Shahlavi, Tala; Ueda, Takahiro; Busch, David B; Inui, Hiroki; Emmert, Steffen; Imoto, Kyoko; Muniz-Medina, Vanessa; Baker, Carl C; DiGiovanna, John J; Schmidt, Deborah; Khadavi, Arash; Metin, Ahmet; Gozukara, Engin; Slor, Hanoch; Sarasin, Alain; Kraemer, Kenneth H

    2006-01-01

    Xeroderma pigmentosum group C (XP-C) is a rare autosomal recessive disorder. Patients with two mutant alleles of the XPC DNA repair gene have sun sensitivity and a 1000-fold increase in skin cancers. Clinically normal parents of XP-C patients have one mutant allele and one normal allele. As a step toward evaluating cancer risk in these XPC heterozygotes we characterized cells from 16 XP families. We identified 15 causative mutations (5 frameshift, 6 nonsense and 4 splicing) in the XPC gene in cells from 16 XP probands. All had premature termination codons (PTC) and absence of normal XPC protein on western blotting. The cell lines from 26 parents were heterozygous for the same mutations. We employed a real-time quantitative reverse transcriptase-PCR assay as a rapid and sensitive method to measure XPC mRNA levels. The mean XPC mRNA levels in the cell lines from the XP-C probands were 24% (P<10(-7)) of that in 10 normal controls. This reduced XPC mRNA level in cells from XP-C patients was caused by the PTC that induces nonsense-mediated mRNA decay. The mean XPC mRNA levels in cell lines from the heterozygous XP-C carriers were intermediate (59%, P=10(-4)) between the values for the XP patients and the normal controls. This study demonstrates reduced XPC mRNA levels in XP-C patients and heterozygotes. Thus, XPC mRNA levels may be evaluated as a marker of cancer susceptibility in carriers of mutations in the XPC gene. PMID:16081512

  3. Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

    PubMed Central

    Weiss, S L; Sunde, R A

    1998-01-01

    Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3

  4. Successful personalized chemotherapy for metastatic gastric cancer based on quantitative BRCA1 mRNA expression level: A case report

    PubMed Central

    HUANG, YING; WU, PUYUAN; LIU, BAORUI; DU, JUAN

    2016-01-01

    Personalized chemotherapy is based on the specific genetic profile of individual patients and is replacing the traditional ‘one size fits all’ medicine. Breast cancer 1 (BRCA1) plays a central role in the chemotherapy-induced DNA damage response. It has been repeatedly demonstrated that BRCA1 mRNA levels were negatively associated with cisplatin sensitivity, but positively associated with docetaxel sensitivity in patients with gastric cancer in experimental and clinical studies. This feature leads to customized chemotherapy based on the BRCA1 mRNA expression level and results in a high efficacy of treatment. The present study describes the case of a 77-year-old patient with metastatic gastric cancer who was treated with personalized chemotherapy based on quantitative BRCA1 mRNA expression level. This study and the available literature data suggest that the expression level of BRCA1 mRNA is dynamic to BRCA1-based chemotherapy. More importantly, de novo assessment of BRCA1 status is a preferable option for ciscisplatin- or docetaxel-resistant patients, since the expression levels of BRCA1 mRNA in certain patients may alter significantly following treatment. Therefore, BRCA1 expression should be assessed for predicting differential chemosensitivity and tailoring chemotherapy in gastric cancer. PMID:27313763

  5. Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions.

    PubMed

    Berman, Y; Devi, L; Spangler, R; Kreek, M J; Carr, K D

    1997-06-01

    It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need. PMID:9191075

  6. Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy.

    PubMed

    Spitali, Pietro; Heemskerk, Hans; Vossen, Rolf H A M; Ferlini, Alessandra; den Dunnen, Johan T; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2010-09-01

    Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model. PMID:20458276

  7. Single nucleotide polymorphisms in CETP, SLC46A1, SLC19A1, CD36, BCMO1, APOA5, and ABCA1 are significant predictors of plasma HDL in healthy adults

    PubMed Central

    2013-01-01

    Background In a marker-trait association study we estimated the statistical significance of 65 single nucleotide polymorphisms (SNP) in 23 candidate genes on HDL levels of two independent Caucasian populations. Each population consisted of men and women and their HDL levels were adjusted for gender and body weight. We used a linear regression model. Selected genes corresponded to folate metabolism, vitamins B-12, A, and E, and cholesterol pathways or lipid metabolism. Methods Extracted DNA from both the Sacramento and Beltsville populations was analyzed using an allele discrimination assay with a MALDI-TOF mass spectrometry platform. The adjusted phenotype, y, was HDL levels adjusted for gender and body weight only statistical analyses were performed using the genotype association and regression modules from the SNP Variation Suite v7. Results Statistically significant SNP (where P values were adjusted for false discovery rate) included: CETP (rs7499892 and rs5882); SLC46A1 (rs37514694; rs739439); SLC19A1 (rs3788199); CD36 (rs3211956); BCMO1 (rs6564851), APOA5 (rs662799), and ABCA1 (rs4149267). Many prior association trends of the SNP with HDL were replicated in our cross-validation study. Significantly, the association of SNP in folate transporters (SLC46A1 rs37514694 and rs739439; SLC19A1 rs3788199) with HDL was identified in our study. Conclusions Given recent literature on the role of niacin in the biogenesis of HDL, focus on status and metabolism of B-vitamins and metabolites of eccentric cleavage of β-carotene with lipid metabolism is exciting for future study. PMID:23656756

  8. The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8

    PubMed Central

    2014-01-01

    Background RNA metabolism, including RNA synthesis and RNA degradation, is one of the most conserved biological systems and has been intensively studied; however, the degradation network of ribonucleases (RNases) and RNA substrates is not fully understood. Results The genome of the extreme thermophile, Thermus thermophilus HB8 includes 15 genes that encode RNases or putative RNases. Using DNA microarray analyses, we examined the effects of disruption of each RNase on mRNA abundance. Disruption of the genes encoding RNase J, RecJ-like protein and RNase P could not be isolated, indicating that these RNases are essential for cell viability. Disruption of the TTHA0252 gene, which was not previously considered to be involved in mRNA degradation, affected mRNA abundance, as did disruption of the putative RNases, YbeY and PhoH-like proteins, suggesting that they have RNase activity. The effects on mRNA abundance of disruption of several RNase genes were dependent on the phase of cell growth. Disruption of the RNase Y and RNase HII genes affected mRNA levels only during the log phase, whereas disruption of the PhoH-like gene affected mRNA levels only during the stationary phase. Moreover, disruption of the RNase R and PNPase genes had a greater impact on mRNA abundance during the stationary phase than the log phase, whereas the opposite was true for the TTHA0252 gene disruptant. Similar changes in mRNA levels were observed after disruption of YbeY or PhoH-like genes. The changes in mRNA levels in the bacterial Argonaute disruptant were similar to those in the RNase HI and RNase HII gene disruptants, suggesting that bacterial Argonaute is a functional homolog of RNase H. Conclusion This study suggests that T. thermophilus HB8 has 13 functional RNases and that each RNase has a different function in the cell. The putative RNases, TTHA0252, YbeY and PhoH-like proteins, are suggested to have RNase activity and to be involved in mRNA degradation. In addition, PhoH-like and Ybe

  9. Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

    PubMed

    Meng, Zhen; Hu, Peng; Lei, Jilin; Jia, Yudong

    2016-09-01

    Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae. PMID:27255364

  10. GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions.

    PubMed

    Shao, Yi Ta; Tseng, Yung Che; Chang, Chia-Hao; Yan, Hong Young; Hwang, Pung Pung; Borg, Bertil

    2015-02-01

    In vertebrates, reproduction is regulated by the brain-pituitary-gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T+POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T+POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response. PMID:25446939

  11. A distinct ERCC1 haplotype is associated with mRNA expression levels in prostate cancer patients.

    PubMed

    Woelfelschneider, Andreas; Popanda, Odilia; Lilla, Carmen; Linseisen, Jakob; Mayer, Claudia; Celebi, Oktay; Debus, Jürgen; Bartsch, Helmut; Chang-Claude, Jenny; Schmezer, Peter

    2008-09-01

    Both genetic variants and messenger RNA (mRNA) expression of DNA repair and tumor suppressor genes have been investigated as molecular markers for therapy outcome. However, the phenotypic impact of genetic variants often remained unclear, thus the rationale of their use in risk prediction may be limited. We therefore analyzed genetic variants together with anthropometric and lifestyle factors to see how these affect mRNA levels of ERCC1, MDM2 and TP53 in primary blood lymphocytes. mRNA expression was measured in 376 prostate cancer patients by quantitative real-time polymerase chain reaction after reverse transcription, and ERCC1 rs11615 T>C, ERCC1 rs3212986 C>A, MDM2 rs2279744 T>G and TP53 rs17878362 (p53PIN3) polymorphisms were determined. Considerable interindividual differences in mRNA expression were found (coefficients of variation: ERCC1, 45%; MDM2, 43% and TP53, 35%). ERCC1 expression was positively correlated with plasma levels of beta-carotene (P = 0.03) and negatively correlated with canthaxanthin (P = 0.02) and lutein (P = 0.02). Overall, the polymorphisms affected mRNA expression only weakly. Carriers of a distinct ERCC1 haplotype (CC) showed, however, significantly lower expression values than non-carriers (P = 0.001). Applying logistic regression, we found that CC haplotype carriers had a 1.69-fold increased odds ratio (95% confidence interval: 1.06-2.71) for reduced ERCC1 mRNA levels. This low ERCC1 expression might be associated with reduced DNA repair and better therapy response. In summary, the association we have found between ERCC1 genotype and mRNA expression supports recent clinical observations that genetic variation in ERCC1 can affect treatment outcome and prognosis. Our study further revealed a modulating effect by nutritional factors. PMID:18332046

  12. Neuroleptics Affect Neuropeptide S and NPSR mRNA Levels in the Rat Brain.

    PubMed

    Pałasz, Artur; Rojczyk, Ewa

    2015-11-01

    Neuropeptide S (NPS) has a multidirectional regulatory activity, especially when considered as a potent endogenous anxiolytic factor. Accumulating data suggests that neuroleptics affect peptidergic signaling in various brain structures. However, there is no information regarding the influence of treatment with antipsychotics on brain NPS expression. In the current study, we assessed the NPS and NPS receptor (NPSR) mRNA levels in the brains of rats shortly and chronically treated with chlorpromazine and olanzapine using quantitative real-time PCR. Both single-dose and long-term (4 months) olanzapine treatment led to the upregulation of NPS expression in the rat hypothalamus. It supports the hypothesis that NPS is involved in the dopamine-dependent anxiolytic actions of selected neuroleptics and possibly also in the pathophysiology of mental disorders. On the other hand, NPSR expression decreased after single-dose and chronic chlorpromazine administration in the hypothalamus, as well as after chronic olanzapine and chlorpromazine administration in the striatum and hippocampus. These results cast a new light on the pharmacology of antipsychotics and contribute to a better understanding of the mechanisms responsible for their action. Furthermore, our findings underline the complex nature of potential interactions between dopamine receptors and brain peptidergic pathways, which has potential clinical applications. PMID:26227793

  13. Circadian oscillations in period gene mRNA levels are transcriptionally regulated.

    PubMed Central

    Hardin, P E; Hall, J C; Rosbash, M

    1992-01-01

    The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator. Images PMID:1465387

  14. Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

    SciTech Connect

    Popovich, B.; Barrieux, A.; Dillmann, W.H.

    1987-05-01

    Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.

  15. Regulation of mRNA and Protein Levels of β1 Integrin Variants in Human Prostate Carcinoma

    PubMed Central

    Perlino, Elda; Lovecchio, Mariarosaria; Vacca, Rosa A.; Fornaro, Mara; Moro, Loredana; Ditonno, Pasquale; Battaglia, Michele; Selvaggi, Francesco P.; Mastropasqua, Mauro G.; Bufo, Pantaleo; Languino, Lucia R.

    2000-01-01

    Alterations of integrin expression levels in cancer cells correlate with changes in invasiveness, tumor progression, and metastatic potential. The β1C integrin, an alternatively spliced form of the human β1 integrin, has been shown to inhibit prostate cell proliferation. Furthermore, β1C protein levels were found to be abundant in normal prostate glandular epithelium and down-regulated in prostatic adenocarcinoma. To gain further insights into the molecular mechanisms underlying abnormal cancer cell proliferation, we have studied β1C and β1 integrin expression at both mRNA and protein levels by Northern and immunoblotting analysis using freshly isolated neoplastic and normal human prostate tissue specimens. Steady-state mRNA levels were evaluated in 38 specimens: 33 prostatic adenocarcinomas exhibiting different Gleason’s grade and five normal tissue specimens that did not show any histological manifestation of benign prostatic hypertrophy. Our results demonstrate that β1C mRNA is expressed in normal prostate and is significantly down-regulated in neoplastic prostate specimens. In addition, using a probe that hybridizes with all β1 variants, mRNA levels of β1 are found reduced in neoplastic versus normal prostate tissues. We demonstrate that β1C mRNA down-regulation does not correlate with either tumor grade or differentiation according to Gleason’s grade and TNM system evaluation, and that β1C mRNA levels are not affected by hormonal therapy. In parallel, β1C protein levels were analyzed. As expected, β1C is found to be expressed in normal prostate and dramatically reduced in neoplastic prostate tissues; in contrast, using an antibody to β1 that recognizes all β1 variants, the levels of β1 are comparable in normal and neoplastic prostate, thus indicating a selective down-regulation of the β1C protein in prostate carcinoma. These results demonstrate for the first time that β1C and β1 mRNA expression is down-regulated in prostate carcinoma

  16. Higher LPA2 and LPA6 mRNA Levels in Hepatocellular Carcinoma Are Associated with Poorer Differentiation, Microvascular Invasion and Earlier Recurrence with Higher Serum Autotaxin Levels.

    PubMed

    Enooku, Kenichiro; Uranbileg, Baasanjav; Ikeda, Hitoshi; Kurano, Makoto; Sato, Masaya; Kudo, Hiroki; Maki, Harufumi; Koike, Kazuhiko; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

    2016-01-01

    Hepatocellular carcinoma (HCC) commonly develops in patients with liver fibrosis; in these patients, the blood levels of lysophosphatidic acid (LPA) and its generating enzyme autotaxin (ATX) increase with the liver fibrosis stage. We aimed to examine the potential relevance of ATX and LPA in HCC. Fifty-eight HCC patients who underwent surgical treatment were consecutively enrolled in the study. Among the LPA receptors in HCC, higher LPA2 mRNA levels correlated with poorer differentiation, and higher LPA6 mRNA levels correlated with microvascular invasion, which suggested a higher malignant potential of HCC with increased LPA2 and LPA6 expression. In patients with primary HCC, neither LPA2 nor LPA6 mRNA levels were associated with recurrence. However, when serum ATX levels were combined for analysis as a surrogate for plasma LPA levels, the cumulative intra-hepatic recurrence rate was higher in patients in whom both serum ATX levels and LPA2 or LPA6 mRNA levels were higher than the median. However, the mRNA level of phosphatidic acid-selective phospholipase A1ɑ, another LPA-generating enzyme, in HCC patients was not associated with pathological findings or recurrence, even in combination with the expression of LPA receptors. Higher LPA2 mRNA levels were associated with poorer differentiation, and higher LPA6 levels were associated with microvascular invasion in HCC; both became a risk factor for recurrence after surgical treatment when combined with increased serum ATX levels. ATX and LPA receptors merit consideration as therapeutic targets of HCC. PMID:27583415

  17. A hairpin within YAP mRNA 3'UTR functions in regulation at post-transcription level.

    PubMed

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping; Ye, Lihong; Zhang, Xiaodong

    2015-04-01

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3'untranslated region (3'UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3'UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3'UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3'UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3'UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3'UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. PMID:25727017

  18. The Prognostic Value of BRCA1 mRNA Expression Levels Following Neoadjuvant Chemotherapy in Breast Cancer

    PubMed Central

    Margeli, Mireia; Cirauqui, Beatriz; Castella, Eva; Tapia, Gustavo; Costa, Carlota; Gimenez-Capitan, Ana; Barnadas, Agusti; Ronco, Maria Sanchez; Benlloch, Susana; Taron, Miquel; Rosell, Rafael

    2010-01-01

    Background A fraction of sporadic breast cancers has low BRCA1 expression. BRCA1 mutation carriers are more likely to achieve a pathological complete response with DNA-damage-based chemotherapy compared to non-mutation carriers. Furthermore, sporadic ovarian cancer patients with low levels of BRCA1 mRNA have longer survival following platinum-based chemotherapy than patients with high levels of BRCA1 mRNA. Methodology/Principal Findings Tumor biopsies were obtained from 86 breast cancer patients who were candidates for neoadjuvant chemotherapy, treated with four cycles of neoadjuvant fluorouracil, epirubicin and cyclophosphamide. Estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 5/6 and vimentin were examined by tissue microarray. HER2 were also assessed by chromogenic in situ hybridization, and BRCA1 mRNA was analyzed in a subset of 41 patients for whom sufficient tumor tissue was available by real-time quantitative PCR. Median time to progression was 42 months and overall survival was 55 months. In the multivariate analysis for time to progression and overall survival for 41 patients in whom BRCA1 could be assessed, low levels of BRCA1 mRNA, positive PR and negative lymph node involvement predicted a significantly lower risk of relapse, low levels of BRCA1 mRNA and positive PR were the only variables associated with significantly longer survival. Conclusions/Significance We provide evidence for a major role for BRCA1 mRNA expression as a marker of time to progression and overall survival in sporadic breast cancers treated with anthracycline-based chemotherapy. These findings can be useful for customizing chemotherapy. PMID:20209131

  19. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  20. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected. PMID:12417619

  1. mRNA degradation: an underestimated factor in steady-state transcript levels of cytochrome c oxidase subunits?

    PubMed

    Bremer, Katharina; Moyes, Christopher D

    2014-06-15

    Steady-state mRNA levels are determined by synthesis and degradation; however, changes in mRNA levels are usually attributed to transcription. For cytochrome c oxidase (COX), cold acclimation typically leads to an increase in COX activity while transcript levels for the nuclear-encoded subunits change non-stoichiometrically. Whether those patterns are caused by differences in subunit transcription rates, decay rates or both was not known. We assessed decay rates of transcripts for COX subunits, including representatives that decreased, increased in parallel with COX or increased in excess of COX. Low temperature reduced the decay rate of all transcripts; however, COX subunits displayed higher thermal sensitivity than housekeeping genes. The lower decay rates for COX transcripts might explain some of their increase in response to cold acclimation. The reason for the exaggerated transcript response of two subunits (COX6B-1 and COX7A-2) may be due to decreased decay. However, decay rate differences could not explain the patterns seen with another subunit that did not change in mRNA level with thermal acclimation (COX6A-2). Further, the decay patterns differed between two thermal acclimation experiments, which may explain some of the heterogeneity seen in fish studies. The differences in decay rates suggest that the lack of stoichiometry in mRNA levels is exacerbated by post-transcriptional mechanisms. Collectively, these results suggest that temperature-induced differences in COX subunit mRNA levels and deviations from stoichiometry between them may partially arise from subunit-specific sensitivities to degradation. We suggest that all subunits are controlled by transcription, and that exaggerated responses of some subunits are due to reduced decay rates. PMID:24737751

  2. Chronic estrogen exposure maintains elevated levels of progesterone receptor mRNA in guinea pig hypothalamus.

    PubMed

    Bayliss, D A; Millhorn, D E

    1991-05-01

    We performed in situ hybridization on hypothalamic sections from ovariectomized guinea pig using a cocktail of three 35S-labeled oligonucleotides complementary to mammalian progesterone receptor (PR) cDNA. PR mRNA was readily detected in hypothalamic neurons from guinea pigs pretreated with 17 beta-estradiol benzoate (E2B), but not from animals which did not receive supplemental E2B. The distribution of PR mRNA-containing cells corresponded well with previous localizations of PR in guinea pig. In contrast to earlier reports of E2B regulation of PR mRNA in rat hypothalamus, however, we found that PR mRNA remained elevated during chronic exposure to E2B (up to 10 days) in guinea pig. PMID:2072827

  3. Alteration of Na,K-ATPase subunit mRNA and protein levels in hypertrophied rat heart.

    PubMed

    Charlemagne, D; Orlowski, J; Oliviero, P; Rannou, F; Sainte Beuve, C; Swynghedauw, B; Lane, L K

    1994-01-14

    To determine if an altered expression of the Na,K-ATPase alpha isoform genes is responsible for an observed increase in cardiac glycoside sensitivity in compensatory hypertrophy, we performed Northern and slot blot analyses of RNA and specific immunological detection of Na,K-ATPase isoforms in rat hearts from normal and pressure overload-treated animals induced by abdominal aortic constriction. During the early phase of hypertrophy, the only alteration is a decrease in the alpha 2 mRNA isoform. In the compensated hypertrophied heart, the levels of the predominant alpha 1 isoform (mRNA and protein) and the beta 1 subunit mRNA are unchanged. In contrast, the alpha 2 isoform (mRNA and protein) is decreased by 35% and up to 61-64% in mild (< 55%) and severe (> 55%) hypertrophy, respectively. The alpha 3 isoform (mRNA and protein), which is extremely low in adult heart, is increased up to 2-fold during hypertrophy but accounts for only approximately equal to 5% of the total alpha isoform mRNA. These findings demonstrate that, in cardiac hypertrophy, the three alpha isoforms of the Na,K-ATPase are independently regulated and that regulation occurs at a pretranslational level. The pattern of expression in hypertrophied adult heart is similar to that of the neonatal heart where the inverse regulation between the alpha 2 and alpha 3 ouabain high affinity isoforms has been reported. This suggests that distinct regulatory mechanisms controlling Na,K-ATPase isoform expression may, at least in part, be involved in the sensitivity to cardiac glycosides. PMID:8288620

  4. Estrogen Affects Levels of Bcl-2 Protein and mRNA in Medial Amygdala of Ovariectomized Rats

    PubMed Central

    Fan, Lu; Pandey, Subhash C.; Cohen, Rochelle S.

    2013-01-01

    The survival factor Bcl-2 is a cyclic AMP response element-binding protein (CREB) gene product implicated in mediating some of estrogen’s effects on neuroprotection. Previously, we showed an effect of estradiol benzoate (E) on numbers of neuron-specific protein (NeuN)- and phosphorylated CREB (pCREB)-positive cells in medial (MeA), but not central (CeA), amygdala of ovariectomized rats. To determine whether these effects are accompanied by an E-induced increase in Bcl-2, we examined the effects of E on levels of Bcl-2 protein and mRNA in MeA and CeA of ovariectomized rats treated with E regimens resulting in moderate (2.5μg E for 4 or 14 days) or high (10μg E for 14 days) plasma estradiol levels. As a physiological control, we showed that all E treatments increased uterine wet weight relative to vehicle; 10μg E for 14 days also increased uterine weight compared to that seen with lower E levels. Western blot analysis revealed that all E groups displayed an increase in uterine Bcl-2 protein levels compared to vehicle. We found that 2.5μg and 10μg E for 14 days increased levels of Bcl-2 gold immunolabeling compared to vehicle and 2.5μg E for 4 days in MeA, but not CeA. We measured Bcl-2 mRNA levels in vehicle and 2.5μg E for 14 days groups. There was a significant increase in Bcl-2 mRNA levels in MeA, but not CeA, of E-treated ovariectomized rats compared with vehicle controls. The E-induced increase in protein and mRNA levels of Bcl-2 in MeA may be important for neuroprotection in this region. PMID:18655204

  5. Preterm birth affects GABAA receptor subunit mRNA levels during the foetal-to-neonatal transition in guinea pigs.

    PubMed

    Shaw, J C; Palliser, H K; Walker, D W; Hirst, J J

    2015-06-01

    Modulation of gamma-aminobutyric acid A (GABAA) receptor signalling by the neurosteroid allopregnanolone has a major role in late gestation neurodevelopment. The objective of this study was to characterize the mRNA levels of GABAA receptor subunits (α4, α5, α6 and δ) that are key to neurosteroid binding in the brain, following preterm birth. Myelination, measured by the myelin basic protein immunostaining, was used to assess maturity of the preterm brains. Foetal guinea pig brains were obtained at 62 days' gestational age (GA, preterm) or at term (69 days). Neonates were delivered by caesarean section, at 62 days GA and term, and maintained until tissue collection at 24 h of age. Subunit mRNA levels were quantified by RT-PCR in the hippocampus and cerebellum of foetal and neonatal brains. Levels of the α6 and δ subunits were markedly lower in the cerebellum of preterm guinea pigs compared with term animals. Importantly, there was an increase in mRNA levels of these subunits during the foetal-to-neonatal transition at term, which was not seen following preterm birth. Myelination was lower in preterm neonatal brains, consistent with marked immaturity. Salivary cortisol concentrations, measured by EIA, were also higher for the preterm neonates, suggesting greater stress. We conclude that there is an adaptive increase in the levels of mRNA of the key GABAA receptor subunits involved in neurosteroid action after term birth, which may compensate for declining allopregnanolone levels. The lower levels of these subunits in preterm neonates may heighten the adverse effect of the premature decline in neurosteroid exposure. PMID:25661827

  6. Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

    USGS Publications Warehouse

    Yada, T.; Hyodo, S.; Schreck, C.B.

    2008-01-01

    Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems. ?? 2008 Elsevier Inc. All rights reserved.

  7. Time course of changes in growth factor mRNA levels in muscle of steroid-implanted and nonimplanted steers.

    PubMed

    Pampusch, M S; Johnson, B J; White, M E; Hathaway, M R; Dunn, J D; Waylan, A T; Dayton, W R

    2003-11-01

    We used a muscle biopsy technique in conjunction with real-time PCR analysis to examine the time course of changes in muscle IGF-I, IGFBP-3, myostatin, and hepatocyte growth factor (HGF) mRNA in the longissimus muscles of Revalor-S-implanted and nonimplanted steers on d 0, 7, 12, and 26 after implantation (nine steers/treatment group). Administration of a Revalor-S implant increased (P < 0.01) ADG and improved (P < 0.05) feed efficiency, 36 and 34%, respectively, compared with steers that received no implant during the 26-d trial. Daily dry matter intake did not differ (P > 0.15) between nonimplanted and implanted steers. Steers receiving the Revalor-S implant had increased (P < 0.001) circulating IGF-I concentrations compared with nonimplanted steers. The longissimus muscles of steers receiving the Revalor-S implant contained increased (P < 0.001) IGF-I mRNA levels compared with longissimus muscles of nonimplanted steers over the 26-d duration of the study. Longissimus muscle IGF-I mRNA levels in implanted steers were increased (P < 0.003) relative to d-0 concentrations on d 7 and 12 (101% and 128%, respectively), and byd 26, longissimus muscle mRNA levels were more than three times (P < 0.0001) those in the longissimus muscles of the same steers on d 0. There was no treatment effect on the level of IGFBP-3, myostatin, or HGF mRNA in the longissimus muscle at any time point; however, levels of IGFBP-3, myostatin, and HGF mRNA increased with time on feed. Based on current and previous studies, we hypothesize that the increased IGF-I level in muscle of implanted steers by d 7 of implantation stimulates satellite cell proliferation and maintains a high number of proliferating satellite cells at a point in the growth curve where satellite cell numbers and activity are normally dropping off. This would prolong the period of rapid muscle growth, resulting in the observed increased rate and efficiency of muscle deposition in implanted steers. PMID:14601876

  8. YB-1 and MTA1 protein levels and not DNA or mRNA alterations predict for prostate cancer recurrence

    PubMed Central

    Sheridan, Christine Moore; Grogan, Tristan R.; Nguyen, Hao G.; Galet, Colette; Rettig, Matthew B.; Hsieh, Andrew C.; Ruggero, Davide

    2015-01-01

    Attempts to identify biomarkers to detect prostate tumorigenesis, and thus minimize prostate cancer progression and inform treatment decisions have primarily focused on alterations at the DNA and mRNA levels, ignoring alterations at the level of protein synthesis control. We have previously shown that the PI3K-AKT-mTOR pathway, frequently deregulated in prostate cancer, specifically induces the synthesis of proteins that contribute to metastasis, most notably YB-1 and MTA1, without altering mRNA levels thereby demonstrating the importance of translation control in driving the expression of these genes in cancer. Here, we analyze genomic sequencing and mRNA expression databases, as well as protein expression employing an annotated tissue microarray generated from 332 prostate cancer patients with 15 years of clinical follow-up to determine the combined prognostic capability of YB-1 and MTA1 alterations in forecasting prostate cancer outcomes. Remarkably, protein abundance, but not genomic or transcriptional alterations of YB-1 and MTA1, is predictive of disease recurrence, exhibiting a dose-dependent effect on time to PSA recurrence, an indicator of tumor relapse. Moreover, high protein levels of YB-1 and MTA1 are associated with a 3-fold increased risk for requiring future hormone therapy or radiation therapy. Importantly, YB-1 and MTA1 protein levels significantly increase the predictive capacity of a clinical model for prostate cancer recurrence. These findings demonstrate that protein abundance of YB-1 and MTA1, irrespective of DNA or mRNA status, can predict for prostate cancer relapse and uncover a vast underappreciated repository of biomarkers regulated at the level of protein expression. PMID:25797255

  9. Increased IL18 mRNA levels in peripheral artery disease and its association with triglyceride and LDL cholesterol levels: a pilot study.

    PubMed

    Deser, Serkan Burc; Bayoglu, Burcu; Besirli, Kazım; Cengiz, Mujgan; Arapi, Berk; Junusbekov, Yerik; Dirican, Ahmet; Arslan, Caner

    2016-06-01

    Peripheral artery disease (PAD) typically refers to lower limb vessel ischemia caused by atherosclerotic stenosis of lower extremity arteries. IL18 is a pleiotropic pro-inflammatory cytokine reported to function as an inflammatory biomarker in cardiovascular diseases. IL18 activity is balanced by high-affinity naturally occurring IL18-binding protein (IL18BP). This study aimed to determine whether IL18, IL18 BP mRNA levels and -137 G/C (rs187238) polymorphism, which was previously associated with IL18 gene transcriptional activity, were associated with PAD etiology. IL18, IL18BP mRNA levels from peripheral blood mononuclear cells and -137 G/C (rs187238) polymorphism were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and RT-PCR, respectively, in 55 PAD patients (26 aorta-iliac, 29 femoro-popliteal) and 61 disease-free controls. IL18 mRNA levels were increased in PAD patients compared with healthy controls (p = 0.09); however, did not reach a statistical significant level, also did not significantly differ between aorta-iliac and femoro-popliteal occlusive PAD subgroups (p = 0.285). However, IL18BP mRNA levels were significantly lower in PAD group compared with controls (p < 0.001). Genotype frequencies of rs187238 polymorphism did not significantly differ between PAD patients and controls (p = 0.385). IL18 mRNA levels were significantly correlated with triglycerides and LDL cholesterol levels in PAD patients (p = 0.003, p = 0.014, respectively). HDL cholesterol levels were negatively correlated with IL18 mRNA levels in controls (p = 0.05). This report is a preliminary study to show an association between IL18, IL18BP mRNA levels and PAD and suggests that the IL18 gene may have a significant relationship with triglyceride and LDL cholesterol levels in PAD patients. PMID:26438531

  10. Irisin mRNA and circulating levels in relation to other myokines in healthy and morbidly obese humans

    PubMed Central

    Vamvini, Maria T.; Aronis, Konstantinos N.; Panagiotou, Grigorios; Huh, Joo Young; Chamberland, John P.; Brinkoetter, Mary T.; Petrou, Michael; Christophi, Costas A.; Kales, Stefanos N.; Christiani, David C.; Mantzoros, Christos S.

    2013-01-01

    Objective Skeletal muscle is considered to be an endocrine organ that secretes a number of myokines including follistatin, myostatin, activin A and the newly identified irisin. Irisin’s biology and function exhibits similarities with the functions of the follistatin-myostatin-activin A axis. It remains unknown whether there is any interplay among these molecules. The aim of this study is to examine potential associations of irisin with the follistatin, myostatin and activin A axis. Material-methods Two observational studies were performed to evaluate the associations of irisin with the other three peptides. Study A included 150 healthy males aged 18.48 ±0.16 years with Body Mass Index (BMI) 23.18± 3.75 kg/m2. Fasting serum samples were used to measure the levels of the molecules of interest. Study B included 14 morbidly obese individuals, candidates for bariatric surgery, aged 53.14±8.93 years with BMI 50.18±10.63 kg/m2. Blood samples were obtained after an overnight fast. Eight out of the fourteen participants consented to an optional thigh biopsy during their bariatric surgery. Using the above blood and tissue samples, we measured circulating levels and muscle mRNA of irisin, follistatin, myostatin and activin A. Results We report that FNDC5 mRNA in muscle is positively correlated with follistatin mRNA expression in morbidly obese subjects (rho=0.93, p<0.001). We also found that circulating irisin is positively correlated with follistatin circulating levels among lean subjects (rho=0.17, p=0.05) while this association was suggestive among the obese (rho=0.56, p=0.07). Conclusion The newly identified myokine irisin may be positively associated with follistatin at both the mRNA and circulating protein level. PMID:24062354

  11. Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats.

    PubMed

    Kawase, Atsushi; Ito, Ayami; Yamada, Ayano; Iwaki, Masahiro

    2015-06-01

    Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats. PMID:24899460

  12. Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

    PubMed

    Harding, Louisa B; Schultz, Irvin R; da Silva, Denis A M; Ylitalo, Gina M; Ragsdale, Dave; Harris, Stephanie I; Bailey, Stephanie; Pepich, Barry V; Swanson, Penny

    2016-09-01

    It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17β-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to

  13. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level.

    PubMed

    Pereverzev, Anton P; Gurskaya, Nadya G; Ermakova, Galina V; Kudryavtseva, Elena I; Markina, Nadezhda M; Kotlobay, Alexey A; Lukyanov, Sergey A; Zaraisky, Andrey G; Lukyanov, Konstantin A

    2015-01-01

    Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos. PMID:25578556

  14. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    SciTech Connect

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping; Ye, Lihong; Zhang, Xiaodong

    2015-04-03

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

  15. The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection.

    PubMed Central

    Hardwicke, M A; Sandri-Goldin, R M

    1994-01-01

    We have previously shown that the herpes simplex virus immediate-early regulatory protein ICP27 acts posttranscriptionally to affect mRNA processing (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992). Specifically, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. Here, we have investigated the effect of ICP27 during herpes simplex virus type 1 (HSV-1) infection on accumulation of spliced cellular mRNAs. ICP27 viral mutants have been shown to be defective in host shutoff (W. R. Sacks, C. C. Greene, D. P. Aschman, and P. A. Schaffer, J. Virol. 55:796-805, 1985). Therefore, we examined whether ICP27 could contribute to this complex process by decreasing cellular mRNA levels through its effects on host cell splicing. It was found that in infections with viral mutants defective in ICP27, the accumulated levels of three spliced host mRNAs were higher than those seen with wild-type HSV-1. The differences occurred posttranscriptionally as shown by nuclear runoff transcription assays. The stabilities of the spliced products during infection with wild-type or ICP27 mutant viruses were similar, and unspliced precursor mRNA for a viral spliced gene was detected in infections with wild-type HSV-1 but not in infections in which ICP27 was not expressed. These results suggest that the reduction in cellular mRNA levels and the accumulation of pre-mRNA are related and may be caused by an impairment in host cell splicing. These data further show that ICP27 is required for these effects to occur. Images PMID:8035480

  16. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  17. Acute Effects of Capsaicin on Proopioimelanocortin mRNA Levels in the Arcuate Nucleus of Sprague-Dawley Rats

    PubMed Central

    Lee, Jin-Seong; Kim, Hyeun-Kyeung; Baek, Sun-Yong; Kim, Cheol-Min

    2012-01-01

    Objective Capsaicin, a noxious stimulant and main component of the hot flavor of red peppers, has an analgesic effect when administered to humans. We investigated the expression of proopioimelanocortin (POMC) mRNA in the arcuate nucleus of Sprague-Dawley (SD) rats after administering capsaicin, hypothesizing that administering capsaicin activates the central opioid system. Methods SD rats were divided randomly into two groups; one group received a saline injection and the other received a capsaicin injection. The POMC mRNA level in the arcuate nucleus of the hypothalamus was measured by the reverse transcription-polymerase chain reaction at 0, 20, 40, 60, and 120 minutes after capsaicin administration. Results Capsaicin administration resulted in a significantly increased POMC mRNA level, compared to that in saline-treated rats at the 20-minute time point (t=-4.445, p=0.001). However, no significant group differences were observed at other times (t=-1.886, p=0.089; t= -0.973, p=0.353; t=-2.193, p=0.053 for 40, 60, and 120 minutes, respectively). Conclusion The analgesic effect of capsaicin might be associated with increased activity of the cerebral opioid system. This finding suggests that capsaicin acted for nociception and analgesia and could affect alcohol-intake behavior, which might further imply that a food culture could affect drinking behavior. PMID:22707971

  18. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure

    SciTech Connect

    Tin-Tin-Win-Shwe Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1{beta}, TNF-{alpha}, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 {mu}g) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 {mu}g of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1{beta} mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-{alpha} mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 {beta} mRNA expressions synergistically with LTA

  19. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure.

    PubMed

    Tin-Tin-Win-Shwe; Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1 beta, TNF-alpha, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1 beta mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-alpha mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 beta mRNA expressions synergistically with LTA in the

  20. Arsenic Trioxide Attenuates NF-κB and Cytokine mRNA Levels in the Livers of Cocks.

    PubMed

    Zhang, Kexin; Zhao, Panpan; Guo, Guangyang; Guo, Ying; Tian, Li; Sun, Xiao; Li, Siwen; He, Ying; Sun, Ying; Chai, Hongliang; Zhang, Wen; Xing, Mingwei

    2016-04-01

    Arsenic (As) is a trace element widely found in nature. It exists in several forms, including organic arsenic, inorganic arsenic, and trivalent arsenic, the most toxic. Arsenic trioxide (As2O3) is widespread in nature. This form tends to accumulate in animals and humans and therefore has a potential harm for them. Cytokines play essential roles in the immune response and inflammation. Although the importance of cytokines in the responses to arsenic exposure has been demonstrated in many types of mammals, the function of these in poultry, especially in chickens, remains unclear. The purpose of the present study was to examine the effect of As2O3 exposure on cytokines in cock livers. In this study, 72 1-day-old male Hy-line cocks were randomly divided into four groups including the control group, low-As group, middle-As group, and high-As group. The livers were collected on days 30, 60, and 90 of the experiment. The levels of nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 beta (IL-12β), and interleukin-1 beta (IL-1β) mRNA in the livers of the cocks were measured using real-time PCR. The results showed that the expression levels of IL-6, IL-8, TNF-α, and NF-κB which seemed to be a critical mediator in the inflammatory response tended to increase in the birds chronically treated with As2O3. However, the mRNA expression levels of IL-4, IL-12β, and IL-1β were decreased in the experiment. The information regarding the effects of As2O3 on cytokine mRNA expression generated in this study will be important information for arsenic toxicology evaluation. PMID:26276563

  1. Overexpression of Tau Downregulated the mRNA Levels of Kv Channels and Improved Proliferation in N2A Cells

    PubMed Central

    Li, Xiantao; Hu, Ximu; Li, Xiaoqing; Hao, Xuran

    2015-01-01

    Microtubule binding protein tau has a crucial function in promoting the assembly and stabilization of microtubule. Besides tuning the action potentials, voltage-gated K+ channels (Kv) are important for cell proliferation and appear to play a role in the development of cancer. However, little is known about the possible interaction of tau with Kv channels in various tissues. In the present study, tau plasmids were transiently transfected into mouse neuroblastoma N2A cells to explore the possible linkages between tau and Kv channels. This treatment led to a downregulation of mRNA levels of several Kv channels, including Kv2.1, Kv3.1, Kv4.1, Kv9.2, and KCNH4, but no significant alteration was observed for Kv5.1 and KCNQ4. Furthermore, the macroscopic currents through Kv channels were reduced by 36.5% at +60 mV in tau-tranfected N2A cells. The proliferation rates of N2A cells were also improved by the induction of tau expression and the incubation of TEA (tetraethylammonium) for 48 h by 120.9% and 149.3%, respectively. Following the cotransfection with tau in HEK293 cells, the mRNA levels and corresponding currents of Kv2.1 were significantly declined compared with single Kv2.1 transfection. Our data indicated that overexpression of tau declined the mRNA levels of Kv channels and related currents. The effects of tau overexpression on Kv channels provided an alternative explanation for low sensitivity to anti-cancer chemicals in some specific cancer tissues. PMID:25590133

  2. Evaluation of Adenosine Triphosphate-Binding Cassette Transporter A1 (ABCA1) R219K and C-Reactive Protein Gene (CRP) +1059G/C Gene Polymorphisms in Susceptibility to Coronary Heart Disease.

    PubMed

    Li, Jing-Fang; Peng, Dian-Ying; Ling, Mei; Yin, Yong

    2016-01-01

    BACKGROUND This meta-analysis investigated the correlation of ABCA1 R219K and C-Reactive Protein Gene (CRP) +1059G/C gene polymorphisms with susceptibility to coronary heart disease (CHD). MATERIAL AND METHODS We searched PubMed, Springer link, Wiley, EBSCO, Ovid, Wanfang database, VIP database, and China National Knowledge Infrastructure (CNKI) databases to retrieve published studies by keyword. Searches were filtered using our stringent inclusion and exclusion criteria. Resultant high-quality data collected from the final selected studies were analyzed using Comprehensive Meta-analysis 2.0 software. Eleven case-control studies involving 3053 CHD patients and 3403 healthy controls met our inclusion criteria. Seven studies were conducted in Asian populations, 3 studies were done in Caucasian populations, and 1 was in an African population. RESULTS Our major finding was that ABCA1 R219K polymorphism increased susceptibility to CHD in allele model (OR=0.729, 95% CI=0.559~0.949, P=0.019) and dominant model (OR=0.698, 95% CI=0.507~0.961, P=0.027). By contrast, we were unable to find any significant association between the CRP +1059G/C polymorphism and susceptibility to CHD (allele model: OR=1.170, 95% CI=0.782~1.751, P=0.444; dominant model: OR=1.175, 95% CI=0.768~1.797, P=0.457). CONCLUSIONS This meta-analysis provides convincing evidence that polymorphism of ABCA1 R219K is associated with susceptibility to CHD while the CRP +1059G/C polymorphism appears to have no correlation with susceptibility to CHD. PMID:27560308

  3. Evaluation of Adenosine Triphosphate-Binding Cassette Transporter A1 (ABCA1) R219K and C-Reactive Protein Gene (CRP) +1059G/C Gene Polymorphisms in Susceptibility to Coronary Heart Disease

    PubMed Central

    Li, Jing-Fang; Peng, Dian-Ying; Ling, Mei; Yin, Yong

    2016-01-01

    Background This meta-analysis investigated the correlation of ABCA1 R219K and CRP +1059G/C gene polymorphisms with susceptibility to coronary heart disease (CHD). Material/Methods We searched PubMed, Springer link, Wiley, EBSCO, Ovid, Wanfang database, VIP database, and China National Knowledge Infrastructure (CNKI) databases to retrieve published studies by keyword. Searches were filtered using our stringent inclusion and exclusion criteria. Resultant high-quality data collected from the final selected studies were analyzed using Comprehensive Meta-analysis 2.0 software. Eleven case-control studies involving 3053 CHD patients and 3403 healthy controls met our inclusion criteria. Seven studies were conducted in Asian populations, 3 studies were done in Caucasian populations, and 1 was in an African population. Results Our major finding was that ABCA1 R219K polymorphism increased susceptibility to CHD in allele model (OR=0.729, 95% CI=0.559~0.949, P=0.019) and dominant model (OR=0.698, 95% CI=0.507~0.961, P=0.027). By contrast, we were unable to find any significant association between the CRP +1059G/C polymorphism and susceptibility to CHD (allele model: OR=1.170, 95% CI=0.782~1.751, P=0.444; dominant model: OR=1.175, 95% CI=0.768~1.797, P=0.457). Conclusions This meta-analysis provides convincing evidence that polymorphism of ABCA1 R219K is associated with susceptibility to CHD while the CRP +1059G/C polymorphism appears to have no correlation with susceptibility to CHD. PMID:27560308

  4. The Promoter Methylation Status and mRNA Expression Levels of CTCF and SIRT6 in Sporadic Breast Cancer

    PubMed Central

    Wang, Da; Zhang, Xuemei

    2014-01-01

    Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. This study was to detect promoter methylation status and mRNA expression levels of CCCTC-binding factor (CTCF) and sirtuin 6 (SIRT6), and to explore the relationship between methylation and mRNA expression in breast cancer patient samples. Promoter methylation analysis and expression profile analysis of two genes were performed by methylation-specific PCR, bisulfite sequencing PCR, and quantitative real-time PCR in cancer lesions and matched normal tissues. The promoter region of CTCF has not been hypermethylated in all patient samples. In contrast, methylation of SIRT6 gene was present in invasive cancers (93.5%) and matched normal tissues (96.8%) from 62 patients. Promoter hypermethylation of SIRT6 was also observed in ductal carcinoma in situ (three of three) and matched normal tissues (two of three). mRNA expression of CTCF and SIRT6 in invasive tumors showed a lower level than that in paired normal tissues (p=0.008 and p=0.030, respectively). The fold change values of CTCF expression were significantly lower in invasive ductal cancer lesions with Ki-67-positive status (p=0.042). In conclusion, our data showed that the methylation status of CTCF and SIRT6 promoter regions was not statistically different in cancer lesions compared with matched normal tissues. No significant association between promoter methylation status and expression profiles of CTCF and SIRT6 was found in invasive breast cancers. PMID:24842653

  5. Correlations among metabolic enzyme activities, their mRNA's and hormone levels in broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ross 708 broiler chickens were fed one of three levels of crude protein (12, 21 or 30%) from 7 to 28 days of age. Birds were then switched to either higher (12 to 30%, 21 to 30%) or lower levels of crude protein (30-12%, 21-12%). Birds were sampled three days following switches to their respective f...

  6. A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA*

    PubMed Central

    Yordanova, Martina M.; Wu, Cheng; Andreev, Dmitry E.; Sachs, Matthew S.; Atkins, John F.

    2015-01-01

    The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3′ end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5′ and 3′ of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5′ of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5′ part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3′ part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3′ of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting. PMID:25998126

  7. Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels

    PubMed Central

    Fadda, Abeer; Ryten, Mark; Droll, Dorothea; Rojas, Federico; Färber, Valentin; Haanstra, Jurgen R; Merce, Clemetine; Bakker, Barbara M; Matthews, Keith; Clayton, Christine

    2014-01-01

    African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5′–3′ exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction. PMID:25145465

  8. Effect of ozone on degradation and mRNA levels of Rubisco in relation to potato leaf age

    SciTech Connect

    Eckardt, N.A.; Pell, E.J. )

    1993-05-01

    Leaf senescence is characterized by loss of the major photosynthetic enzyme, Ribulose bisphosphate carboxylase (Rubisco). Exposure to ozone (O[sub 3]) is often associated with a premature decline in the quantity of this enzyme. Declines in Rubisco quantity could arise through inhibition of synthesis or enhancement of degradation. Several experiments were conducted to investigate the effect of O[sub 3] on these events in immature and mature leaves of potato. The effect of O[sub 3] on Rubisco synthesis was investigated indirectly by measuring the relative quantities of mRNA for the rubisco large (rbcL) and small (rbcS) subunits following a 5 hour exposure to 0.309 [mu]L L[sup [minus]1] O[sup 3] or charcoal-filtered air. O[sup 3] treatment was associated with a significant loss in rbcS mRNA in immature and mature potato leaves sampled immediately following the exposure. After the O[sup 3] exposure, a set of plants was placed in the dark at 30 C for two days. Levels of rbcS mRNA declined rapidly during the first twelve hours of dark incubation, thus declines in Rubisco quantity following two days of dark incubation were ascribed to degradation. Enhanced degradation due to O[sub 3] during the dark incubation was observed in the mature leaves, but not in the immature leaves. We conclude that O[sub 3] can cause both inhibited synthesis and enhanced degradation of Rubisco, and the response in dependent on leaf age.

  9. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase

    PubMed Central

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity. PMID:27478477

  10. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase.

    PubMed

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana; Leos-Rivas, Catalina

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity. PMID:27478477

  11. Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots

    NASA Technical Reports Server (NTRS)

    Wu, L. L.; Mitchell, J. P.; Cohn, N. S.; Kaufman, P. B.

    1993-01-01

    The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

  12. Cholesteryl Ester Transfer Protein (CETP) polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk.

    PubMed

    Papp, Audrey C; Pinsonneault, Julia K; Wang, Danxin; Newman, Leslie C; Gong, Yan; Johnson, Julie A; Pepine, Carl J; Kumari, Meena; Hingorani, Aroon D; Talmud, Philippa J; Shah, Sonia; Humphries, Steve E; Sadee, Wolfgang

    2012-01-01

    Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex

  13. Effects of glucose on collagen mRNA levels and collagen secretion in EAhy 926 endothelial cell line.

    PubMed

    Kössi, J; Muona, P; Tuukkanen, J; Ylä-Outinen, H; Kalliomäki, M; Risteli, J; Oikarinen, A; Laato, M; Peltonen, J

    2001-01-01

    Diabetes mellitus (DM) is a complex metabolic disease associated with increased accumulation of extracellular matrix by endothelial cells and contributing to vascular complications of long-standing diabetes. On the other hand, DM is also associated with decreased accumulation of extracellular matrix in granulation tissue, which is suggested to be a consequence of impaired angiogenesis. The role of hyperglycemia in these situations is not fully understood. We examined the effects of high glucose concentrations on the gene expression and secretion of various collagens in cultured EAhy 926 endothelial cells. EAhy 926 endothelial cells expressed alpha1(I) collagen mRNA at a low level and small amount of the corresponding peptide was secreted from the cells; mRNA was not affected but peptide secretion was increased by elevated glucose concentration. mRNAs for type III and VI collagens were not detected in the endothelial cells. Furthermore, high glucose concentration in long term had no morphological effects on cultured endothelial cells but increased the expression of type IV collagen, which could rather be beneficial for angiogenesis in a healing wound. Our results suggest that high glucose concentration per se may contribute to increased accumulation of extracellular matrix in blood vessels but probably is not responsible for decreased angiogenesis and granulation tissue formation in diabetic patients. PMID:12041927

  14. Effects of glucose on collagen mRNA levels and collagen secretion in EAhy 926 endothelial cell line.

    PubMed

    Kössi, J; Muona, P; Tuukkanen, J; Ylä-Outinen, H; Kalliomäki, M; Risteli, J; Oikarinen, A; Laato, M; Peltonen, J

    2001-01-01

    Diabetes mellitus (DM) is a complex metabolic disease associated with increased accumulation of extracellular matrix by endothelial cells and contributing to vascular complications of long-standing diabetes. On the other hand, DM is also associated with decreased accumulation of extracellular matrix in granulation tissue, which is suggested to be a consequence of impaired angiogenesis. The role of hyperglycemia in these situations is not fully understood. We examined the effects of high glucose concentrations on the gene expression and secretion of various collagens in cultured EAhy 926 endothelial cells. EAhy 926 endothelial cells expressed alpha 1(I) collagen mRNA at a low level and small amount of the corresponding peptide was secreted from the cells; mRNA was not affected but peptide secretion was increased by elevated glucose concentration. mRNAs for type III and VI collagens were not detected in the endothelial cells Furthermore, high glucose concentration in long term had no morphological effects on cultured endothelial cells but increased the expression of type IV collagen, which could rather be beneficial for angiogenesis in a healing wound. Our results suggest that high glucose concentration per se may contribute to increased accumulation of extracellular matrix in blood vessels but probably is not responsible for decreased angiogenesis and granulation tissue formation in diabetic patients. PMID:12016747

  15. Elevated level of renal xanthine oxidase mRNA transcription after nephropathogenic infectious bronchitis virus infection in growing layers

    PubMed Central

    Lin, Huayuan; Huang, Qiqi; Liu, Weilian; Zou, Yuelong; Zhu, Shuliang; Deng, Guangfu; Kuang, Jun; Zhang, Caiying; Cao, Huabin; Hu, Guoliang

    2015-01-01

    To assess relationships between xanthine oxidase (XOD) and nephropathogenic infectious bronchitis virus (NIBV) infection, 240 growing layers (35 days old) were randomly divided into two groups (infected and control) of 120 chickens each. Each chicken in the control and infected group was intranasally inoculated with 0.2 mL sterile physiological saline and virus, respectively, after which serum antioxidant parameters and renal XOD mRNA expression in growing layers were evaluated at 8, 15 and 22 days post-inoculation (dpi). The results showed that serum glutathione peroxidase and superoxide dismutase activities in the infected group were significantly lower than in the control group at 8 and 15 dpi (p < 0.01), while serum malondialdehyde concentrations were significantly higher (p < 0.01). The serum uric acid was significantly higher than that of the control group at 15 dpi (p < 0.01). In addition, the kidney mRNA transcript level and serum activity of XOD in the infected group was significantly higher than that of the control group at 8, 15 and 22 dpi (p < 0.05). The results indicated that NIBV infection could cause the increases of renal XOD gene transcription and serum XOD activity, leading to hyperuricemia and reduction of antioxidants in the body. PMID:26119168

  16. Interactions of several lipid-related gene polymorphisms and cigarette smoking on blood pressure levels.

    PubMed

    Yin, Rui-Xing; Wu, Dong-Feng; Wu, Jin-Zhen; Cao, Xiao-Li; Aung, Lynn Htet Htet; Miao, Lin; Long, Xing-Jiang; Liu, Wan-Ying; Zhang, Lin; Li, Meng

    2012-01-01

    The interactions of single nucleotide polymorphisms (SNPs) and cigarette smoking on blood pressure levels are limited. The present study was undertaken to detect nine lipid-related SNPs and their interactions with cigarette smoking on blood pressure levels. Genotyping of ATP-binding cassette transporter A1 (ABCA-1) V825I, acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) rs1044925, low density lipoprotein receptor (LDL-R) AvaⅡ, hepatic lipase gene (LIPC) -250G>A, endothelial lipase gene (LIPG) 584C>T, methylenetetrahydrofolate reductase (MTHFR) 677C>T, proprotein convertase subtilisin-like kexin type 9 (PCSK9) E670G, peroxisome proliferator-activated receptor delta (PPARD) +294T>C, and Scavenger receptor class B type 1 (SCARB1) rs5888 was performed in 935 nonsmokers and 845 smokers. The interactions were detected by factorial regression analysis. The frequencies of genotypes (ACAT-1 and LIPG), alleles (ABCA-1), and both genotypes and alleles (LDL-R, LIPC, PPARD and SCARB1) were different between nonsmokers and smokers (P < 0.05-0.001). The levels of pulse pressure (PP, ABCA-1), and systolic, diastolic blood pressure (SBP, DBP) and PP (LIPC) in nonsmokers were different among the genotypes (P < 0.01-0.001). The levels of SBP (ABCA-1, ACAT-1, LIPG and PCSK9), DBP (ACAT-1, LDL-R, LIPC, PCSK9 and PPARD), and PP (LIPC, LIPG, MTHFR and PCSK9) in smokers were different among the genotypes (P < 0.01-0.001). The SNPs of ABCA-1, ACAT-1 and PCSK9; ACAT-1, LDL-R, MTHFR and PCSK9; and ABCA-1, LIPC, PCSK9 and PPARD were shown interactions with cigarette smoking to influence SBP, DBP and PP levels (P < 0.05-0.001); respectively. The differences in blood pressure levels between the nonsmokers and smokers might partly result from different interactions of several SNPs and cigarette smoking. PMID:22606049

  17. Significance of the BRAF mRNA Expression Level in Papillary Thyroid Carcinoma: An Analysis of The Cancer Genome Atlas Data

    PubMed Central

    Jee, Hyeon-Gun; Kim, Young A; Kim, Ju Han; Xing, Mingzhao; Lee, Kyu Eun

    2016-01-01

    Background BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC), and it is associated with high-risk prognostic factors. However, the significance of the BRAF mRNA level in PTC remains unknown. We evaluated the significance of BRAF mRNA expression level by analyzing PTC data from The Cancer Genome Atlas (TCGA) database. Methods Data from 499 patients were downloaded from the TCGA database. After excluding other PTC variants, we selected 353 cases of classic PTC, including 193 cases with BRAFV600E and 160 cases with the wild-type BRAF. mRNA abundances were measured using RNA-Seq with the Expectation Maximization algorithm. Results The mean BRAF mRNA level was significantly higher in BRAFV600E patients than in patients with wild-type BRAF (197.6 vs. 179.3, p = 0.031). In wild-type BRAF patients, the mean BRAF mRNA level was higher in cases with a tumor > 2 cm than those with a tumor ≤ 2.0 cm (189.4 vs. 163.8, p = 0.046), and was also higher in cases with lymph node metastasis than in those without lymph node metastasis (188.5 vs. 157.9, p = 0.040). Within BRAFV600E patients, higher BRAF mRNA expression was associated with extrathyroidal extension (186.4 vs. 216.4, p = 0.001) and higher T stage (188.1 vs. 210.2, p = 0.016). Conclusions A higher BRAF mRNA expression level was associated with tumor aggressiveness in classic PTC regardless of BRAF mutational status. Evaluation of BRAF mRNA level may be helpful in prognostic risk stratification of PTC. PMID:27410688

  18. Penicillin-Binding Protein 5 Sequence Alteration and Levels of plp5 mRNA Expression in Clinical Isolates of Enterococcus faecium with Different Levels of Ampicillin Resistance.

    PubMed

    Belhaj, Mondher; Boutiba-Ben Boubaker, Ilhem; Slim, Amin

    2016-04-01

    Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF. PMID:26618475

  19. Enterostatin decreases postprandial pancreatic UCP2 mRNA levels and increases plasma insulin and amylin.

    PubMed

    Arsenijevic, Denis; Gallmann, Eva; Moses, William; Lutz, Thomas; Erlanson-Albertsson, Charlotte; Langhans, Wolfgang

    2005-07-01

    This study investigated the chronic effect of enterostatin on body weight and some of the associated changes in postprandial metabolism. Rats were adapted to 6 h of food access/day and a choice of low-fat and high-fat (HF) food and then given enterostatin or vehicle by an intraperitoneally implanted minipump delivering 160 nmol enterostatin/h continuously over a 5-day infusion period. Enterostatin resulted in a slight but significant reduction of HF intake and body weight. After the last 6-h food access period, enterostatin-treated animals had lower plasma triglyceride and free fatty acid but higher plasma glucose and lactate levels than control animals. Enterostatin infusion resulted in increased uncoupling protein-2 (UCP2) expression in various tissues, including epididymal fat and liver. UCP2 was reduced in the pancreas of enterostatin-treated animals, and this was associated with increased plasma levels of insulin and amylin. Whether these two hormones are involved in the observed decreased food intake due to enterostatin remains to be determined. As lipid metabolism appeared to be altered by enterostatin, we measured peroxisome proliferator-activated receptor (PPAR) expression in tissues and observed that PPARalpha, -beta, -gamma1, and -gamma2 expression were modified by enterostatin in epididymal fat, pancreas, and liver. This further links altered lipid metabolism with body weight loss. Our data suggest that alterations in UCP2 and PPARgamma2 play a role in the control of insulin and amylin release from the pancreas. This implies that enterostatin changes lipid and carbohydrate metabolic pathways in addition to its effects on food intake and energy expenditure. PMID:15713687

  20. The Key Proteins of Dopaminergic Neurotransmission of Human Peripheral Blood Lymphocytes: Changed mRNA Level in Alcohol Dependence Syndrome.

    PubMed

    Taraskina, A E; Grunina, M N; Zabotina, A M; Nasyrova, R F; Ivanov, M V; Krupitsky, E M; Schwartzman, A L

    2015-12-01

    The expression of dopamine receptor (DRD), Nurr1 transcription factor (NR4A2), and α-sinucleine (SNCA) genes in peripheral blood lymphocytes is evaluated. The results indicate that alcohol dependence is associated with high expression of SNCA and DRD4 (signifi cantly higher than in the control group) and is not associated with changes in the work of NR4A2 and DRD3 genes. The levels of DRD3 and DRD4 mRNA form a positive linear correlation (p≤0.05). The expression of SNCA and DRD4 genes can serve as an important peripheral marker of alcohol dependence development, which is essential for antipsychotic therapy. PMID:26621272

  1. Ochratoxin a lowers mRNA levels of genes encoding for key proteins of liver cell metabolism.

    PubMed

    Hundhausen, Christoph; Boesch-Saadatmandi, Christine; Matzner, Nicole; Lang, Florian; Blank, Ralf; Wolffram, Siegfried; Blaschek, Wolfgang; Rimbach, Gerald

    2008-01-01

    Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin that frequently contaminates food and feedstuffs. Although recent studies have indicated that OTA modulates renal gene expression, little is known regarding its impact on differential gene expression in the liver. Therefore a microarray study of the HepG2 liver cell transcriptome in response to OTA exposure (0, 0.25, 2.5 micromol/l for 24 h) was performed using Affymetrix GeneChip technology. Selected microarray results were verified by real-time PCR and Western blotting as independent methods. Out of 14,500 genes present on the microarray, 13 and 250 genes were down-regulated by 0.25 and 2.5 micromol/l OTA, respectively. Reduced mRNA levels of calcineurin A beta (PPP3CB), which regulates inflammatory signalling pathways in immune cells, and of the uncoupling protein 2 (UCP2), which has been suggested to control the production of reactive oxygen species (ROS), were observed in response to 0.25 micromol/l OTA. A particularly strong down-regulation due to 2.5 micromol/l OTA was evident for the mRNA levels of insulin-like growth factor binding protein 1 (IGFBP1) and tubulin beta 1 (TUBB1) which have been demonstrated to function as a pro-survival factor in hepatocytes and as an important cytoskeletal component, respectively. In addition, many genes involved in energy and xenobiotic metabolism, including phosphoglycerate kinase 1 (PGK1), stearoyl-Coenzyme A desaturase 1 (SCD), and glutathione S-transferase omega 1 (GSTO1), were down-regulated by OTA. Furthermore, OTA significantly inhibited the capacitative calcium entry into the HepG2 cells, indicating an alteration of calcium homeostasis. Overall, OTA dose-dependently affects multiple genes encoding for key proteins of liver cell metabolism. PMID:19287073

  2. Polymorphisms of the IL8 gene correlate with milking traits, SCS and mRNA level in Chinese Holstein.

    PubMed

    Chen, Renjin; Yang, Zhangping; Ji, Dejun; Mao, Yongjiang; Chen, Ying; Li, Yunlong; Wu, Haitao; Wang, Xiaolong; Chang, Lingling

    2011-08-01

    To explore the relation of Interleukin-8 (IL8) gene polymorphism with immunity against mastitis in dairy cow, the polymorphism of IL8 gene was investigated in 610 Chinese Holstein cow from 30 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, and milk yield, milk fat percentage, milk protein percentage, 305 day corrected milk yield, 305 day milk fat yield, 305 day milk protein yield and somatic cell score (SCS) were measured and analyzed, and the mRNA levels of IL8 genotypes in blood were detected by real-time PCR. The results showed that three genotypes, KK, KA and AA were detected with frequencies of 0.187, 0.451, and 0.362, respectively. The gene frequencies of K and A were 0.412 and 0.588, respectively. The significant association of the IL8 mutations with milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield, SCS, and significant association with milk protein percentage were identified, while their association with milk fat percentage were not significant. KK had higher milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield than AA or KA, and the least square mean of SCS of KK was significantly lower than that of AA or KA. AA had significant lower milk protein yield than KK or KA. The relative IL8 mRNA level of KK in blood was the highest. These findings demonstrated that IL8 genotype significantly correlated with mastitis resistance and the locus could be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein. PMID:21113675

  3. A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA.

    PubMed

    Voisey, J; Gomez-Cabrera, M Del C; Smit, D J; Leonard, J H; Sturm, R A; van Daal, A

    2006-06-01

    To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A --> G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production. PMID:16704456

  4. β-glucuronidase mRNA levels are correlated with gait and working memory in premutation females: understanding the role of FMR1 premutation alleles

    PubMed Central

    Kraan, C. M.; Cornish, K. M.; Bui, Q. M.; Li, X.; Slater, H. R.; Godler, D. E.

    2016-01-01

    Fragile X tremor ataxia syndrome (FXTAS) is a late-onset disorder manifesting in a proportion of FMR1 premutation individuals (PM: 55-199 CGG triplet expansions). FXTAS is associated with elevated levels of FMR1 mRNA which are toxic. In this study, relationships between neurocognitive and intra-step gait variability measures with mRNA levels, measured in blood samples, were examined in 35 PM and 35 matched control females. The real-time PCR assays measured FMR1 mRNA, and previously used internal control genes: β-Glucuronidase (GUS), Succinate Dehydrogenase 1 (SDHA) and Eukaryotic Translation Initiation Factor 4A (EI4A2). Although there was significant correlation of gait variability with FMR1 mRNA levels (p = 0.004) when normalized to GUS (FMR1/GUS), this was lost when FMR1 was normalized to SDHA and EI4A2 (2IC). In contrast, GUS mRNA level normalized to 2IC showed a strong correlation with gait variability measures (p < 0.007), working memory (p = 0.001) and verbal intelligence scores (p = 0.008). PM specific changes in GUS mRNA were not mediated by FMR1 mRNA. These results raise interest in the role of GUS in PM related disorders and emphasise the importance of using appropriate internal control genes, which have no significant association with PM phenotype, to normalize FMR1 mRNA levels. PMID:27387142

  5. β-glucuronidase mRNA levels are correlated with gait and working memory in premutation females: understanding the role of FMR1 premutation alleles.

    PubMed

    Kraan, C M; Cornish, K M; Bui, Q M; Li, X; Slater, H R; Godler, D E

    2016-01-01

    Fragile X tremor ataxia syndrome (FXTAS) is a late-onset disorder manifesting in a proportion of FMR1 premutation individuals (PM: 55-199 CGG triplet expansions). FXTAS is associated with elevated levels of FMR1 mRNA which are toxic. In this study, relationships between neurocognitive and intra-step gait variability measures with mRNA levels, measured in blood samples, were examined in 35 PM and 35 matched control females. The real-time PCR assays measured FMR1 mRNA, and previously used internal control genes: β-Glucuronidase (GUS), Succinate Dehydrogenase 1 (SDHA) and Eukaryotic Translation Initiation Factor 4A (EI4A2). Although there was significant correlation of gait variability with FMR1 mRNA levels (p = 0.004) when normalized to GUS (FMR1/GUS), this was lost when FMR1 was normalized to SDHA and EI4A2 (2IC). In contrast, GUS mRNA level normalized to 2IC showed a strong correlation with gait variability measures (p < 0.007), working memory (p = 0.001) and verbal intelligence scores (p = 0.008). PM specific changes in GUS mRNA were not mediated by FMR1 mRNA. These results raise interest in the role of GUS in PM related disorders and emphasise the importance of using appropriate internal control genes, which have no significant association with PM phenotype, to normalize FMR1 mRNA levels. PMID:27387142

  6. Elevated Levels of FMR1 mRNA in Granulosa Cells Are Associated with Low Ovarian Reserve in FMR1 Premutation Carriers

    PubMed Central

    Elizur, Shai E.; Lebovitz, Oshrit; Derech-Haim, Sanaz; Dratviman-Storobinsky, Olga; Feldman, Baruch; Dor, Jehoshua; Orvieto, Raoul; Cohen, Yoram

    2014-01-01

    Aim To assess the role of mRNA accumulation in granulosa cells as the cause of low ovarian response among FMR1 premutation carriers undergoing pre-implantation genetic diagnosis (PGD). Design Case control study in an academic IVF unit. Twenty-one consecutive FMR1 premutation carriers and 15 control women were included. After oocyte retrieval the granulosa cells mRNA levels of FMR1 was measured using RT-PCR. Results In FMR1 premutation carriers, there was a significant non-linear association between the number of CGG repeats and the number of retrieved oocytes (p<0.0001) and a trend to granulosa cells FMR1 mRNA levels (p = 0.07). The lowest number of retrieved oocytes and the highest level of mRNA were seen in women with mid-size CGG repeats (80–120). A significant negative linear correlation was observed between the granulosa cells FMR1 mRNA levels and the number of retrieved oocytes (R2 linear = 0.231, P = 0.02). Conclusion We suggest that there is a no-linear association between the number of CGG repeats and ovarian function, resulting from an increased granulosa cells FMR1 mRNA accumulation in FMR1 carriers in the mid-range (80–120 repeats). PMID:25153074

  7. Phytochrome Control of Specific mRNA levels in Developing Pea Buds 1

    PubMed Central

    Kaufman, Lon S.; Roberts, Linda L.; Briggs, Winslow R.; Thompson, William F.

    1986-01-01

    We have examined the time course for accumulation of each of 12 different nuclear gene transcripts in pea buds after irradiating dark grown seedlings with a single pulse low fluence red light (103 micromoles per square meter delivered in 100 seconds). The 12 time courses can be grouped into four general classes. Six transcripts (including RNAs coding for the chlorophyll a/b binding protein and ribulose-1,5-bisphosphate carboxylase) accumulate at a linear rate during 24 hours in darkness following the light pulse. Two transcripts increase rapidly at first but then reach a plateau after 3 hours and remain at that level for the next 21 hours. Another two transcripts exhibit a prolonged lag period before beginning to accumulate, and do not reach significant accumulation rates until 12 to 16 hours after the red light pulse. One transcript appears to undergo a transient increase in abundance in response to red light, but this is superimposed on a background of slowly increasing abundance of this RNA in control plants. This response, unlike all the others, exhibits reciprocity failure in experiments in which the same fluence of light is given over periods ranging between 50 and 4000 seconds. We have also examined the kinetics with which each of these 12 responses escapes from phytochrome-far-red absorbing form control by attempting to reverse the induction with far-red light given at various times after the red light pulse. Again, several different patterns are apparent for the different transcripts. The time at which far red reversibility first begins to be lost, the rate at which it is lost, and the final extent of reversibility remaining after 7 hours in the dark all differ for different transcripts. In addition, we have observed that some responses retain virtually complete photoreversibility for at least 7 hours. In some cases, a comparison of the time course and escape kinetic data indicates that relatively rapid turnover of the RNA must occur. It is not clear

  8. Akt isoform-dependent regulation of ATP-Binding cassette A1 expression by apolipoprotein E.

    PubMed

    Okoro, Emmanuel U; Guo, Zhongmao; Yang, Hong

    2016-08-12

    We previously reported that apolipoprotein E (apoE) upregulates ATP-binding cassette transporter A1 (ABCA1) transcription through phosphatidylinositol 3-kinase (PI3K). Here we demonstrate that treatment of murine macrophages with human apoE3 enhanced Akt phosphorylation, and upregulated ABCA1 protein and mRNA expression. Inhibition of PI3K weakened apoE3-induced Akt phosphorylation, and ABCA1 protein and mRNA increase. In contrast, inhibition of Akt only diminished apoE-induced ABCA1 protein but not the mRNA level. Suppression of protein synthesis did not erase the ability of apoE3 to increase ABCA1 protein level. Further, apoE3 increased the resistance of ABCA1 protein to calpain-mediated degradation without affecting calpain activity. Treatment of macrophages with apoE3 selectively enhanced the phosphorylation of Akt1 and Akt2, but not Akt3. Knockdown of Akt1 or Akt2 increased and decreased ABCA1 protein level, respectively; while overexpression of these Akt isoenzymes caused changes in ABCA1 protein level opposite to those induced by knockdown of the corresponding Akt. These data imply that apoE3 guards against calpain-mediated ABCA1 degradation through Akt2. PMID:27297104

  9. Explore the dynamic alternation of gene PLAC4 mRNA expression levels in maternal plasma in second trimester for nonivasive detection of trisomy 21

    PubMed Central

    Sun, Hai-Yan; Chen, Dao-Zhen; Lu, Mu-Dan; Tang, Ye; Xiao, Jian-Pin

    2015-01-01

    Objective Noninvasive prenatal detection of trisomy 21 (T21) has been achieved by measuring the ratio of two alleles of a single nucleotide polymorphism in circulating placenta specific 4 (PLAC4) mRNA in maternal plasma with a few assays in recent years. Our research is to explore the variations of PLAC4 mRNA expression level in maternal plasma with normal pregnancies in second trimester, which can provide pregnant women deeper insights with suitable detection period for the non-invasive prenatal detection of T21. Methods We measured a serial plasma PLAC4 mRNA concentrations weekly from the same 25 singleton normal pregnant women. We recruited maternal plasma samples from 45 singleton pregnant women, comprising of 25 euploid pregnancies (control group; range, 17 to 21 weeks) and 20 T21 pregnancies (T21 group; range, 19 to 24 weeks). With the application of reverse transcription polymerase chain reaction, we achieved an insight of PLAC4 mRNA expression levels in maternal plasma during second trimester with euploid pregnancies. Results Among the control group, the levels of PLAC4 mRNA expression in the gestation of 17 to 18 weeks were significantly less than those in the gestation of 18 to 21 weeks (P<0.05). The average PLAC4 mRNA concentration of the normal pregnant women was not higher than that of the T21 group (P>0.05). Conclusion The PLAC4 mRNA showed a higher level of expression in the gestation of 18 to 21 weeks with an euploid pregnancy of pregnant women. We also found that there was no significant difference in plasma PLAC4 mRNA concentration between the normal and the T21 pregnancies in second trimester. PMID:26217595

  10. Blood glutathione peroxidase-1 mRNA levels can be used as molecular biomarkers to determine dietary selenium requirements in rats.

    PubMed

    Sunde, Roger A; Thompson, Kevin M; Evenson, Jacqueline K; Thompson, Britta M

    2009-11-01

    Transcript (mRNA) levels are increasingly being used in medicine as molecular biomarkers for disease and disease risk, including use of whole blood as a target tissue for analysis. Development of blood molecular biomarkers for nutritional status, too, has potential application that parallels opportunities in medicine, including providing solid data for individualized nutrition. We previously reported that blood glutathione peroxidase-1 (Gpx1) mRNA was expressed at levels comparable to major tissues in rats and humans. To determine the efficacy of using blood Gpx1 mRNA to assess selenium (Se) status and requirements, we fed graded levels of Se (0-0.3 microg Se/g as selenite) to weanling male rats. Se status was determined by liver Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver and blood was determined by ribonuclease protection analysis. Liver Se and plasma glutathione peroxidase-3 and liver Gpx1 activities indicated that minimal Se requirements were at 0.08 microg Se/g diet. When total RNA was isolated from whole blood, Gpx1 mRNA in Se-deficient rats decreased to 10% of levels in Se-adequate (0.2 microg Se/g diet) rats. With Se supplementation, blood Gpx1 mRNA levels increased sigmoidally to a plateau with a minimum Se requirement of 0.08 microg Se/g diet, whereas glutathione peroxidase-4 mRNA levels were unaffected. Similarly, Gpx1 mRNA in RNA isolated from fractionated red blood cells decreased in Se-deficient rats to 23% of Se-adequate levels, with a minimum Se requirement of 0.09 microg Se/g diet. Additional studies showed that the preponderance of whole blood Gpx1 mRNA arises from erythroid cells, most likely reticulocytes and young erythrocytes. In summary, whole blood selenoprotein mRNA levels can be used as molecular biomarkers for assessing Se requirements, illustrating that whole blood has potential as a target tissue in development of molecular biomarkers for use in nutrition as well as in medicine. PMID:19855070

  11. Increased serum hepcidin-25 level and increased tumor expression of hepcidin mRNA are associated with metastasis of renal cell carcinoma

    PubMed Central

    2009-01-01

    Background Hepcidin has an important role in iron metabolism. We investigated whether hepcidin was involved in renal cell carcinoma (RCC). Methods We measured serum hepcidin-25 levels in 32 patients by liquid chromatograpy (LC)-mass spectrometry (MS)/MS, and assessed hepcidin mRNA expression in paired tumor and non-tumor tissue samples from the surgical specimens of 53 consecutive patients with RCC by real-time reverse transcription polymerase chain reaction. Results The serum hepcidin-25 level was higher in patients with metastatic RCC than nonmetastatic RCC (P < 0.0001), and was positively correlated with the serum interleukin-6 and C-reactive protein levels (P < 0.001). Expression of hepcidin mRNA was lower in tumor tissues than in non-tumor tissues (P < 0.0001). The serum hepcidin-25 level was not correlated with the expression of hepcidin mRNA in the corresponding tumor tissue specimens from 32 patients. Hepcidin mRNA expression in tumor tissue was correlated with metastatic potential, but not with histological differentiation or tumor stage. Kaplan-Meier analysis showed that over expression of hepcidin mRNA was related to shorter overall survival in RCC patients. Univariate analysis (Cox proportional hazards model) showed that the hepcidin mRNA level was an independent prognostic factor for overall survival. Conclusion Our findings suggest that a high serum hepcidin-25 level may indicate the progression of RCC, and that upregulation of hepcidin mRNA expression in tumor tissue may be related to increased metastatic potential. PMID:19656379

  12. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1

    PubMed Central

    Kilchert, Cornelia; Wittmann, Sina; Passoni, Monica; Shah, Sneha; Granneman, Sander; Vasiljeva, Lidia

    2015-01-01

    Summary In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation. PMID:26670050

  13. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1.

    PubMed

    Kilchert, Cornelia; Wittmann, Sina; Passoni, Monica; Shah, Sneha; Granneman, Sander; Vasiljeva, Lidia

    2015-12-22

    In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these "decay-promoting" introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation. PMID:26670050

  14. Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.

    PubMed Central

    L'Huillier, P J; Davis, S R; Bellamy, A R

    1992-01-01

    Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell. Images PMID:1425576

  15. Green tea polyphenols improve cardiac muscle mRNA, and protein levels of signal pathways related to insulin and lipid metabolism and inflammation in insulin-resistant rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epidemiologic studies indicate that the consumption of green tea polyphenols (GTP) may reduce the risk of coronary artery disease. To explore the underlying mechanisms of action at the molecular level, we examined the effects of GTP on cardiac mRNA and protein levels of genes involved in insulin an...

  16. FGF15/19 protein levels in the portal blood do not reflect changes in the ileal FGF15/19 or hepatic CYP7A1 mRNA levels

    PubMed Central

    Shang, Quan; Guo, Grace L.; Honda, Akira; Saumoy, Monica; Salen, Gerald; Xu, Guorong

    2013-01-01

    It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway fibroblast growth factor (FGF)15/19-fibroblast growth factor receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/19 protein levels in the portal blood reflected changes in FGF15/19 mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF19 rose 16-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF19 protein levels changed in the portal blood to correspond with the marked increase of FGF15/19 mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF19 mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/19 protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/19 in the ileum and CYP7A1 in the liver change significantly. PMID:23852734

  17. Positive correlation between patency and mRNA levels for cyclooxygenase-2 and prostaglandin E synthase in the uterine cervix of bitches with pyometra.

    PubMed

    Tamada, Hiromichi; Adachi, Nahoko; Kawate, Noritoshi; Inaba, Toshio; Hatoya, Shingo; Sawada, Tsutomu

    2016-03-01

    Factors involved in patency of uterine cervices in the bitch with pyometra remain to be clarified. This study examined relationship between patency and mRNA levels for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2 and prostaglandin E synthase (PGES) in the uterine cervix of bitches with pyometra. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Levels of mRNA expression were determined by semi-quantitative reverse transcription-polymerase chain reaction. The cervical patency was positively correlated with mRNA levels for COX-2 and PGES, but not those for iNOS and COX-1. The results suggest that gene expression of COX-2 and PGES may be involved in the regulation of patency in the uterine cervix of bitches with pyometra. PMID:26596635

  18. Positive correlation between patency and mRNA levels for cyclooxygenase-2 and prostaglandin E synthase in the uterine cervix of bitches with pyometra

    PubMed Central

    TAMADA, Hiromichi; ADACHI, Nahoko; KAWATE, Noritoshi; INABA, Toshio; HATOYA, Shingo; SAWADA, Tsutomu

    2015-01-01

    Factors involved in patency of uterine cervices in the bitch with pyometra remain to be clarified. This study examined relationship between patency and mRNA levels for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2 and prostaglandin E synthase (PGES) in the uterine cervix of bitches with pyometra. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Levels of mRNA expression were determined by semi-quantitative reverse transcription-polymerase chain reaction. The cervical patency was positively correlated with mRNA levels for COX-2 and PGES, but not those for iNOS and COX-1. The results suggest that gene expression of COX-2 and PGES may be involved in the regulation of patency in the uterine cervix of bitches with pyometra. PMID:26596635

  19. Global miRNA expression and correlation with mRNA levels in primary human bone cells

    PubMed Central

    Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Pastinen, Tomi; Grundberg, Elin; Kindmark, Andreas

    2015-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA–mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA–mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression. PMID:26078267

  20. Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts.

    PubMed

    Brenneisen, P; Briviba, K; Wlaschek, M; Wenk, J; Scharffetter-Kochanek, K

    1997-01-01

    Reactive oxygen species (ROS) have been shown to be important messenger molecules in the induction of several genes. In human dermal fibroblasts the herbicide paraquat (PQ2+) was used to induce intracellular oxidative stress that was modulated by the inhibition of copper, zinc superoxide dismutase (Cu,ZnSOD), glutathione peroxidase (GSHPx), catalase, and blocking of the Fenton reaction. Interstitial collagenase (MMP-1) mRNA increased time dependently for up to 72 h following paraquat treatment. A correlation with the translation of MMP-1 could, however, only be detected up to 24 h, indicating an uncoupling of transcription and translation. Interleukin-1 alpha and beta mRNA showed two peaks at 6 h and 72 h. The inhibition of catalase by aminotriazol (ATZ), inhibition of GSHPx by buthionine sulfoximine (BSO), and blocking the Fenton reaction by the iron chelator desferrioxamine (DFO) in concert led to an increase in steady-state MMP-1 mRNA levels, possibly dependent on intracellular H2O2 increase. This combined treatment potentiated MMP-1 mRNA induction up to 6.5-fold compared to paraquat treated controls. Furthermore, exogenously added H2O2 caused an increase in MMP-1 mRNA levels. In contrast, inhibition of Cu,ZnSOD by diethyldithiocarbamate (DDC), leading to diminished H2O2 production from O2.-, decreased MMP-1 mRNA induction. Collectively, our data provide evidence that H2O2 is an important intermediate in the downstream signalling pathway finally leading to the induction of increased steady state MMP-1 mRNA levels. The synthesis of MMPs may contribute to connective tissue damage in vivo related to photoaging, inflammatory diseases, and tumor invasion. PMID:8981044

  1. AFP mRNA level in enriched circulating tumor cells from hepatocellular carcinoma patient blood samples is a pivotal predictive marker for metastasis.

    PubMed

    Jin, Junhua; Niu, Xiaojuan; Zou, Lihui; Li, Lin; Li, Shugang; Han, Jingli; Zhang, Peiying; Song, Jinghai; Xiao, Fei

    2016-08-01

    Circulating tumor cells (CTCs) quantification may be helpful for evaluating cancer dissemination, predicting prognosis and assessing therapeutic effectiveness and safety. In the present study, CTCs from blood samples of 72 patients with hepatocellular carcinoma (HCC) were enriched with anti-EpCAM nanoparticles. AFP mRNA level was detected by nested polymerase chain reaction (PCR) after enrichment of CTCs from HCC blood samples at 0, 3, 6, 9 and 12 months after hepatectomy, respectively. AFP mRNA expression in CTCs was positive in 43 patients (59.7%) and negative in 29 patients (40.3%) before hepatectomy. Among 43 patients with positive AFP mRNA expression in CTCs before hepatectomy, 10 and 11 were diagnosed as intrahepatic/extrahepatic metastasis before and after hepatectomy, respectively. In addition, these 21 patients with metastasis had persisting positive AFP mRNA of CTCs during the whole tested year. Specifically, 3 patients with AFP mRNA negative in CTCs before hepatectomy changed to be positive at 6 and 9 months, and 2 of them were diagnosed as metastasis 12 months after hepatectomy. We conclude that the positive AFP mRNA of CTCs can be a pivotal predictor for HCC metastasis before and after hepatectomy. The release of AFP expression from hepatocellular carcinoma cells into circulation must be a major source of HCC metastasis. PMID:27160647

  2. mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

    PubMed

    Gupta, Rajani; Bhar, Kaushik; Sen, Nandini; Bhowmick, Debajit; Mukhopadhyay, Satinath; Panda, Koustubh; Siddhanta, Anirban

    2016-02-01

    Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas. PMID:26934777

  3. Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans.

    PubMed

    Zaragoza, Oscar; González-Párraga, Pilar; Pedreño, Yolanda; Alvarez-Peral, Francisco J; Argüelles, Juan-Carlos

    2003-06-01

    Growing cells of the Candida albicans trehalose-deficient mutant tps1/tps1 were extremely sensitive to severe oxidative stress exposure (H2O2). However, their viability was not affected after saline stress or heat-shock treatments, being roughly equivalent to that of the parental strain. In wild-type cells, these adverse conditions induced the intracellular accumulation of trehalose together with activation of trehalose-6P synthase, whereas the endogenous trehalose content and the corresponding biosynthetic activity were barely detectable in the tps1/tps1 mutant. The addition of cycloheximide did not prevent the marked induction of trehalose-6P synthase activity. Furthermore, the presence of H2O2 decreased the level of TPS1 mRNA expression. Hence, the conspicuous trehalose accumulation in response to oxidative stress is not induced by increased transcription of TPS1. Our results are consistent with a specific requirement of trehalose in order to withstand a severe oxidative stress in C. albicans, and suggest that trehalose accumulation observed under these conditions is a complex process that most probably involves post-translational modifications of the trehalose synthase complex. PMID:12783274

  4. A single infusion of MDCO-216 (ApoA-1 Milano/POPC) increases ABCA1-mediated cholesterol efflux and pre-beta 1 HDL in healthy volunteers and patients with stable coronary artery disease

    PubMed Central

    Kallend, D.G.; Reijers, J.A.A.; Bellibas, S.E.; Bobillier, A.; Kempen, H.; Burggraaf, J.; Moerland, M.; Wijngaard, P.L.J.

    2016-01-01

    Aims Apolipoprotein A-1 (ApoA-1), based on epidemiology, is inversely associated with cardiovascular (CV) events. Human carriers of the ApoA-1 Milano variant have a reduced incidence of CV disease. Regression of atherosclerotic plaque burden was previously observed on intravascular ultrasound (IVUS) with ETC-216, a predecessor of MDCO-216. MDCO-216, a complex of dimeric ApoA-1 Milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, is being developed to reduce atherosclerotic plaque burden and CV events. We investigated the efficacy and safety of a single infusion of MDCO-216 in healthy volunteers and in patients with coronary artery disease (CAD). Methods and results Twenty-four healthy volunteers and 24 patients with documented CAD received a 2-h infusion of MDCO-216 in a randomized, placebo controlled, single ascending dose study. Five cohorts of healthy volunteers and four cohorts of CAD patients received ApoA-1 Milano doses ranging from 5 to 40 mg/kg. Subjects were followed for 30 days. Dose-dependent increases in ApoA-1, phospholipid, and pre-beta 1 HDL and decreases in ApoE were observed. Prominent and sustained increases in triglyceride, and decreases in HDL-C, endogenous ApoA-1 and ApoA-II occurred at doses >20 mg/kg and profound increases in ABCA1-mediated cholesterol efflux were observed. Other lipid and lipoprotein parameters were generally unchanged. MDCO-216 was well tolerated. Conclusions MDCO-216-modulated lipid parameters profoundly increased ABCA1-mediated cholesterol efflux and was well tolerated. These single-dose data support further development of this agent for reducing atherosclerotic disease and subsequent CV events. PMID:27418968

  5. Vanadate and selenium inhibit the triiodothyronine induced enzyme activity and mRNA level for both fatty acid synthase and malic enzyme

    SciTech Connect

    Zhu, Y.; Mirmiran, R.; Goodridge, A.G.; Stapleton, S.R. Western Michigan Univ., Kalamazoo )

    1991-03-15

    In chick-embryo hepatocytes in culture, triiodothyronine stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone has no effect but amplifies the induction by T3. Recent evidence has demonstrated the insulin-mimicking action of vanadate and selenium on various physiological processes. Little information, however, is available on the affects of vanadate and selenium on the expression of genes that are regulated by insulin. These studies were initiated to test the potential of vanadate and selenium to mimic the amplification affect of insulin on the T3 induction of FAS and ME. In chick-embryo hepatocytes incubated in a chemically defined medium, addition of T3 for 48h causes an increase in the enzyme activity and mRNA level for both FAS and ME. Addition of sodium vanadate or sodium selenate (20 {mu}M) coincident with the T3 almost completely inhibited the stimulation of FAS and ME activity and accumulation of their respective mRNA's. Fifty percent maximal inhibition occurred at about 3-40{mu}M vanadate or 5-10{mu}M selenium. Vanadate and selenium similarity inhibited FAS and ME enzyme activity and mRNA level when the cells were incubated in the presence of insulin and T3. The effect of these metals was selective; isocitrate dehydrogenase activity as well as the level of glyceraldehyde 3-phosphate mRNA were not affected by any of the additions made to the cells in culture. This effect by vanadate and selenium also does not appear to be a generalized effect of metals on lipogenic enzymes as molydate under similar experimental conditions has no effect on either the enzyme activity or mRNA level of FAS or ME. Studies are continuing to determine the mechanism of action of these agents on the regulation of lipogenic enzymes.

  6. EDNRA variants associate with smooth muscle mRNA levels, cell proliferation rates, and cystic fibrosis pulmonary disease severity

    PubMed Central

    Darrah, Rebecca; McKone, Edward; O'Connor, Clare; Rodgers, Christine; Genatossio, Alan; McNamara, Sharon; Gibson, Ronald; Stuart Elborn, J.; Ennis, Madeleine; Gallagher, Charles G.; Kalsheker, Noor; Aitken, Moira; Wiese, Dawn; Dunn, John; Smith, Paul; Pace, Rhonda; Londono, Douglas; Goddard, Katrina A. B.; Knowles, Michael R.

    2010-01-01

    Airway inflammation and pulmonary disease are heterogeneous phenotypes in cystic fibrosis (CF) patients, even among patients with the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Endothelin, a proinflammatory peptide and smooth muscle agonist, is increased in CF airways, potentially contributing to the pulmonary phenotype. Four cohorts of CF patients were screened for variants in endothelin pathway genes to determine whether any of these variants associated with pulmonary function. An initial cohort of 808 CF patients homozygous for the common CF mutation, ΔF508, showed significant association for polymorphisms in the endothelin receptor A gene, EDNRA (P = 0.04), but not in the related endothelin genes (EDN1, EDN2, EDN3, or EDNRB) or NOS1, NOS2A, or NOS3. Variants within EDNRA were examined in three additional cohorts of CF patients, 238 patients from Seattle, WA, 303 from Ireland and the U.K., and 228 from Cleveland, OH, for a total of 1,577 CF patients. The three additional groups each demonstrated a significant association between EDNRA 3′-untranslated region (UTR) variant rs5335 and pulmonary function (P = 0.002). At the molecular level, single nucleotide primer extension assays suggest that the effect of the variants is quantitative. EDNRA mRNA levels from cultured primary tracheal smooth muscle cells are greater for the allele that appears to be deleterious to lung function than for the protective allele, suggesting a mechanism by which increased receptor function is harmful to the CF airway. Finally, cell proliferation studies using human airway smooth muscle cells demonstrated that cells homozygous for the deleterious allele proliferate at a faster rate than those homozygous for the protective allele. PMID:20028935

  7. Analysis of hepatic deiodinase 2 mRNA levels in natural fish lake populations exposed to different levels of putative thyroid disrupters.

    PubMed

    Jarque, Sergio; Bosch, Carme; Casado, Marta; Grimalt, Joan O; Raldúa, Demetrio; Piña, Benjamin

    2014-04-01

    Hepatic mRNA levels of the dio2 gene (deiodinase 2), implicated in thyroid hormone homeostasis, were analyzed in trout from six remote lakes in the Pyrenees (Spain) and the Tatra Mountains (Slovakia). Highest levels corresponded to fish from the two coldest lakes in Pyrenees, whereas relatively low levels were found in the Tatra lakes. These values correlated with the presence of highly-brominated polybrominated diphenyl ethers (PBDE) congeners in the muscle of the same animals, reflecting the distribution of these compounds across European mountain ranges. In contrast, cyp1a expression levels, diagnostic for the presence of dioxin-like pollutants, mirrored the distribution of semi-volatile organochlorine compounds, indicating the specificity of the two types of biological responses. Exposure to PDBEs is known to increase transcription of dio2 and other thyroid-related genes in laboratory experiments; we propose that our data reflects the same phenomenon in natural populations, driven by anthropogenic pollutants at the environmental concentrations. PMID:24530182

  8. Comparable mRNA expression of inflammatory markers but lower claudin-1 mRNA levels in foreskin tissue of HSV-2 seropositive versus seronegative asymptomatic Kenyan young men

    PubMed Central

    Röhl, Maria; Tjernlund, Annelie; Mehta, Supriya D; Pettersson, Pernilla; Bailey, Robert C; Broliden, Kristina

    2015-01-01

    Objectives Skin biopsies from local sites of herpes simplex virus 2 (HSV-2)-induced ulcers can show infiltrates of inflammatory cells several months after macroscopic healing. We hypothesise that foreskin tissue samples of asymptomatic HSV-2 seropositive men had remaining signs of inflammation at the molecular level. Even in the absence of clinical lesions, genital inflammation may contribute to increased HIV susceptibility on sexual exposure to the virus. Setting Foreskin tissue samples were collected from men undergoing elective circumcision in Kisumu, Kenya. Participants The foreskin tissue samples (n=86) were stratified into study groups based on HSV-2 serology and assessed for mRNA expression of inflammatory markers. Markers of interest were further assessed by immunohistochemical staining within the tissue samples. Results The two study groups had comparable levels of all molecular markers (CD3, CD4, CD8, CD69, CCR5, HLA-DR, Langerin, DC-SIGN, Mannose Receptor 1, IL-1, IL-6, TNF-α, β7, IgA, IFN-α, CCL5, E-cadherin, ZO-1 and occludin), except for lower mRNA levels of the epithelial junction protein claudin-1 in the HSV-2 seropositive group (p=0.008). Although mRNA levels of claudin-1 were lower in HSV-2 seropositive individuals, the corresponding protein could be visualised in the foreskin epithelium of all samples tested. Conclusions Whereas no general inflammation was demonstrated in the foreskin of asymptomatic HSV-2 seropositive individuals, a decreased expression of claudin-1 indicates a less robust genital epithelial barrier. An intact epithelial barrier is essential for blocking mucosal entry of genital infections, including HIV. PMID:25694458

  9. Chronic toxicity of pesticides to the mRNA expression levels of metallothioneins and cytochrome P450 1A genes in rainbow trout.

    PubMed

    Ceyhun, Saltuk Bugrahan; Aksakal, Ercüment; Kirim, Birsen; Atabeyoglu, Kübra; Erdogan, Orhan

    2012-03-01

    The hazardous effects of pesticides on various metabolic pathways are a great problem for environmental health and should be well determined. In the present study, the authors treated rainbow trout with 0.6 μg/L deltamethrin for 28 days and 1.6 mg/L 2,2-dichlorovinyl dimethyl phosphate for 21 days. After this time period, the authors observed alterations in mRNA expression levels of MT-A, MT-B and CYP-1A. Chronic exposure to low levels of pesticides may have a more significant effect on fish populations than acute poisoning. While both pesticides caused a significant increase on mRNA levels of MT-A and CYP-1A, MT-B mRNA levels were increased significantly only upon deltamethin administration. The significant increase in mRNA levels of the corresponding genes may be considered as a defence mechanism in addition to the antioxidants against oxidative stress, as well as a detoxification mechanism against adverse effects of pesticides. PMID:21665904

  10. Involvement of microRNA214 and transcriptional regulation in reductions in mevalonate pyrophosphate decarboxylase mRNA levels in stroke-prone spontaneously hypertensive rat livers.

    PubMed

    Michihara, Akihiro; Ide, Norie; Mizutani, Yurika; Okamoto, Manami; Uchida, Maya; Matsuoka, Hiroshi; Akasaki, Kenji

    2015-01-01

    Hypocholesterolemia has been epidemiologically identified as one of the causes of stroke (cerebral hemorrhage). We previously reported that lower protein levels of mevalonate pyrophosphate decarboxylase (MPD), which is responsible for reducing serum cholesterol levels in stroke-prone spontaneously hypertensive rats (SHRSP), in the liver were caused by a reduction in mRNA levels. However, the mechanism responsible for reducing MPD expression levels in the SHRSP liver remains unclear. Thus, we compared microRNA (miR)-214 combined with the 3'-untranslated region of MPD mRNA and heterogeneous nuclear RNA (hnRNA) between SHRSP and normotensive Wistar Kyoto rats (WKY). miR-214 levels in the liver were markedly higher in SHRSP than in WKY, whereas hnRNA levels were significantly lower. These results indicate that the upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP. PMID:26158200

  11. Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment.

    PubMed

    Rioja, I; Bush, K A; Buckton, J B; Dickson, M C; Life, P F

    2004-07-01

    Biomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1beta, TNF-alpha and IL-6 mRNA and protein expression levels in joints from DBA/1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1beta and IL-6, but not for TNF-alpha, in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1beta and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-alpha in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs. PMID:15196245

  12. Effect of sulfide, osmotic, and thermal stresses on taurine transporter mRNA levels in the gills of the hydrothermal vent-specific mussel Bathymodiolus septemdierum.

    PubMed

    Nakamura-Kusakabe, Ikumi; Nagasaki, Toshihiro; Kinjo, Azusa; Sassa, Mieko; Koito, Tomoko; Okamura, Kei; Yamagami, Shosei; Yamanaka, Toshiro; Tsuchida, Shinji; Inoue, Koji

    2016-01-01

    Hydrothermal vent environmental conditions are characterized by high sulfide concentrations, fluctuating osmolality, and irregular temperature elevations caused by vent effluents. These parameters represent potential stressors for organisms that inhabit the area around hydrothermal vents. Here, we aimed to obtain a better understanding of the adaptation mechanisms of marine species to hydrothermal vent environments. Specifically, we examined the effect of sulfide, osmolality, and thermal stress on the expression of taurine transporter (TAUT) mRNA in the gill of the deep-sea mussel Bathymodiolus septemdierum, which is a dominant species around hydrothermal vent sites. We analyzed TAUT mRNA levels by quantitative real-time polymerase chain reaction (PCR) in the gill of mussels exposed to sulfide (0.1 or 1mg/L Na2S·9H2O), hyper- (115% seawater) and hypo- (97.5%, 95.5%, and 85% seawater) osmotic conditions, and thermal stresses (12°C and 20°C) for 24 and 48h. The results showed that mussels exposed to relatively low levels of sulfide (0.1mg/L) and moderate heat stress (12°C) exhibited higher TAUT mRNA levels than the control. Although hyper- and hypo-osmotic stress did not significantly change TAUT mRNA levels, slight induction was observed in mussels exposed to low osmolality. Our results indicate that TAUT is involved in the coping mechanism of mussels to various hydrothermal vent stresses. PMID:26431911

  13. Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

    PubMed Central

    Ferrero, Rut; Torres, Magdalena

    2002-01-01

    Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

  14. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR.

    PubMed

    DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong; Losick, Richard

    2016-01-01

    Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We also show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex. PMID:26434553

  15. Prognostic Impact of mRNA Expression Levels of HER1–4 (ERBB1–4) in Patients with Locally Advanced Rectal Cancer

    PubMed Central

    Kripp, Melanie; Merx, Kirsten; Wirtz, Ralph Markus; Gaiser, Timo; Eidt, Sebastian; Schwaab, Juliana; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas

    2016-01-01

    Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC) undergoing perioperative chemoradiotherapy (CRT). Members of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases EGFR (HER1, ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC. Patients and Methods. ERBB1–4 mRNA expression and tumor proliferation using Ki-67 (MKI67) mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63) treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial. Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression of ERBB1 (EGFR, HER1) had significantly increased risk for recurrence and death. Patients with high mRNA expression of MKI67 had reduced risk for relapse. Conclusion. This analysis suggests a prognostic impact of both ERBB1 and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy. PMID:27610130

  16. Changes in Denitrifier Abundance, Denitrification Gene mRNA Levels, Nitrous Oxide Emissions, and Denitrification in Anoxic Soil Microcosms Amended with Glucose and Plant Residues▿

    PubMed Central

    Henderson, Sherri L.; Dandie, Catherine E.; Patten, Cheryl L.; Zebarth, Bernie J.; Burton, David L.; Trevors, Jack T.; Goyer, Claudia

    2010-01-01

    In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N2O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirSP) increased only in glucose-amended soil: the nirSP guild abundance increased 5-fold over the 72-h incubation period (P < 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P < 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 × 107 gene copies and 6.9 × 107 transcripts g−1 dry soil. Cumulative N2O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirSP and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios. PMID:20154105

  17. Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

    PubMed

    Staroń, Robert; Van Swelm, Rachel P L; Lipiński, Paweł; Gajowiak, Anna; Lenartowicz, Małgorzata; Bednarz, Aleksandra; Gajewska, Małgorzata; Pieszka, Marek; Laarakkers, Coby M M; Swinkels, Dorine W; Starzyński, Rafał R

    2015-01-01

    Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status. PMID:26323096

  18. Effect of Helicobacter pylori on NFKB1, p38α and TNF-α mRNA expression levels in human gastric mucosa

    PubMed Central

    SULZBACH DE OLIVEIRA, HENRIQUE SULZBACH; BIOLCHI, VANDERLEI; RICHARDT MEDEIROS, HELOUISE RICHARDT; BIZERRA GANDOR JANTSCH, DAIANE BIZERRA GANDOR; KNABBEN DE OLIVEIRA BECKER DELVING, LUCIANA KNABBEN; RECKZIEGEL, ROBERTO; GOETTERT, MÁRCIA INÊS; BRUM, ILMA SIMONI; POZZOBON, ADRIANE

    2016-01-01

    Helicobacter pylori infects ~50% of the world population, causing chronic gastritis and other forms of cellular damage. The present study assessed the influence of H. pylori on the mRNA expression levels of nuclear factor-κB1 (NFKB1), p38α and tumor necrosis factor-α (TNF-α) in human gastric mucosa in a southern Brazilian population. Human gastric tissue was collected by upper endoscopy and H. pylori diagnosis was performed using a rapid urease test and histological analysis. Total RNA was extracted and purified for subsequent cDNA synthesis and analysis by quantitative polymerase chain reaction (qPCR). The gastric tissue samples were divided into four groups as follows: Normal, inactive chronic gastritis, active chronic gastritis and intestinal metaplasia. The SDHA gene was classified as the most stable when compared with ACTB, GAPDH, B2M and HPRT1 genes, and was therefore selected as the reference gene for qPCR data normalization. TNF-α mRNA expression was significantly higher in samples that were positive for H. pylori and with active chronic gastritis. However, no difference was detected in the mRNA expression levels of NFKB1 and p38α between the groups. The present study concluded that the presence of H. pylori is associated with TNF-α upregulation in human gastric mucosa, but had no effect on NFKB1 and p38α mRNA expression levels. PMID:27284322

  19. Acute "binge" cocaine increases mu-opioid receptor mRNA levels in areas of the rat mesolimbic mesocortical dopamine system.

    PubMed

    Yuferov, V; Zhou, Y; Spangler, R; Maggos, C E; Ho, A; Kreek, M J

    1999-01-01

    Autoradiography studies demonstrated that chronic "binge" cocaine administration increased mu-opioid receptor density in dopaminergically innervated rat brain regions, including the cingulate cortex, the nucleus accumbens, and the basolateral amygdala. The present study investigated the effects of a single day of binge-pattern cocaine administration (3 x 15 mg/kg, intraperitoneally [i.p.] at hourly intervals) on mu-opioid receptor mRNA levels in selected brain regions. Rats were sacrificed 30 min after the third injection and mRNA levels were measured by a quantitative solution hybridization RNase protection assay. Acute binge cocaine administration significantly increased mu-opioid receptor mRNA levels in the frontal cortex, nucleus accumbens, and amygdala, but not in the caudate-putamen, thalamus, hippocampus, and hypothalamus. As has been suggested for other G-protein coupled receptors, the rapid increase of MOR mRNA reported in this study might represent an adaptive response to compensate for a decrease in number of receptors following cocaine-induced opioid peptide release. PMID:10210176

  20. Production of mRNA from the cry1Ac transgene differs among Bollgard lines which correlates to the level of subsequent protein.

    PubMed

    Adamczyk, John J; Perera, Omaththage; Meredith, William R

    2009-02-01

    Commercial cultivars of Bollgard cotton, Gossypium hirsutum L., differ in the amount of expressed Cry1Ac protein. However, the plant-mechanism for which this occurs is still unknown. Using quantitative real-time polymerase chain reaction (qPCR), we developed a method to determine if differences in the overall level of Cry1Ac among Bollgard lines could be correlated to the mRNA transcripts. Our data shows that the cry1Ac mRNA transcript differs among Bollgard lines and are correlated with corresponding Cry1Ac protein levels. In addition, qPCR based methods can efficiently be employed to quantify Cry1Ac protein expression levels in transgenic cotton cultivars. We postulate that qPCR based methods could be successfully employed for quantifying expression levels of transgenes in plants carrying different Bt toxins. PMID:18594999

  1. Green tea increases the antiinflammatory tristetraprolin and decreases the proinflammatory tumor necrosis factor mRNA levels in rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tristetraprolin (TTP) family proteins have antiinflammatory activity by binding to and destabilizing proinflammatory mRNAs such as TNF mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has antiinflammatory properties but the molecular mechanism has not been e...

  2. Hepatitis C virus genotype and HIV coinfection affect cytokine mRNA levels in unstimulated PBMC but do not shift the T1/T2 balance.

    PubMed

    Lee, Silvia; Watson, Mark W; Clark, Ben; Flexman, James P; Cheng, Wendy; French, Martyn A H; Price, Patricia

    2006-08-01

    Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability. PMID:16834574

  3. Developmental changes in the hypothalamic mRNA expression levels of PACAP and its receptor PAC1 and their sensitivity to fasting in male and female rats.

    PubMed

    Iwasa, Takeshi; Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Munkhzaya, Munkhsaikhan; Yiliyasi, Maira; Kato, Takeshi; Kuwahara, Akira; Irahara, Minoru

    2016-08-01

    The actions and responses of hypothalamic appetite regulatory and factors change markedly during the neonatal to pre-pubertal period. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to play pivotal roles in the regulation of metabolic and nutritional status through its specific receptor PAC1. PACAP/PAC1 have anorectic roles, and their functions are regulated by leptin in adulthood. In the present study, we showed that hypothalamic PACAP mRNA expression decreases during the neonatal to pre-pubertal period (from postnatal day 10-30) in both male and female rats. During this period, hypothalamic PACAP mRNA expression was not affected by 24h fasting in either sex, while the serum leptin levels (leptin is a positive regulator of hypothalamic PACAP expression in adulthood) of both sexes were decreased by fasting. On the other hand, hypothalamic PAC1 mRNA expression did not change during the neonatal to pre-pubertal period in either sex; however, its levels were consistently higher in males than in females. Hypothalamic PAC1 mRNA expression was decreased by 24h fasting in males, but no such changes were observed in females. These results indicate while hypothalamic PACAP expression is sensitive to a negative energy state and the serum leptin level in adulthood, no such relationships are seen in the pre-pubertal period. In addition, we speculate that differences in the gonadal steroidal milieu might induce sexual dimorphism in the basal hypothalamic PAC1 mRNA level and its response to fasting. The mechanisms responsible for and the physiological effects of such changes in hypothalamic PACAP and PAC1 expression during the developmental period remain to be clarified. PMID:27181029

  4. Molecular analysis of methylmalonic acidemia: Identification of novel mutations in the methylmalonyl-CoA mutase gene with decreased level of mutant mRNA

    SciTech Connect

    Ogasawara, M.; Matsubara, Y.; Mikami, H.; Narisawa, K.

    1994-09-01

    Deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive trait and clinically characterized by metabolic ketoacidosis. Previous studies of Caucasian and African American patients identified seven MCM mutations, and we also detected four missense substitutions (Ala197Thr, Val368Asp, Arg369His and Val669Glu). However, mutations with decreased level of MCM mRNA, which accounts for at least 25% of mutations among Caucasian patients, have not been reported. Our study on eight Japanese patients indicated that 13 of 16 mutant alleles (81%) showed decreased level of MCM mRNA, suggesting that these {open_quotes}low message{close_quotes} alleles are likely to be common contributors to MCM deficiency. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of these mutant mRNAs was less than 1% controls. We were able to amplify such mutant mRNAs by nested PCR and directly determine the primary structure. Sequence analysis revealed three novel mutations: a G-to-T substitution at nucleotide position 425, a 2 bp deletion at nt 769 and 770, and a G-to-T substitution at nt 326. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The analysis of 17 Japanese patients revealed the presence of G425T in 7 alleles (21%), suggesting a relatively high incidence of the mutation among Japanese patients. This observation is in sharp contrast to previous reports describing diverse heterogeneity of MCM mutations among Caucasians. Our report is the first to identify MCM mutations that decrease the stability of MCM mRNA. Amplification of trace amount of mRNA followed by sequencing analysis may provide useful tool for identifying such mutations.

  5. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians.

    PubMed

    Ogunkolade, Babatunji-William; Boucher, Barbara J; Prahl, Jean M; Bustin, Stephen A; Burrin, Jacky M; Noonan, Kate; North, Bernard V; Mannan, Nassima; McDermott, Michael F; DeLuca, Hector F; Hitman, Graham A

    2002-07-01

    Associations have been reported between vitamin D receptor (VDR) gene polymorphisms, type 1 diabetes, insulin secretion, and the insulin resistance syndrome. As VDR polymorphisms have no known functional significance, these findings may implicate a variant of the VDR gene or a locus in linkage disequilibrium with the VDR. We have examined VDR mRNA and VDR protein levels in relation to VDR polymorphisms (41 Bangladeshi subjects) and analyzed insulin secretory capacity (143 Bangladeshi subjects), allowing for other known determinants. Peripheral blood mononuclear cells (PBMCs) from subjects who had been genotyped for BsmI, ApaI, TaqI, and FokI VDR restriction fragment length polymorphisms were used for both total VDR mRNA quantitation (using TaqMan) and measurement of VDR protein levels (using a specific micro-immunoassay). Stepwise multiple regression analyses were used (to P < 0.05) to analyze the data. For the insulin secretion index, the best-fit model (n = 143, P < 0.0001) gave age (P = 0.002), TaqI (P < 0.0001), and BMI (P = 0.001) as independent determinants; with the inclusion of VDR mRNA and VDR protein levels, VDR mRNA was the sole independent determinant (n = 41, P = 0.024). However, the best-fit model for VDR mRNA (P = 0.004) gave FokI (P = 0.044) and TaqI (P = 0.04) genotypes and insulin secretory capacity (P = 0.042) as independent determinants. For VDR protein levels, the best-fit model (P = 0.006) gave TaqI genotype (P = 0.005) and circulating 1,25-dihydroxyvitamin-D levels (P = 0.03) as independent determinants. In conclusion, these studies confirm an association between VDR polymorphisms and insulin secretory capacity and demonstrate the VDR genotype to be a significant determinant of VDR mRNA and VDR protein levels in PBMCs, providing functional support to previously described genetic associations with the VDR gene. Furthermore, VDR expression has been shown to be a determinant of insulin secretory capacity. PMID:12086963

  6. An evaluation of the effects of acute and chronic L-tyrosine administration on BDNF levels and BDNF mRNA expression in the rat brain.

    PubMed

    Ferreira, Gabriela K; Scaini, Giselli; Jeremias, Isabela C; Carvalho-Silva, Milena; Gonçalves, Cinara L; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

    2014-04-01

    Tyrosinemia type II, which is also known as Richner-Hanhart syndrome, is an inborn error of metabolism that is due to a block in the transamination reaction that converts tyrosine to p-hydroxyphenylpyruvate. Because the mechanisms of neurological dysfunction in hypertyrosinemic patients are poorly known and the symptoms of these patients are related to the central nervous system, the present study evaluated brain-derived neurotrophic factor (BDNF) levels and bdnf mRNA expression in young rats and during growth. In our acute protocol, Wistar rats (10 and 30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old), and the rats were killed 12 h after the last injection. The brains were rapidly removed, and we evaluated the BDNF levels and bdnf mRNA expression. The present results showed that the acute administration of L-tyrosine decreased both BDNF and bdnf mRNA levels in the striatum of 10-day-old rats. In the 30-day-old rats, we observed decreased BDNF levels without modifications in bdnf transcript level in the hippocampus and striatum. Chronic administration of L-tyrosine increased the BDNF levels in the striatum of rats during their growth, whereas bdnf mRNA expression was not altered. We hypothesize that oxidative stress can interact with the BDNF system to modulate synaptic plasticity and cognitive function. The present results enhance our knowledge of the pathophysiology of hypertyrosinemia. PMID:24091827

  7. Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-related peptides in the rat adrenal medulla.

    PubMed Central

    Kanamatsu, T; Unsworth, C D; Diliberto, E J; Viveros, O H; Hong, J S

    1986-01-01

    The effect of reflex splanchnic nerve stimulation on proenkephalin A biosynthesis was investigated in the rat adrenal medulla. Tissue levels of native [Met5]enkephalin-like immunoreactivity (IR) (measured by direct RIA of tissue extracts), cryptic [Met5]enkephalin-like IR (calculated as the increase in [Met5]enkephalin-like IR detected in tissue extracts after sequential digestion with trypsin and carboxypeptidase B), and proenkephalin A mRNA were determined in adrenal medulla from rats sacrificed at various times after a period of insulin-induced hypoglycemia. Two hours of insulin hypoglycemia, which produced intense reflex stimulation of the splanchnic nerves as evidenced by a 55% decrease in the adrenal medulla catecholamine levels, resulted in a 3-fold increase in proenkephalin A mRNA levels in this tissue. The proenkephalin A mRNA levels reached a maximum 15-fold increase over control values 24 hr after this period of hypoglycemic stress and then gradually declined with an approximate half-life of 4 days. Native and cryptic [Met5]enkephalin-like IR had increased 9-fold and 12-fold, respectively, 24 hr after this period of hypoglycemia, and both demonstrated maximum increases of 130-fold and 50-fold, respectively, after 96 hr. Combined pretreatment (i.p. administration) with the ganglionic and muscarinic blocking agents chlorisondamine (5 mg/kg of body weight) and atropine (1 mg/kg) blocked the increase in levels of proenkephalin A mRNA seen in the rat adrenal medulla following insulin hypoglycemia. These data indicate that reflex splanchnic nerve discharge stimulates proenkephalin biosynthesis, probably at the level of gene expression. Images PMID:3538020

  8. Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

    PubMed Central

    Lee-Kwon, W; Park, D; Bernier, M

    1998-01-01

    Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase. PMID:9531502

  9. In ovo effects of perfluorohexane sulfonate and perfluorohexanoate on pipping success, development, mRNA expression, and thyroid hormone levels in chicken embryos.

    PubMed

    Cassone, Cristina G; Vongphachan, Viengtha; Chiu, Suzanne; Williams, Kim L; Letcher, Robert J; Pelletier, Eric; Crump, Doug; Kennedy, Sean W

    2012-05-01

    Perfluoroalkyl acids (PFAAs), specifically perfluorinated sulfonates and carboxylates, are synthetic substances known for their chemical stability, resistance to degradation, and potential to biomagnify in food chains. The toxicological and biological effects of PFAAs in avian species are not well characterized, although there is some evidence to suggest that they can impact neurodevelopment and hatching success. Our laboratory recently reported significant effects of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) on messenger RNA (mRNA) levels of thyroid hormone (TH)-responsive genes in chicken embryonic neuronal cells. In this study, we determined in ovo effects of PFHxS and PFHxA exposure (maximum dose = 38,000 and 9700 ng/g egg, respectively) on embryonic death, developmental endpoints, tissue accumulation, mRNA expression in liver and cerebral cortex, and plasma TH levels. Pipping success was reduced to 63% at the highest dose of PFHxS; no effects were observed for PFHxA. PFHxS exposure (38,000 ng/g) decreased tarsus length and embryo mass. PFHxS and PFHxA accumulated in the three tissue compartments analyzed as follows: yolk sac > liver > cerebral cortex. Type II and type III 5'-deiodinases (D2 and D3) and cytochrome P450 3A37 mRNA levels were induced in liver tissue of chicken embryos exposed to PFHxS, whereas D2, neurogranin (RC3), and octamer motif binding factor 1 mRNA levels were upregulated in cerebral cortex. Plasma TH levels were reduced in a concentration-dependent manner following PFHxS exposure; PFHxA had no effect. This in ovo study successfully validated previous in vitro results concerning the modulation of TH-responsive genes and identified adverse effects associated with TH homeostasis in response to PFHxS treatment. PMID:22302310

  10. RNA/DNA ratio and LPL and MyoD mRNA expressions in muscle of Oreochromis niloticus fed with elevated levels of palm oil

    NASA Astrophysics Data System (ADS)

    Ayisi, Christian Larbi; Zhao, Jinliang

    2016-02-01

    Palm oil is of great potential as one of the sustainable alternatives to fish oil (FO) in aquafeeds. In this present study, five isonitrogenous diets (32% crude protein) with elevated palm oil levels of 0%, 2%, 4%, 6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase (LPL) and MyoD mRNA expressions in muscle of Oreochromis niloticus. The results showed that RNA, DNA content as well as ratio of RNA to DNA were significantly affected ( P < 0.05), in each case the highest was recorded in fish group subjected to 6% palm oil level. There was a strong positive correlation between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and specific growth rate (SGR), protein efficiency ratio (PER), while a negative correlation existed between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and feed conversion ratio (FCR). The mRNA expressions of LPL and MyoD in muscle were not significantly affected by the different palm oil levels, although the highest expression was observed in fish fed with 6% palm oil level. There also existed a strong positive correlation between the mRNA expression of LPL, MyoD and SGR, PER, while their correlation with FCR was negative. In conclusion, elevated palm oil affected the RNA, DNA concentration as well as RNA/DNA ratio significantly, although the mRNA expression of LPL and MyoD were not affected significantly by elevated palm oil levels.

  11. Increased TRPV1 and PAR2 mRNA expression levels are associated only with the esophageal reflux symptoms, but not with the extraesophageal reflux symptoms

    PubMed Central

    Kim, Jin Joo; Kim, Nayoung; Choi, Yoon Jin; Kim, Joo Sung; Jung, Hyun Chae

    2016-01-01

    Abstract Transient receptor potential vanilloid-1 (TRPV1) receptor and proteinase-activated receptor 2 (PAR2) have been implicated in the mechanism of acid-induced inflammation in gastroesophageal reflux disease (GERD). We aimed to evaluate TRPV1 and PAR2 mRNA expression levels in the GERD patients and their relationship with endoscopic findings and reflux symptoms. Sixteen healthy controls, 45 patients with erosive reflux disease (ERD), and 14 nonerosive reflux disease (NERD) patients received endoscopy and completed questionnaires. Quantitative real-time polymerase chain reactions (qPCR) of TRPV1, glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), PAR2, and interleukin (IL)-8 were performed in the distal esophagus specimen. The levels of TRPV1, GDNF, NGF, PAR2, and IL-8 mRNA expression were highest in the ERD group followed by NERD and control groups and the differences between control and ERD groups were statistically significant. Within the ERD group, patients with grade B in Los Angeles (LA) classification showed significantly higher levels of TRPV1, GDNF, and NGF mRNA expression than those with grade A. Presence of reflux symptoms was associated with significant higher levels of TRPV1, PAR2, and IL-8. Notably not extraesophageal but esophageal reflux symptoms were significantly associated with them. Upregulation of TRPV1 and PAR2 pathways might play a role in the development of distal esophageal inflammation and reflux symptoms. And extraesophageal reflux symptoms might not be associated with these processes. PMID:27512850

  12. Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence

    PubMed Central

    Uranbileg, Baasanjav; Ikeda, Hitoshi; Kurano, Makoto; Enooku, Kenichiro; Sato, Masaya; Saigusa, Daisuke; Aoki, Junken; Ishizawa, Takeaki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

    2016-01-01

    Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment. PMID:26886371

  13. Performance, organ zinc concentration, jejunal brush border membrane enzyme activities and mRNA expression in piglets fed with different levels of dietary zinc.

    PubMed

    Martin, Lena; Pieper, Robert; Schunter, Nadine; Vahjen, Wilfried; Zentek, Jürgen

    2013-06-01

    This study aimed at investigating the effect of dietary zinc on performance, jejunal brush border membrane enzyme activities and mRNA levels of enzymes and two zinc transporters in piglets. A total of 126 piglets were weaned at 26 ±1 days of age and randomly allocated into three groups fed with diets 50, 150 and 2500 mg zinc/kg. Performance was recorded and at weekly intervals, eight piglets per group were killed. The activities of isolated brush border membrane enzymes including lactase, maltase, sucrase, aminopeptidase-N and intestinal alkaline phosphatase (IAP), and the relative transcript abundance of aminopeptidase-N (APN), sucrase-isomaltase (SUC), IAP and the two zinc transporters SLC39A4 (ZIP4) and SLC30A1 (ZnT1) were investigated in the jejunum. Feeding pharmacological zinc levels increased weight gain (p < 0.001) during the first week, but performance was lower (p < 0.05) in the third week. Organ zinc concentrations were increased by high dietary zinc level. The activity of IAP was higher (p < 0.05) with the highest dietary zinc level, no effects were determined for other enzymes. Dietary zinc level had no effect on transcript abundance of digestive enzymes. The mRNA levels decreased (p < 0.001) for ZIP4, and increased for ZnT1 (p < 0.05) with pharmacological zinc levels. In conclusion, pharmacological zinc levels improved performance in the short-term. Intestinal mRNA level of zinc transporters changed with high zinc supply, but this did not prevent zinc accumulation in tissues, suggesting hampered homoeostatic regulation. This might cause impaired performance during longer supply. PMID:23742645

  14. Methylation and mRNA expression levels of P15, death-associated protein kinase, and suppressor of cytokine signaling-1 genes in multiple myeloma

    PubMed Central

    Liu, Lin; Tan, Lin; He, Zhenxin

    2016-01-01

    Objective(s): The aim of this study was to investigate the methylation status and mRNA expression levels of P15, death-associated protein kinase (DAPK), and suppressor of cytokine signaling-1 (SOCS1) genes in multiple myeloma (MM). Materials and Methods: The bone marrow samples of 54 MM patients were collected and the methylation status of the P15, DAPK, and SOCS1 gene promoter regions was determined by methylation-specific polymerase chain reaction. Automated sequencing technology was used to sequence the amplified products in order to analyze the base methylation sites. mRNA expression levels were determined using real-time fluorescent quantitative polymerase chain reaction. Results: Among the 54 MM patients, the positive methylation rates of the P15, DAPK, and SOCS1 genes were 27.78%, 18.52%, and 16.67%, respectively. The methylation results were confirmed by sequencing. The positive methylation rates of the P15, DAPK, and SOCS1 genes showed no correlation with patient gender, age, typing, staging, and grouping (P>0.05). There was no significant difference in the mRNA expression levels of the P15, DAPK, and SOCS1 genes between the MM patient group and the control group (P>0.05). Conclusions: Aberrant methylation of the P15, DAPK, and SOCS1 genes exists in MM, and these genes may play certain roles in pathogenesis of MM. There was no significant difference in mRNA expression levels between the methylated group and the non-methylated group, suggesting that these genes are regulated by other mechanisms during their transcription.

  15. Correlation of Cyfra 21-1 levels in saliva and serum with CK19 mRNA expression in oral squamous cell carcinoma.

    PubMed

    Malhotra, Rewa; Urs, Aadithya B; Chakravarti, Anita; Kumar, Suman; Gupta, V K; Mahajan, Bhawna

    2016-07-01

    Oral squamous cell carcinoma (OSCC) accounts for 90 % of malignant lesions of oral cavity. The study assessed the potential of Cyfra 21-1 as a tumor marker in OSCC. The study included 50 patients of OSCC to evaluate levels of Cyfra 21-1 in serum and saliva by electrochemiluminescent immunoassay (ECLIA) and CK19 messenger RNA (mRNA) expression in tissue by florescent quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) along with healthy individuals as control. The salivary and serum Cyfra 21-1 levels in patients of OSCC were significantly higher compared to controls (p value < 0.01). There was a 2.75-fold increase in CK19 mRNA expression in OSCC cases compared to controls. A significant positive correlation was found between serum and salivary Cyfra 21-1, serum Cyfra 21-1, and CK19 mRNA expression and between salivary Cyfra 21-1 and CK19 mRNA expression. Among these, correlation between serum and salivary Cyfra 21-1 was highly significant. Salivary and serum Cyfra 21-1 showed significantly elevated levels in grade II OSCC compared to grade I histopathologically. Elevated levels of salivary Cyfra 21-1 were associated with recurrence in OSCC patients. Reverse operating curve constructed using 3 ng/ml as a cutoff for serum Cyfra 21-1 revealed the sensitivity and specificity to be 88 and 78.2 %, respectively. Using a cutoff value of 8.5 ng/ml for salivary Cyfra 21-1, the sensitivity was found to be 93.8 % and specificity 84.3 %. We advocate salivary Cyfra 21-1 as a better diagnostic marker over serum Cyfra 21-1 as well as a potential marker in the prognosis of OSCC. PMID:26779624

  16. Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos

    SciTech Connect

    Egloff, Caroline; Crump, Doug; Porter, Emily; Williams, Kim L.; Letcher, Robert J.; Gauthier, Lewis T.; Kennedy, Sean W.

    2014-09-15

    The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism. - Highlights: • TBOEP is not embryolethal to chicken embryos. • TEP affected embryonic viability, morphometric endpoints, and thyroid hormone levels. • TEP altered mRNA levels of xenobiotic and lipid metabolism genes. • TEP increased plasma bile acids and caused gallbladder hypotrophy

  17. [Analysis of HSP70 mRNA level and association between linked microsatellite loci and heat tolerance traits in dairy cows].

    PubMed

    Liu, Yan-Xin; Li, Da-Qi; Cui, Qun-Wei; Shi, Hong-Xia; Wang, Gen-Lin

    2010-09-01

    The objective of this study was to investigate the variation of HSP70 mRNA level in dairy cows and relationships of its closely linked microsatellite loci with heat tolerance traits. Blood samples were collected from ten healthy Holstein cows with the same age and milking stage at different temperatures-humid-index (THI) (86.2, high temperature; 70.9, critical high temperature, and 56.8, optimum temperature). The mRNA levels of HSP70 of lymphocytes in peripheral blood were analyzed using real-time RT-PCR. The mRNA level of HSP70 was increased with the THI; the mRNA level of HSP70 at high temperature was higher than others (P<0.01). This indicated that the bovine HSP70 gene may act as a potential can-didate gene for response to heat shock. Genetic variation of three microsatellite loci BMS468, BM1258, and BM1815, which were closely linked to HSP70 gene on chromosome 23, was analyzed in 160 Holstein cows with non-denaturing poly-acrylamide gel electrophoresis. The association between these microsatellite loci and heat tolerance traits were analyzed by least square linear model. The results showed that 134 bp/128 bp at BMS468 and 186 bp/148 bp at BM1815 were the most favorable genotypes for HTC, red cell potassium, and decrement rate of milk yield in high temperature (P<0.05); 101 bp/99 bp at BM1258 was the most favorable genotype for decrement rate of milk yield in high temperature (P<0.05). PMID:20870615

  18. The FLT3ITD mRNA level has a high prognostic impact in NPM1 mutated, but not in NPM1 unmutated, AML with a normal karyotype.

    PubMed

    Schneider, Friederike; Hoster, Eva; Unterhalt, Michael; Schneider, Stephanie; Dufour, Annika; Benthaus, Tobias; Mellert, Gudrun; Zellmeier, Evelyn; Kakadia, Purvi M; Bohlander, Stefan K; Feuring-Buske, Michaela; Buske, Christian; Braess, Jan; Heinecke, Achim; Sauerland, Maria C; Berdel, Wolfgang E; Büchner, Thomas; Wörmann, Bernhard J; Hiddemann, Wolfgang; Spiekermann, Karsten

    2012-05-10

    The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level--measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3+1)--on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML. PMID:22374696

  19. Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle.

    PubMed

    Laville, M; Auboeuf, D; Khalfallah, Y; Vega, N; Riou, J P; Vidal, H

    1996-07-01

    We have investigated the acute regulation by insulin of the mRNA levels of nine genes involved in insulin action, in muscle biopsies obtained before and at the end of a 3-h euglycemic hyperinsulinemic clamp. Using reverse transcription-competitive PCR, we have measured the mRNAs encoding the two insulin receptor variants, the insulin receptor substrate-1, the p85alpha subunit of phosphatidylinositol-3-kinase, Ras associated to diabetes (Rad), the glucose transporter Glut 4, glycogen synthase, 6-phosphofructo-l-kinase, lipoprotein lipase, and the hormone-sensitive lipase. Insulin infusion induced a significant increase in the mRNA level of Glut 4 (+56 +/- 13%), Rad (+96 +/- 25%), the p85alpha subunit of phosphatidylinositol-3-kinase (+92 +/- 18%) and a decrease in the lipoprotein lipase mRNA level (-49 +/- 5%), while the abundance of the other mRNAs was unaffected. The relative expression of the two insulin receptor variants was not modified. These results demonstrate an acute coordinated regulation by insulin of the expression of genes coding key proteins involved in its action in human skeletal muscle and suggest that Rad and the p85alpha regulatory subunit of phosphatidylinositol-3-kinase can be added to the list of the genes controlled by insulin. PMID:8690802

  20. Immune-Stimulatory Effects of a Bacteria-Based Probiotic on Peripheral Leukocyte Subpopulations and Cytokine mRNA Expression Levels in Scouring Holstein Calves

    PubMed Central

    QADIS, Abdul Qadir; GOYA, Satoru; YATSU, Minoru; KIMURA, Atsushi; ICHIJO, Toshihiro; SATO, Shigeru

    2014-01-01

    ABSTRACT Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3+ T cells; CD4+, CD8+ and WC1+ γδ T cell subsets; and CD14+, CD21+ and CD282+ (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves. PMID:24451928

  1. Decreased mRNA levels of cardiac Cx43 and ZO1 in sudden cardiac death related to coronary atherosclerosis: a pilot study.

    PubMed

    Xue, Ye; Zhao, Rui; Du, Si-Hao; Zhao, Dong; Li, Dong-Ri; Xu, Jing-Tao; Xie, Xiao-Li; Wang, Qi

    2016-07-01

    Sudden cardiac death (SCD) is the most frequent cause of sudden unexplained death in forensic practice. The most common cause of SCD is coronary artery disease related to coronary atherosclerosis. Previous study suggested the possible application of connexin 43 (Cx43) and zonula occludens-1 (ZO1) immunostaining in the early diagnosis of myocardial ischemia. However, there appears to be insufficient data with regard to their mRNA levels. The present study investigated the cardiac mRNA levels of Cx43 and ZO1, using forensic autopsy materials consisting of 41 control cases without any disease or structural abnormality of the heart (group 1), 32 deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis (group 2), and 29 traumatic deaths with coronary atherosclerosis (group 3). Ten candidate reference genes were evaluated in the left ventricles of 10 forensic autopsy cases. EEF1A1, PPIA, TPT1, and RPL13A were identified as the most stable reference genes. Using these validated reference genes, mRNA levels of Cx43 and ZO1 were examined in the bilateral ventricles and atria of the heart. Relative mRNA quantification demonstrated decreased calibrated normalized relative quantity (CNRQ) values of Cx43 and ZO1 in bilateral ventricles of group 2. When using one conventional reference gene (GAPDH or ACTB) for normalization, nearly no difference was detected among the three groups. These findings indicate that ventricular gap junction remodeling may be a key contributor to rhythm disturbances. Analysis of cardiac Cx43 and ZO1 using real-time PCR is useful in diagnosis of SCD, and validation of reference genes is crucial. PMID:26972693

  2. MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

    PubMed Central

    2012-01-01

    Background In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6) in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta) treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment), six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs) against IFN-beta were analyzed by the cytopathic effect assay. Results We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment) than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012); in all the positive samples we only found variant A. Furthermore, 58/99 (58.6%) MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8%) among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004); no differences were found between patients with and without NAbs. Conclusions MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment. PMID:23009575

  3. Immunohistochemistry as a valuable tool to assess CD30 expression in peripheral T-cell lymphomas: high correlation with mRNA levels.

    PubMed

    Bossard, Céline; Dobay, Maria Pamela; Parrens, Marie; Lamant, Laurence; Missiaglia, Edoardo; Haioun, Corinne; Martin, Antoine; Fabiani, Bettina; Delarue, Richard; Tournilhac, Olivier; Delorenzi, Mauro; Gaulard, Philippe; de Leval, Laurence

    2014-11-01

    The extended use of brentuximab-vedotin was reported for CD30(+) nonanaplastic peripheral T-cell lymphomas (PTCLs) with promising efficacy. CD30 status assessment is thus a critical factor for therapeutic decision, but the reliability of immunohistochemistry (IHC) in evaluating its expression remains to be defined. This prompted us to investigate the correlation between semiquantitative CD30 protein assessment by IHC and messenger RNA (mRNA) assessment by microarrays in a cohort of 376 noncutaneous PTCLs representative of the main entities. By IHC, CD30 expression was heterogeneous across and within entities and significantly associated with large tumor cell size. In addition to 100% anaplastic large-cell lymphomas, 57% of other PTCL entities were CD30-positive at a 5% threshold. CD30 protein expression was highly correlated to mRNA levels. mRNA levels were bimodal, separating high from low CD30-expressing PTCL cases. We conclude that IHC is a valuable tool in clinical practice to assess CD30 expression in PTCLs. PMID:25224410

  4. Nonphotic entrainment by 5-HT1A/7 receptor agonists accompanied by reduced Per1 and Per2 mRNA levels in the suprachiasmatic nuclei.

    PubMed

    Horikawa, K; Yokota, S; Fuji, K; Akiyama, M; Moriya, T; Okamura, H; Shibata, S

    2000-08-01

    In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock gene, Period (Per), are involved in photic entrainment. Therefore, we examined whether phase advances elicited by 8-OH DPAT were associated with a change of Period mRNA levels in the SCN. In this experiment, we cloned partial cDNAs encoding hamster Per1, Per2, and Per3 and observed both circadian oscillation and the light responsiveness of Period. Furthermore, we found that the inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurred only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT. PMID:10908630

  5. Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production

    PubMed Central

    Isabelle, Georis; Tate, Jennifer J; Vierendeels, Fabienne; Cooper, Terrance G; Dubois, Evelyne

    2015-01-01

    The first step in executing the genetic program of a cell is production of mRNA. In yeast, almost every gene is transcribed as multiple distinct isoforms, differing at their 5′ and/or 3′ termini. However, the implications and functional significance of the transcriptome-wide diversity of mRNA termini remains largely unexplored. In this paper, we show that the GAT1 gene, encoding a transcriptional activator of nitrogen-responsive catabolic genes, produces a variety of mRNAs differing in their 5′ and 3′ termini. Alternative transcription initiation leads to the constitutive, low level production of 2 full length proteins differing in their N-termini, whereas premature transcriptional termination generates a short, highly nitrogen catabolite repression- (NCR-) sensitive transcript that, as far as we can determine, is not translated under the growth conditions we used, but rather likely protects the cell from excess Gat1. PMID:26259534

  6. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number. PMID:24866763

  7. Phytoestrogens regulate mRNA and protein levels of guanine nucleotide-binding protein, beta-1 subunit (GNB1) in MCF-7 cells.

    PubMed

    Naragoni, Srivatcha; Sankella, Shireesha; Harris, Kinesha; Gray, Wesley G

    2009-06-01

    Phytoestrogens (PEs) are non-steroidal ligands, which regulate the expression of number of estrogen receptor-dependent genes responsible for a variety of biological processes. Deciphering the molecular mechanism of action of these compounds is of great importance because it would increase our understanding of the role(s) these bioactive chemicals play in prevention and treatment of estrogen-based diseases. In this study, we applied suppression subtractive hybridization (SSH) to identify genes that are regulated by PEs through either the classic nuclear-based estrogen receptor or membrane-based estrogen receptor pathways. SSH, using mRNA from genistein (GE) treated MCF-7 cells as testers, resulted in a significant increase in GNB1 mRNA expression levels as compared with 10 nM 17beta estradiol or the no treatment control. GNB1 mRNA expression was up regulated two- to fivefold following exposure to 100.0 nM GE. Similarly, GNB1 protein expression was up regulated 12- to 14-fold. GE regulation of GNB1 was estrogen receptor-dependent, in the presence of the anti-estrogen ICI-182,780, both GNB1 mRNA and protein expression were inhibited. Analysis of the GNB1 promoter using ChIP assay showed a PE-dependent association of estrogen receptor alpha (ERalpha) and beta (ERbeta) to the GNB1 promoter. This association was specific for ERalpha since association was not observed when the cells were co-incubated with GE and the ERalpha antagonist, ICI. Our data demonstrate that the levels of G-protein, beta-1 subunit are regulated by PEs through an estrogen receptor pathway and further suggest that PEs may control the ratio of alpha-subunit to beta/gamma-subunits of the G-protein complex in cells. J. Cell. Physiol. 219: 584-594, 2009. (c) 2009 Wiley-Liss, Inc. PMID:19170076

  8. Alterations in trace element levels and mRNA expression of Hsps and inflammatory cytokines in livers of duck exposed to molybdenum or/and cadmium.

    PubMed

    Cao, Huabin; Gao, Feiyan; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-03-01

    To evaluate the effects of dietary Molybdenum (Mo) or/and Cadmium (Cd) on trace elements and the mRNA expression levels of heat shock proteins (Hsps) and inflammatory cytokines in duck livers. 240 healthy 11-day-old ducks were randomly divided into six groups with 40 ducks in each group, which were treated with Mo or/and Cd at different doses on the basal diet for 120 days. On days 30, 60, 90 and 120, 10 birds in each group were randomly selected and euthanized and then the livers were collected to determine the contents of Mo, Cd, copper (Cu), iron (Fe), zine (Zn), Selenium (Se) and the mRNA expression levels of Hsps, inflammatory cytokines. In addition, liver tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results showed that the mRNA expression of Hsp60, Hsp70, Hsp90, tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and cyclooxygenase-2 (COX-2) were significantly (P<0.01) upregulated in combination groups; Contents of Cu, Fe, Zn, and Se decreased in combined groups (P<0.05) in the later period of the test while contents of Mo and Cd significantly increased (P<0.01); Furthermore severe hepatocyte diffuse fatty, hepatic cords swelling, hepatic sinusoid disappeared, and inflammatory cells infiltrated around the hepatic central vein were observed in Mo combined with Cd groups. The results indicated that dietary Mo or/and Cd might lead to stress, inflammatory response, tissue damage and disturb homeostasis of trace elements in duck livers. Moreover the two elements showed a possible synergistic relationship. And the high mRNA expression of HSPs and inflammatory cytokines may play a role in the resistance of liver toxicity induced by Mo and Cd. PMID:26682514

  9. Effects of footshocks on anxiety-like behavior and mRNA levels of precursor peptides for corticotropin releasing factor and opioids in the forebrain of the rat.

    PubMed

    Wang, Huiying; Li, Sa; Kirouac, Gilbert J

    2015-12-01

    Corticotropin releasing factor (CRF) and dynorphin are neuropeptides that are associated with the negative emotional states. Experimental evidence indicates that dynorphin neurons located in the nucleus accumbens and CRF neurons in the bed nucleus of the stria terminalis (BST) and the central nucleus of the amygdala (CeA) mediate anxiety-like behaviors immediately after the stressful experience (24-48h). The present study was done to evaluate if changes in the levels of the mRNA for these peptides in the striatum, BST, and CeA were associated with the long-lasting avoidance of novelty, a measure of an anxiety-like state, in a subset of rats exposed to unpredictable and moderately intense footshocks (5×2s of 1.5mA). Shocked rats with enhanced fear to a novel tone 24h after the footshocks (high responders; HR) displayed long-lasting avoidance in the elevated T-maze whereas shocked rats with low levels of acute fear (low responders; LR) had low levels of avoidance similar to nonshocked rats. An increase in the level of proCRF mRNA was detected in the CeA of the HR compared to LR and nonshocked rats but not in other areas of the brain sampled. In contrast, prodynorphin and proenkephalin mRNA levels in the striatum, BST and CeA were not different between HR, LR and nonshocked rats. This study provides evidence that CRF neurons in the CeA may play a role in the anxiety-like state produced in a subset of rats exposed to footshocks. PMID:26363852

  10. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    PubMed

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  11. Changes in expression levels of ERCC1, DPYD, and VEGFA mRNA after first-line chemotherapy of metastatic colorectal cancer: results of a multicenter study

    PubMed Central

    Uemoto, Shinji; Yoshida, Kazuhiro; Saiura, Akio; Watanabe, Masayuki; Maehara, Yoshihiko; Oki, Eiji; Ikeda, Yasuharu; Matsuda, Hiroyuki; Yamamoto, Masakazu; Shimada, Mitsuo; Taketomi, Akinobu; Unno, Michiaki; Sugihara, Kenichi; Ogata, Yutaka; Eguchi, Susumu; Kitano, Seigo; Shirouzu, Kazuo; Saiki, Yasumitsu; Takamori, Hiroshi; Mori, Masaki; Hirata, Toshihiko; Wakabayashi, Go; Kokudo, Norihiro

    2015-01-01

    Our previous study showed that administering oxaliplatin as first-line chemotherapy increased ERCC1 and DPD levels in liver colorectal cancers (CRCs) metastases. Second, whether the anti-VEGF monoclonal antibody bevacizumab alters tumoral VEGFA levels is unknown. We conducted this multicenter observational study to validate our previous findings on ERCC1 and DPD, and clarify the response of VEGFA expression to bavacizumab administration. 346 CRC patients with liver metastases were enrolled at 22 Japanese institutes. Resected liver metastases were available for 175 patients previously treated with oxaliplatin-based chemotherapy (chemotherapy group) and 171 receiving no previous chemotherapy (non-chemotherapy group). ERCC1, DPYD, and VEGFA mRNA levels were measured by real-time RT-PCR. ERCC1 mRNA expression was significantly higher in the chemotherapy group than in the non-chemotherapy group (P = 0.033), and were significantly correlated (Spearman's correlation coefficient = 0.42; P < 0.0001). VEGFA expression level was higher in patients receiving bevacizumab (n = 51) than in those who did not (n = 251) (P = 0.007). This study confirmed that first-line oxaliplatin-based chemotherapy increases ERCC1 and DPYD expression levels, potentially enhancing chemosensitivity to subsequent therapy. We also found that bevacizumab induces VEGFA expression in tumor cells, suggesting a biologic rationale for extending bevacizumab treatment beyond first progression. PMID:26372896

  12. Evaluation of mRNA Expression Levels of cyp51A and mdr1, Candidate Genes for Voriconazole Resistance in Aspergillus flavus

    PubMed Central

    Fattahi, Azam; Zaini, Farideh; Kordbacheh, Parivash; Rezaie, Sasan; Safara, Mahin; Fateh, Roohollah; Farahyar, Shirin; Kanani, Ali; Heidari, Mansour

    2015-01-01

    Background: Voriconazole Resistance (VRC-R) in Aspergillus flavus isolates impacts the management of aspergillosis, since azoles are the first choice for prophylaxis and therapy. However, to the best of our knowledge, the mechanisms underlying voriconazole resistance are poorly understood. Objectives: The present study was designed to evaluate mRNA expression levels of cyp51A and mdr1 genes in voriconazole resistant A. flavus by a Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique. Materials and Methods: Five A. flavus isolates with resistance to VRC were examined by a RT-PCR approach. Results: Four out of five isolates revealed cyp51A and mdr1 mRNA overexpression. Interestingly, the isolate, which was negative for cyp51A and mdr1 mRNA expression showed a high voriconazole Minimum Inhibitory Concentration (MIC). Furthermore, a computational-based analysis predicted that voriconazole resistance could be mediated through cooperation with a network protein interaction. Conclusions: Our experimental and in silico findings may provide new insight in the complex molecular pathways of drug resistance and also could assist design an efficient therapeutic strategy for aspergillosis treatment. PMID:26865941

  13. Pathogenesis of Aleutian mink disease parvovirus infection: effects of suppression of antibody response on viral mRNA levels and on development of acute disease.

    PubMed Central

    Alexandersen, S; Storgaard, T; Kamstrup, N; Aasted, B; Porter, D D

    1994-01-01

    We suppressed the B-cell development and antibody response in mink by using treatment with polyclonal anti-immunoglobulin M (anti-IgM) to study the effects of antiviral antibodies on development of Aleutian mink disease parvovirus (ADV)-induced disease in more detail. Newborn mink kits were injected intraperitoneally with 1 mg of either anti-IgM or a control preparation three times a week for 30 to 34 days. At 21 days after birth, groups of mink kits were infected with the highly virulent United isolate of ADV. At selected time points, i.e., postinfection days 9, 13, 29, and 200, randomly chosen mink kits were sacrificed, and blood and tissues were collected for analyses. The efficacy of immunosuppressive treatment was monitored by electrophoretic techniques and flow cytometry. Effects of treatment on viral replication, on viral mRNA levels, and on development of acute or chronic disease were determined by histopathological, immunoelectrophoretic, and molecular hybridization techniques. Several interesting findings emerged from these studies. First, antiviral antibodies decreased ADV mRNA levels more than DNA replication. Second, suppression of B-cell development and antibody response in mink kits infected at 21 days of age resulted in production of viral inclusion bodies in alveolar type II cells. Some of these kits showed mild clinical signs of respiratory disease, and one kit died of respiratory distress; however, clinical signs were seen only after release of immunosuppression, suggesting that the production of antiviral antibodies, in combination with the massive amounts of free viral antigen present, somehow is involved in the induction of respiratory distress. It is suggested that the antiviral antibody response observed in mink older than approximately 14 days primarily, by a yet unknown mechanism, decreases ADV mRNA levels which, if severe enough, results in restricted levels of DNA replication and virion production. Furthermore, such a restricted ADV

  14. Increased TRPV1 and PAR2 mRNA expression levels are associated only with the esophageal reflux symptoms, but not with the extraesophageal reflux symptoms.

    PubMed

    Kim, Jin Joo; Kim, Nayoung; Choi, Yoon Jin; Kim, Joo Sung; Jung, Hyun Chae

    2016-08-01

    Transient receptor potential vanilloid-1 (TRPV1) receptor and proteinase-activated receptor 2 (PAR2) have been implicated in the mechanism of acid-induced inflammation in gastroesophageal reflux disease (GERD). We aimed to evaluate TRPV1 and PAR2 mRNA expression levels in the GERD patients and their relationship with endoscopic findings and reflux symptoms.Sixteen healthy controls, 45 patients with erosive reflux disease (ERD), and 14 nonerosive reflux disease (NERD) patients received endoscopy and completed questionnaires. Quantitative real-time polymerase chain reactions (qPCR) of TRPV1, glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), PAR2, and interleukin (IL)-8 were performed in the distal esophagus specimen.The levels of TRPV1, GDNF, NGF, PAR2, and IL-8 mRNA expression were highest in the ERD group followed by NERD and control groups and the differences between control and ERD groups were statistically significant. Within the ERD group, patients with grade B in Los Angeles (LA) classification showed significantly higher levels of TRPV1, GDNF, and NGF mRNA expression than those with grade A. Presence of reflux symptoms was associated with significant higher levels of TRPV1, PAR2, and IL-8. Notably not extraesophageal but esophageal reflux symptoms were significantly associated with them.Upregulation of TRPV1 and PAR2 pathways might play a role in the development of distal esophageal inflammation and reflux symptoms. And extraesophageal reflux symptoms might not be associated with these processes. PMID:27512850

  15. Tudor-SN, a component of stress granules, regulates growth under salt stress by modulating GA20ox3 mRNA levels in Arabidopsis

    PubMed Central

    Yan, Chunxia; Yan, Zongyun; Wang, Yizheng; Yan, Xiaoyuan; Han, Yuzhen

    2014-01-01

    The Tudor-SN protein (TSN) is universally expressed and highly conserved in eukaryotes. In Arabidopsis, TSN is reportedly involved in stress adaptation, but the mechanism involved in this adaptation is not understood. Here, we provide evidence that TSN regulates the mRNA levels of GA20ox3, a key enzyme for gibberellin (GA) biosynthesis. The levels of GA20ox3 transcripts decreased in TSN1/TSN2 RNA interference (RNAi) transgenic lines and increased in TSN1 over-expression (OE) transgenic lines. The TSN1 OE lines displayed phenotypes that may be attributed to the overproduction of GA. No obvious defects were observed in the RNAi transgenic lines under normal conditions, but under salt stress conditions these lines displayed slower growth than wild-type (WT) plants. Two mutants of GA20ox3, ga20ox3-1 and -2, also showed slower growth under stress than WT plants. Moreover, a higher accumulation of GA20ox3 transcripts was observed under salt stress. The results of a western blot analysis indicated that higher levels of TSN1 accumulated after salt treatment than under normal conditions. Subcellular localization studies showed that TSN1 was uniformly distributed in the cytoplasm under normal conditions but accumulated in small granules and co-localized with RBP47, a marker protein for stress granules (SGs), in response to salt stress. The results of RNA immunoprecipitation experiments indicated that TSN1 bound GA20ox3 mRNA in vivo. On the basis of these findings, we conclude that TSN is a novel component of plant SGs that regulates growth under salt stress by modulating levels of GA20ox3 mRNA. PMID:25205572

  16. Risk of childhood asthma is associated with CpG-site polymorphisms, regional DNA methylation and mRNA levels at the GSDMB/ORMDL3 locus

    PubMed Central

    Acevedo, Nathalie; Reinius, Lovisa E.; Greco, Dario; Gref, Anna; Orsmark-Pietras, Christina; Persson, Helena; Pershagen, Göran; Hedlin, Gunilla; Melén, Erik; Scheynius, Annika; Kere, Juha; Söderhäll, Cilla

    2015-01-01

    Single-nucleotide polymorphisms (SNPs) in GSDMB (Gasdermin B) and ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) are strongly associated with childhood asthma, but the molecular alterations contributing to disease remain unknown. We investigated the effects of asthma-associated SNPs on DNA methylation and mRNA levels of GSDMB and ORMDL3. Genetic association between GSDMB/ORMDL3 and physician-diagnosed childhood asthma was confirmed in the Swedish birth-cohort BAMSE. CpG-site SNPs (rs7216389 and rs4065275) showed differences in DNA methylation depending on carrier status of the risk alleles, and were significantly associated with methylation levels in two CpG sites in the 5′ UTR (untranslated region) of ORMDL3. In the Swedish Search study, we found significant differences in DNA methylation between asthmatics and controls in five CpG sites; after adjusting for lymphocyte and neutrophil cell counts, three remained significant: one in IKZF3 [IKAROS family zinc finger 3 (Aiolos); cg16293631] and two in the CpG island (CGI) of ORMDL3 (cg02305874 and cg16638648). Also, cg16293631 and cg02305874 correlated with mRNA levels of ORMDL3. The association between methylation and asthma was independent of the genotype in rs7216389, rs4065275 and rs12603332. Both SNPs and CpG sites showed significant associations with ORMDL3 mRNA levels. SNPs influenced expression independently of methylation, and the residual association between methylation and expression was not mediated by these SNPs. We found a differentially methylated region in the CGI shore of ORMDL3 with six CpG sites less methylated in CD8+ T-cells. In summary, this study supports that there are differences in DNA methylation at this locus between asthmatics and controls; and both SNPs and CpG sites are independently associated with ORMDL3 expression. PMID:25256354

  17. Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress, hepatic stellate cell activation, cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs.

    PubMed

    Abhilash, P A; Harikrishnan, R; Indira, M

    2012-02-01

    Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β(1), TNF-α and α(1) (I) collagen in hepatic tissues. PMID:22149461

  18. Changes in brain seabream GnRH mRNA and pituitary seabream GnRH peptide levels during ovarian maturation in female barfin flounder.

    PubMed

    Amano, Masafumi; Pham, Ky Xuan; Amiya, Noriko; Yamanome, Takeshi; Yamamori, Kunio

    2008-09-01

    The pleuronectid barfin flounder Verasper moseri expresses three forms of gonadotropin-releasing hormones (GnRHs), i.e., seabream GnRH (sbGnRH), salmon GnRH, and chicken GnRH-II. Among these, sbGnRH is the dominant form in the pituitary, indicating that sbGnRH regulates gonadal maturation. In order to clarify the physiological roles of sbGnRH during ovarian maturation in reared female barfin flounder, the changes in brain sbGnRH mRNA levels and pituitary sbGnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. The fish hatched in April 2002. The gonadosomatic index remained low until August 2004 and increased thereafter until April 2005 when the fish began to ovulate. The sbGnRH mRNA levels per brain increased significantly from April 2004 to April 2005. Pituitary sbGnRH peptide levels also increased significantly during this period. These results indicate that sbGnRH is involved in ovarian maturation and ovulation in the barfin flounder. PMID:18662692

  19. A novel long non-coding RNA in the rheumatoid arthritis risk locus TRAF1-C5 influences C5 mRNA levels.

    PubMed

    Messemaker, T C; Frank-Bertoncelj, M; Marques, R B; Adriaans, A; Bakker, A M; Daha, N; Gay, S; Huizinga, T W; Toes, R E M; Mikkers, H M M; Kurreeman, F

    2016-03-01

    Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis. PMID:26673966

  20. Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel.

    PubMed

    Pelster, Bernd; Schneebauer, Gabriel; Dirks, Ron P

    2016-01-01

    Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories "response to

  1. Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel

    PubMed Central

    Pelster, Bernd; Schneebauer, Gabriel; Dirks, Ron P.

    2016-01-01

    Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories “response to

  2. HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer

    PubMed Central

    2013-01-01

    Introduction Recent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial. Methods HER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro. Results Only 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, P <0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, P <0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (P = 0.004), but not in HER2-positive/ESR1-negative tumors. Conclusions Only patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group. Introduction The human epidermal growth factor receptor 2 (HER2) is the prototype of a predictive biomarker for targeted treatment [1-8]. International initiatives have established the

  3. Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver

    SciTech Connect

    Houldsworth, J.; Attardi, G.

    1988-01-01

    Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH/sub 2/-end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones.

  4. The feathering gene is linked to degranulation and oxidative burst not cytokine/chemokine mRNA expression levels or Salmonella enteritidis organ invasion in broilers.

    PubMed

    Swaggerty, Christina L; He, Haiqi; Genovese, Kenneth J; Kaiser, Pete; Pevzner, Igal Y; Kogut, Michael H

    2006-12-01

    In the past, we showed differences in heterophil function between parental broilers (A [fast feathering] > B [slow feathering]) and their F1 reciprocal crosses (D [fast feathering] > C [slow feathering]). In the present study, we evaluated the linkage of the feathering gene to heterophil function, pro-inflammatory cytokine/chemokine mRNA expression levels, and resistance to Salmonella enteritidis organ invasion. Heterophils were isolated from 2-day-old chickens (C and D) separated into males and females - slow males and females (SM and SF), and fast males and females (FM and FF). Heterophil functions of degranulation and oxidative burst were measured. Heterophils from FF chickens (183+/-8.9) released more (P < 0.05) beta-d-glucuronidase (microM) than heterophils from SF chickens (149+/-3.7); FF heterophils (4.6 x 10(4)) generated a significantly (P < 0.05) greater oxidative burst (mean relative fluorescent units) compared with SF heterophils (4.2 x 10(4)). Interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were quantitated by real-time quantitative reverse transcriptase-polymerase chain reaction. No differences were observed between SM and FM or between SF and FF heterophils. Finally, 1-day-old chickens were administered S. enteritidis and liver/spleen organ invasion was quantitated. No differences were observed between the number of S. enteritidis-positive FF and SF chickens, but FM were significantly (P < 0.05) more resistant to S. enteritidis organ invasion than SM chickens. The data indicate degranulation and oxidative burst were linked with the feathering gene; however, interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were not. Furthermore, susceptibility to in vitro S. enteritidis organ invasion was not linked to the feathering gene. PMID:17121735

  5. VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level

    PubMed Central

    Arcondéguy, Tania; Lacazette, Eric; Millevoi, Stefania; Prats, Hervé; Touriol, Christian

    2013-01-01

    Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3′-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3′ untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation. PMID:23851566

  6. Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.

    PubMed Central

    Mansoor, O; Beaufrere, B; Boirie, Y; Ralliere, C; Taillandier, D; Aurousseau, E; Schoeffler, P; Arnal, M; Attaix, D

    1996-01-01

    The cellular mechanisms responsible for enhanced muscle protein breakdown in hospitalized patients, which frequently results in lean body wasting, are unknown. To determine whether the lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways are activated, we measured mRNA levels for components of these processes in muscle biopsies from severe head trauma patients. These patients exhibited negative nitrogen balance and increased rates of whole-body protein breakdown (assessed by [13C]leucine infusion) and of myofibrillar protein breakdown (assessed by 3-methylhistidine urinary excretion). Increased muscle mRNA levels for cathepsin D, m-calpain, and critical components of the ubiquitin proteolytic pathway (i.e., ubiquitin, the 14-kDa ubiquitin-conjugating enzyme E2, and proteasome subunits) paralleled these metabolic adaptations. The data clearly support a role for multiple proteolytic processes in increased muscle proteolysis. The ubiquitin proteolytic pathway could be activated by altered glucocorticoid production and/or increased circulating levels of interleukin 1beta and interleukin 6 observed in head trauma patients and account for the breakdown of myofibrillar proteins, as was recently reported in animal studies. Images Fig. 1 Fig. 1 Fig. 3 Fig. 4 PMID:8610106

  7. Ginkgo biloba extract and its flavonol and terpenelactone fractions do not affect beta-secretase mRNA and enzyme activity levels in cultured neurons and in mice.

    PubMed

    Augustin, Sabine; Huebbe, Patricia; Matzner, Nicole; Augustin, Kay; Schliebs, Reinhard; Cermak, Rainer; Wolffram, Siegfried; Rimbach, Gerald

    2008-01-01

    Numerous clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer's disease (AD). Although neuroprotective properties of EGb761 have been consistently reported, the molecular mechanisms of EGb761 and the specific role of its major constituents, the flavonols and terpenlactones, are largely unknown. One major hallmark of AD is the deposition of amyloid-beta (A beta) as amyloid plaques in the brain. A beta is a cleavage product of amyloid precursor protein (APP). Certain proteases, called beta-secretases (BACE), are crucial in the formation of A beta. The purpose of the present study was to investigate the efficacy of EGb761 and its flavonol and terpenelactone fraction to modulate BACE-1 enzyme activity and mRNA levels in vitro and in vivo. Neither EGb761 nor its fractions affected BACE-1 activity in vitro. Furthermore, also in Neuro-2a cells and wild-type as well as transgenic (Tg2576) laboratory mice, no significant effect of EGb761 on BACE-1 enzyme activity and mRNA levels were observed. Current findings suggest that BACE-1 may not be a major molecular target of EGb761 and its flavonol and terpenelactone fraction. PMID:18186016

  8. mRNA Levels of Imprinted Genes in Bovine In Vivo Oocytes, Embryos and Cross Species Comparisons with Humans, Mice and Pigs

    PubMed Central

    Jiang, Zongliang; Dong, Hong; Zheng, Xinbao; Marjani, Sadie L.; Donovan, David M.; Chen, Jingbo; Tian, Xiuchun (Cindy)

    2015-01-01

    Twenty-six imprinted genes were quantified in bovine in vivo produced oocytes and embryos using RNA-seq. Eighteen were detectable and their transcriptional patterns were: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); peaked at a specific stage (PHLDA2, SGCE, PEG10, PEG3, GNAS, MEG3, DGAT1, ASCL2, NNAT, and NAP1L5); or constantly low (DIRAS3, IGF2, H19 and RTL1). These patterns reflect mRNAs that are primarily degraded, important at a specific stage, or only required at low quantities. The mRNAs for several genes were surprisingly abundant. For instance, transcripts for the maternally imprinted MEST and PLAGL1, were high in oocytes and could only be expressed from the maternal allele suggesting that their genomic imprints were not yet established/recognized. Although the mRNAs detected here were likely biallelically transcribed before the establishment of imprinted expression, the levels of mRNA during these critical stages of development have important functional consequences. Lastly, we compared these genes to their counterparts in mice, humans and pigs. Apart from previously known differences in the imprinting status, the mRNA levels were different among these four species. The data presented here provide a solid reference for expression profiles of imprinted genes in embryos produced using assisted reproductive biotechnologies. PMID:26638780

  9. Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae).

    PubMed

    Grünewald, K; Eckert, M; Hirschberg, J; Hagen, C

    2000-04-01

    The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated. PMID:10759523

  10. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  11. The mRNA level of Charcot-Leyden crystal protein/galectin-10 is a marker for CRTH2 activation in human whole blood in vitro.

    PubMed

    Lin, Tai-An; Kourteva, Galina; Hilton, Holly; Li, Hongli; Tare, Nadine S; Carvajal, Valerie; Hang, Julie S; Wei, Xin; Renzetti, Louis M

    2010-11-01

    CRTH2 is one of the prostaglandin D₂ receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD₂. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD₂, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD₂-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents. PMID:20858065

  12. Differential regulation of polysome mRNA levels in mouse Hepa-1C1C7 cells exposed to dioxin.

    PubMed

    Thornley, Jessica A; Trask, Heidi W; Ridley, Christian J A; Korc, Murray; Gui, Jiang; Ringelberg, Carol S; Wang, Sinny; Tomlinson, Craig R

    2011-10-01

    The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a "tagging" of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology. PMID:21570461

  13. DIFFERENTIAL REGULATION OF POLYSOME mRNA LEVELS IN MOUSE HEPA-1C1C7 CELLS EXPOSED TO DIOXIN

    PubMed Central

    Thornley, Jessica A.; Trask, Heidi W.; Ridley, Christian J. A.; Korc, Murray; Gui, Jiang; Ringelberg, Carol S.; Wang, Sinny; Tomlinson, Craig R.

    2011-01-01

    The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a “tagging” of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology. PMID:21570461

  14. Influence of functional polymorphisms in TNF-α, IL-8, and IL-10 cytokine genes on mRNA expression levels and risk of gastric cancer.

    PubMed

    de Oliveira, Juliana Garcia; Rossi, Ana Flávia Teixeira; Nizato, Daniela Manchini; Cadamuro, Aline Cristina Targa; Jorge, Yvana Cristina; Valsechi, Marina Curado; Venâncio, Larissa Paola Rodrigues; Rahal, Paula; Pavarino, Érika Cristina; Goloni-Bertollo, Eny Maria; Silva, Ana Elizabete

    2015-12-01

    Functional polymorphisms in promoter regions can produce changes in the affinity of transcription factors, thus altering the messenger ribonucleic acid (mRNA) expression levels of inflammatory cytokines associated with the risk of cancer development. The goal of this study was to evaluate the influence that polymorphisms in the cytokine genes known as TNF-α-308 G/A (rs1800629), TNF-α-857 C/T (rs1799724), IL-8-251 T/A (rs4073), IL-8-845 T/C (rs2227532), and IL-10-592 C/A (rs1800872) have on changes to mRNA expression levels and on the risks of chronic gastritis (CG) and gastric cancer (GC). A sample of 723 individuals was genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Relative mRNA expression levels were measured using quantitative real-time PCR (qPCR). Polymorphisms TNF-α-308 G/A and IL-8-251 A/T were not associated with risks of these gastric lesions. However, TNF-α-857 C/T, IL-8-845 T/C, and IL-10-592 C/A were found to be associated with a higher risk of GC, and IL-10-592 C/A was found to be associated with a higher risk of CG. The relative mRNA expression levels (RQ) of TNF-α, IL-8, and IL-10 were markedly downregulated in the CG group (median RQs = 0.128, 0.247, and 0.614, respectively), while the RQ levels of TNF-α in the GC group were upregulated (RQ = 2.749), but were basal for IL-8 (RQ = 1.053) and downregulated for IL-10 (RQ = 0.179). When the groups were stratified according to wild-type and polymorphic alleles, only for IL-8-845 T/C the polymorphic allele was found to influence the expression levels of this cytokine. IL-8-845 C allele carriers were significantly upregulated in both groups (GC and CG; RQ = 3.138 and 2.181, respectively) when compared to TT homozygotes (RQ = -0.407 and 0.165, respectively). In silico analysis in the IL-8 promoter region revealed that the presence of the variant C allele in position -845 is responsible for the presence of the binding

  15. Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus.

    PubMed Central

    Vestergaard, H; Lund, S; Larsen, F S; Bjerrum, O J; Pedersen, O

    1993-01-01

    In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM. Images PMID:8514849

  16. Effects of Dietary Selenium Against Lead Toxicity on mRNA Levels of 25 Selenoprotein Genes in the Cartilage Tissue of Broiler Chicken.

    PubMed

    Gao, H; Liu, C P; Song, S Q; Fu, J

    2016-07-01

    The interactions between the essential element selenium (Se) and the toxic element lead (Pb) have been reported extensively; however, little is known about the effect of Se on Pb toxicity and the expression pattern of selenoproteins in the cartilage of chicken. To investigate the effects of Se on Pb toxicity and the messenger RNA (mRNA) expressions of selenoproteins in cartilage tissue, an in vitro study was performed on 1-day-old broiler chickens (randomly allocated into four groups) with diet of different concentration of Se and Pb. After 90 days, the meniscus cartilage and sword cartilage tissue were examined for the mRNA levels of 25 selenoprotein genes. The results showed that Se and Pb influenced the expression of selenoprotein genes in the chicken cartilage tissue. In detail, Se could alleviate the downtrend of the expression of Gpx1, Gpx2, Gpx4, Txnrd2, Txnrd3, Dio1, Dio2, Seli, Selu, Sepx1, Selk, Selw, Selo, Selm, Sep15, Sepnn1, Sels, and Selt induced by Pb exposure in the meniscus cartilage. In the sword cartilage, Se alleviated the downtrend of the expression of Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Dio2, Dio3, Seli, Selh, SPS2, Sepx1, Selk, Selw, Selo, Selm, Sep15, Selpb, Sepn1, and Selt induced by Pb exposure. The present study provided some compensated data about the roles of Se against Pb toxicity in the regulation of selenoprotein expression. PMID:26643179

  17. Elevated serum levels of IGFBP-2 found in children suffering from acute leukaemia is accompanied by the occurrence of IGFBP-2 mRNA in the tumour clone.

    PubMed Central

    Wex, H.; Vorwerk, P.; Mohnike, K.; Bretschneider, D.; Kluba, U.; Aumann, V.; Blum, W. F.; Mittler, U.

    1998-01-01

    Insulin-like growth factor-binding proteins (IGFBPs) are important modulators of IGF action. In 50 children suffering from acute lymphoblastic leukaemia (ALL), we studied the serum levels of IGFBP-1,-2 and-3. The mean standard deviation score (SDS) values were estimated to be 0.7, 3.1 and -1.7 for the IGFBP-1,-2 and-3, respectively, compared with the normal range defined by a SDS from -2 to +2. IGFBP-1 and-3 were normal, but for IGFBP-2 we found a significantly elevated serum level compared with control groups (P < 0.05). However, during chemotherapy this increased serum IGFBP-2 normalized. In addition, we found a correlation between higher serum levels and the detection rate of the IGFBP-2 transcript in corresponding cells. In patients with ALL, the detection rates of IGFBP-2 mRNA were estimated to be 72% and 35% at the time of diagnosis and at day 33 of chemotherapy respectively; in the control groups (healthy children and children at their initial presentation of diabetes mellitus), the values were 28% and 33% respectively. Based on the correlation between IGFBP-2 serum levels and the corresponding gene expression as well as the normalization of IGFBP-2 levels during chemotherapy, we concluded that the increased serum level mainly originated from the tumour clone itself. Furthermore, possible functional consequences of elevated IGFBP-2 were outlined. Images Figure 2 Figure 3 PMID:9716037

  18. Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels

    PubMed Central

    Kloiber, Stefan; Ripke, Stephan; Kohli, Martin A.; Reppermund, Simone; Salyakina, Daria; Uher, Rudolf; McGuffin, Peter; Perlis, Roy H.; Hamilton, Steven P.; Pütz, Benno; Hennings, Johannes; Brückl, Tanja; Klengel, Torsten; Bettecken, Thomas; Ising, Marcus; Uhr, Manfred; Dose, Tatjana; Unschuld, Paul G.; Zihl, Josef; Binder, Elisabeth; Müller-Myhsok, Bertram; Holsboer, Florian; Lucae, Susanne

    2013-01-01

    Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9 × 10−5) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant

  19. Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels.

    PubMed

    Kloiber, Stefan; Ripke, Stephan; Kohli, Martin A; Reppermund, Simone; Salyakina, Daria; Uher, Rudolf; McGuffin, Peter; Perlis, Roy H; Hamilton, Steven P; Pütz, Benno; Hennings, Johannes; Brückl, Tanja; Klengel, Torsten; Bettecken, Thomas; Ising, Marcus; Uhr, Manfred; Dose, Tatjana; Unschuld, Paul G; Zihl, Josef; Binder, Elisabeth; Müller-Myhsok, Bertram; Holsboer, Florian; Lucae, Susanne

    2013-07-01

    Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9×10(-5)) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR(⁎)D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant

  20. Effect of chemical and metallic compounds on biomass, mRNA levels and laccase activity of Phlebia brevispora BAFC 633.

    PubMed

    Fonseca, María Isabel; Ramos-Hryb, Ana Belén; Fariña, Julia Inés; Afanasiuk, Silvana Soledad Sawostjanik; Villalba, Laura Lidia; Zapata, Pedro Darío

    2014-08-01

    Nine aromatic compounds (caffeic acid, syringaldehyde, vanillic acid, guaiacol, vanillin, sinapic acid, syringol, syringic acid and ferulic acid) and four metallic compounds (CuSO4, AgNO3, MnSO4, and CaCl2) were tested for their ability to increase laccase (Lac) activity in the ligninolytic basidiomycete Phlebia brevispora BAFC 633. The addition of syringaldehyde, syringol, guaiacol, sinapic acid, vanillin, ferulic acid and CuSO4 showed a positive effect on fungal growth; however, it decreased dramatically with the addition of AgNO3 and did not undergo changes in the presence of CaCl2 or MnSO4. Lac activity increased with the addition of all the compounds tested, depending on the concentration and the day of culture. P. brevispora BAFC 633 produced two isoenzymes, a constitutively expressed of 60 kDa and another of 75 kDa expressed upon induction by sinapic acid, MnSO4 or CuSO4. Lac secretion capacity of P. brevispora BAFC 633 can be increased 27 times higher than the control with the highest levels detected in the presence of 0.3 mM CuSO4 at day 14. The action is affected at pre-transcriptional level regulating at the onset of the process, however it does not rule out the effect at the post-transcriptional and post-translational levels, for which is necessary to deepen in the knowledge of all possible regulation points of gene expression. PMID:24682954

  1. Manganese peroxidase mRNA and enzyme activity levels during bioremediation of polycyclic aromatic hydrocarbon-contaminated soil with Phanerochaete chrysosporium.

    PubMed Central

    Bogan, B W; Schoenike, B; Lamar, R T; Cullen, D

    1996-01-01

    mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of three manganese peroxidase (MnP) genes during removal of polycyclic aromatic hydrocarbons from cultures of Phanerochaete chrysosporium grown in presterilized soil. Periods of high mnp transcript levels and extractable MnP enzyme activity were temporally correlated, although separated by a short (1- to 2-day) lag period. This time frame also coincided with maximal rates of fluorene oxidation and chrysene disappearance in soil cultures, supporting the hypothesis that high ionization potential polycyclic aromatic hydrocarbons are oxidized in soil via MnP-dependent mechanisms. The patterns of transcript abundance over time in soil-grown P. chrysosporium were similar for all three of the mnp mRNAs studied, indicating that transcription of this gene family may be coordinately regulated under these growth conditions. PMID:8779576

  2. Lysophosphatidic acid can support the formation of membranous structures and an increase in MBP mRNA levels in differentiating oligodendrocytes

    PubMed Central

    Nogaroli, Luciana; Yuelling, Larra M.; Dennis, Jameel; Gorse, Karen; Payne, Shawn G.; Fuss, Babette

    2009-01-01

    During development, differentiating oligodendrocytes progress in distinct maturation steps from premyelinating to myelinating cells. Such maturing oligodendrocytes express both receptors mediating signaling via extracellular lysophosphatidic acid (LPA) and the major enzyme generating extracellular LPA, namely phosphodiesterase-Iα/autotaxin (PD-Iα/ATX). However, the biological role of extracellular LPA during the maturation of differentiating oligodendrocytes is currently unclear. Here, we demonstrate that application of exogenous LPA induced an increase in the area occupied by the oligodendrocytes’ process network, but only when PD-Iα/ATX expression was down-regulated. This increase in network area was caused primarily by the formation of membranous structures. In addition, LPA increased the number of cells positive for myelin basic protein (MBP). This effect was associated by an increase in the mRNA levels coding for MBP but not myelin oligodendrocyte glycoprotein (MOG). Taken together, these data suggest that LPA may play a crucial role in regulating the later stages of oligodendrocyte maturation. PMID:18594965

  3. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  4. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  5. Changes of Antioxidant Function and the mRNA Expression Levels of Apoptosis Genes in Duck Ovaries Caused by Molybdenum or/and Cadmium.

    PubMed

    Cao, Huabin; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-06-01

    To investigate the effects of molybdenum (Mo) combined with cadmium (Cd) on the antioxidant function and the mRNA expression levels of apoptosis-related genes in duck ovaries, 60 healthy 11-old-day female ducks were treated with hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) or/and cadmium sulfate (3CdSO4·8H2O) at different doses on a daily basis for 120 days. On the 120th day, ten female birds in each group were euthanized, and the ovaries and blood were collected to determine the antioxidant indexes and the mRNA expression levels of Bak-1, Bcl-2, and caspase-3 in ovaries. In addition, ovary tissues were subjected to histopathological analysis with optical microscope. The total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity decreased significantly (P < 0.01) in treated groups comparing with control while the nitric oxide synthase (NOS) activity increased (P < 0.01) both in ovary tissue and serum. The Bak-1 and caspase-3 expressions were upregulated while the Bcl-2 was downgraded by Mo or/and Cd. Biomolecules were affected in all metal-treated groups, whereas combined-treated animals showed greater effects. What is more, pathological damage in Mo and Cd combination treated groups was more severe. The results from the present study indicated that Mo or/and Cd caused oxidative stress and apoptosis in duck ovaries. Combination of Mo and Cd showed additive or synergistic effect leading to apoptosis and oxidative stress, and the pathway might be the mitochondrial pathway. PMID:26446861

  6. Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

    PubMed Central

    Perez, Marco; Muñoz-Galván, Sandra; Jiménez-García, Manuel P.; Marín, Juan J.; Carnero, Amancio

    2015-01-01

    Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors. PMID:26528855

  7. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  8. Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress?

    PubMed Central

    2013-01-01

    Background The harbour porpoise is exposed to increasing pressure caused by anthropogenic activities in its marine environment. Numerous offshore wind farms are planned or under construction in the North and Baltic Seas, which will increase underwater noise during both construction and operation. A better understanding of how anthropogenic impacts affect the behaviour, health, endocrinology, immunology and physiology of the animals is thus needed. The present study compares levels of stress hormones and mRNA expression of cytokines and acute-phase proteins in blood samples of harbour porpoises exposed to different levels of stress during handling, in rehabilitation or permanent human care. Free-ranging harbour porpoises, incidentally caught in pound nets in Denmark, were compared to harbour porpoises in rehabilitation at SOS Dolfijn in Harderwijk, the Netherlands, and individuals permanently kept in human care in the Dolfinarium Harderwijk and Fjord & Belt Kerteminde, Denmark. Blood samples were investigated for catecholamines, adrenaline, noradrenaline and dopamine, as well as for adrenocorticotropic hormone (ACTH), cortisol, metanephrine and normetanephrine. mRNA expression levels of relevant cell mediators (cytokines IL-10 and TNFα, acute-phase proteins haptoglobin and C-reactive protein and the heat shock protein HSP70) were measured using real-time PCR. Results Biomarker expression levels varied between free-ranging animals and porpoises in human care. Hormone and cytokine ranges showed correlations to each other and to the health status of investigated harbour porpoises. Hormone concentrations were higher in free-ranging harbour porpoises than in animals in human care. Adrenaline can be used as a parameter for the initial reaction to acute stress situations; noradrenaline, dopamine, ACTH and cortisol are more likely indicators for the following minutes of acute stress. There is evidence for different correlations between production of normetanephrine

  9. Decreased GAD65 mRNA levels in select subpopulations in the cerebellar dentate nuclei in autism: an in situ hybridization study

    PubMed Central

    Yip, Jane; Soghomonian, Jean Jacques; Blatt, Gene J.

    2009-01-01

    The laterally positioned dentate nuclei lie in a key position in the cerebellum to receive input from Purkinje cells in the lateral cerebellar hemisphere participating in both motor and cognitive functions. Although neuropathology of the four cerebellar nuclei using Nissl staining has been qualitatively reported in children and adults with autism, surprisingly the dentate nuclei appeared less affected despite reported reductions in Purkinje cells in the posterolateral cerebellar hemisphere. To determine any underlying abnormalities in the critically important GABAergic system, the rate-limiting GABAsynthesizing enzyme, glutamic acid decarboxylase (GAD) type 65 was measured via in situ hybridization histochemistry in dentate somata. GAD65 mRNA labeling revealed two distinct subpopulations of neurons in adult control and autism post-mortem brains: small-sized cells (about 10–12 µm in diameter, presumed interneurons) and larger-sized neurons (about 18–20 µm in diameter, likely feedback to IO neurons). A mean 51% reduction in GAD65 mRNA levels was found in the larger labeled cells in the autistic group compared to the control group (p=0.009; independent t-test) but not in the smaller cell subpopulation. This suggests a disturbance in the intrinsic cerebellar circuitry in the autism group potentially interfering with the synchronous firing of inferior olivary neurons, and the timing of Purkinje cell firing and inputs to the dentate nuclei. Disturbances in critical neural substrates within these key circuits could disrupt afferents to motor and/or cognitive cerebral association areas in the autistic brain likely contributing to the marked behavioral consequences characteristic of autism. PMID:19358307

  10. Increased MET Gene Copy Number but Not mRNA Level Predicts Postoperative Recurrence in Patients with Non–Small Cell Lung Cancer1

    PubMed Central

    Kowalczuk, Oksana; Kozlowski, Miroslaw; Niklinska, Wiesława; Kisluk, Joanna; Niklinska, Barbara Joanna; Niklinski, Jacek

    2014-01-01

    The aim of the present study was to investigate the relationship of MET copy number (CN) and MET mRNA expression to other molecular alterations, clinicopathologic characteristics, and survival of patients with resected non–small cell lung cancer. One hundred fifty-one paired surgical samples of tumor and tumor-distant normal lung tissues were analyzed by comparative quantitative polymerase chain reaction (PCR) methods with commercially available assays and the CopyCaller software v. 1.0 for post-PCR data processing (downloadable from www.appliedbiosystems.com). MET copy gain (set as more than 3.0 copies per cell) was found in 18.5% of the samples and occurred more frequently in the adenocarcinomas (ADCs) with an increased epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) CN (P = .001 and .030 for EGFR and HER2, respectively) and in the ADCs with EGFR activating mutations (P = .051) but did not correlate with KRAS dosage or mutational status. MET mRNA level was 1.76-fold higher [95% confidence interval (CI), 1.29-2.40] in the tumor compared to unaffected lung tissue and associated significantly with MET CN (beta coefficient, 1.51; 95% CI, 1.22-1.87; P < .001). In the multivariable analysis, patients diagnosed with ADC with increased MET CN had a significantly higher risk of disease recurrence (hazard ratio, 1.76; 95% CI, 1.20-2.57; P = .004). An increased MET CN in combination with histologic type appears to be a prognostic factor in patients with ADC after a curative surgery. PMID:25389455

  11. Associations of Haplotypes Upstream of IRS1 with Insulin Resistance, Type 2 Diabetes, Dyslipidemia, Preclinical Atherosclerosis, and Skeletal Muscle LOC646736 mRNA Levels

    PubMed Central

    Soyal, Selma M.; Felder, Thomas; Auer, Simon; Oberkofler, Hannes; Iglseder, Bernhard; Paulweber, Bernhard; Dossena, Silvia; Nofziger, Charity; Paulmichl, Markus; Esterbauer, Harald; Krempler, Franz; Patsch, Wolfgang

    2015-01-01

    The genomic region ~500 kb upstream of IRS1 has been implicated in insulin resistance, type 2 diabetes, adverse lipid profile, and cardiovascular risk. To gain further insight into this chromosomal region, we typed four SNPs in a cross-sectional cohort and subjects with type 2 diabetes recruited from the same geographic region. From 16 possible haplotypes, 6 haplotypes with frequencies >0.01 were observed. We identified one haplotype that was protective against insulin resistance (determined by HOMA-IR and fasting plasma insulin levels), type 2 diabetes, an adverse lipid profile, increased C-reactive protein, and asymptomatic atherosclerotic disease (assessed by intima media thickness of the common carotid arteries). BMI and total adipose tissue mass as well as visceral and subcutaneous adipose tissue mass did not differ between the reference and protective haplotypes. In 92 subjects, we observed an association of the protective haplotype with higher skeletal muscle mRNA levels of LOC646736, which is located in the same haplotype block as the informative SNPs and is mainly expressed in skeletal muscle, but only at very low levels in liver or adipose tissues. These data suggest a role for LOC646736 in human insulin resistance and warrant further studies on the functional effects of this locus. PMID:26090471

  12. Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

    PubMed

    Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M

    2014-07-01

    Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP

  13. Altered mRNA Levels of Glucocorticoid Receptor, Mineralocorticoid Receptor, and Co-Chaperones (FKBP5 and PTGES3) in the Middle Frontal Gyrus of Autism Spectrum Disorder Subjects.

    PubMed

    Patel, Neil; Crider, Amanda; Pandya, Chirayu D; Ahmed, Anthony O; Pillai, Anilkumar

    2016-05-01

    Although stress has been implicated in the pathophysiology of autistic spectrum disorder (ASD), it is not known whether glucocorticoid receptor (GR) levels are altered in the brain of subjects with ASD. The messenger RNA (mRNA) levels of GR isoforms (GRα, GRβ, GRγ, and GRP), mineralocorticoid receptor (MR), GR co-chaperones (FKBP5, PTGES3, and BAG1), and inflammatory cytokines (IL-6, IL-1β, and IFN-γ) were examined in the postmortem middle frontal gyrus tissues of 13 ASD and 13 age-matched controls by qRT-PCR. The protein levels were examined by Western blotting. We found significant decreases in GRα (64 %), GRγ (48 %), GRP (20 %) and MR (46 %) mRNA levels in ASD subjects as compared to controls. However, significant increases in FKBP5 (42 %) and PTGES3 (35 %) mRNA levels were observed in ASD subjects. There were no differences in the mRNA levels of GRβ and BAG1 in ASD subjects as compared to controls. MR mRNA was found to be negatively correlated with the diagnostic score for abnormality of development. On the protein level, significant reductions in GR and MR, but no change in FKBP5 and PTGES3 were found in ASD subjects as compared to controls. Moreover, we observed significant increases in IL-1β and IFN-γ mRNA levels in ASD subjects, and these cytokines were negatively associated with GR levels. Our data, for the first time, reports dysregulation of GR, MR, FKBP5, and PTGES3 in ASD and suggest a possible role of inflammation in altered GR function in ASD. PMID:25912394

  14. D-4F reduces EO6 immunoreactivity, SREBP-1c mRNA levels, and renal inflammation in LDL receptor-null mice fed a Western diet.

    PubMed

    Buga, Georgette M; Frank, Joy S; Mottino, Giuliano A; Hakhamian, Ashkan; Narasimha, Ajay; Watson, Andrew D; Yekta, Babak; Navab, Mohamad; Reddy, Srinivasa T; Anantharamaiah, G M; Fogelman, Alan M

    2008-01-01

    LDL receptor-null (LDLR(-/-)) mice on a Western diet (WD) develop endothelial dysfunction and atherosclerosis, which are improved by the apolipoprotein A-I (apoA-I) mimetic peptide D-4F. Focusing on the kidney, LDLR(-/-)mice were fed a WD with D-4F or the inactive control peptide scrambled D-4F (ScD-4F) added to their drinking water. The control mice (ScD-4F) developed glomerular changes, increased immunostaining for MCP-1/CCL2 chemokine, increased macrophage CD68 and F4/80 antigens, and increased oxidized phospholipids recognized by the EO6 monoclonal antibody in both glomerular and tublo-interstitial areas. All of these parameters were significantly reduced by D-4F treatment, approaching levels found in wild-type C57BL/6J or LDLR(-/-) mice fed a chow diet. Sterol-regulatory element binding protein-1c (SREBP-1c) mRNA levels and triglyceride levels were elevated in the kidneys of the control mice (ScD-4F) fed the WD compared with C57BL/6J and LDLR(-/-) mice on chow (P < 0.001 and P < 0.001, respectively) and compared with D-4F-treated mice on the WD (P < 0.01). There was no significant difference in plasma lipids, lipoproteins, glucose, blood pressure, or renal apoB levels between D-4F- and ScD-4F-treated mice. We conclude that D-4F reduced renal oxidized phospholipids, resulting in lower expression of SREBP-1c, which, in turn, resulted in lower triglyceride content and reduced renal inflammation. PMID:17925450

  15. Amphetamine Up-Regulates AGS1 mRNA and Protein Levels in Rat Frontal Cortex: The Role of Dopamine and Glucocorticoid Receptors

    PubMed Central

    Schwendt, Marek; McGinty, Jacqueline F.

    2010-01-01

    Acute and chronic exposure to psychostimulants results in altered function of G-protein-coupled receptors in the forebrain. It is believed that neuroadaptations in G-protein signaling contribute to behavioral sensitivity to psychostimulants that persists over a prolonged drug-free period. Proteins termed activators of G-protein signaling (AGS) have been characterized as potent modulators of both receptor-dependent and receptor-independent G-protein signaling. Nevertheless, the regulation of AGS gene and protein expression by psychostimulants remains poorly understood. In the present study, we investigated amphetamine (AMPH)-induced changes in expression patterns of several forebrain-enriched AGS proteins. A single exposure to AMPH (2.5 mg/kg i.p.) selectively induced gene expression of AGS1, but not Rhes or AGS3 proteins, in the rat prefrontal cortex (PFC) as measured 3h later. Induction of AGS1 mRNA in the PFC by acute AMPH was transient and dose-dependent. Even repeated treatment with AMPH for 5 days did not produce lasting changes in AGS1 mRNA and protein levels in the PFC as measured three weeks post treatment. However, at this time point, a low dose AMPH challenge (1 mg/kg, i.p.) induced a robust behavioral response and up-regulated AGS1 expression in the PFC selectively in animals with an AMPH history. The effects of AMPH on AGS1 expression in the PFC were blocked by a D2, but not D1, dopamine receptor antagonist and partially by a glucocorticoid receptor antagonist. Collectively, the present study suggests that (1) AGS1 represents a regulator of G-protein signaling that is rapidly inducible by AMPH in the frontal cortex, (2) AGS1 regulation in the PFC parallels behavioral activation by acute AMPH in drug-naïve animals and hypersensitivity to AMPH challenge in sensitized animals, and (3) D2 dopamine and glucocorticoid receptors regulate AMPH effects on AGS1 in the PFC. Changes in AGS1 levels in the PFC may result in abnormal receptor-to-G-protein coupling

  16. Simulated microgravity reduces mRNA levels of multidrug resistance genes 4 and 5 in non-metastatic human melanoma cells

    NASA Astrophysics Data System (ADS)

    Eiermann, Peter; Tsiockas, Wasiliki; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

    mRNA levels of sGC α and β were down-regulated by about 31% and 22%, respectively. Thus, the reduced expression of MRP4/5 could be related to the decrease in mRNA levels for the sGC subunits. In addition, the long-term exposure to simulated microgravity did not alter cellular morphology. Taken together, the results of our studies indicate that the expression of MRP4/5 in non-metastatic melanoma cells is inversely regulated by hypergravity and simulated microgravity. Finally, a reduced expression of MRP4 and MRP5 may increase the availability of drugs in cells and influence astronaut medication.

  17. Effects of different dietary intake on mRNA levels of MSTN, IGF-I, and IGF-II in the skeletal muscle of Dorper and Hu sheep hybrid F1 rams.

    PubMed

    Xing, H J; Wang, Z Y; Zhong, B S; Ying, S J; Nie, H T; Zhou, Z R; Fan, Y X; Wang, F

    2014-01-01

    MSTN, IGF-І(insulin-like growth factor-І) and IGF-II (insulin-like growth factor-II) regulate skeletal muscle growth. This study investigated the effects of different dietary intake levels on skeletal muscles. Sheep was randomly assigned to 3 feeding groups: 1) the maintenance diet (M), 2) 1.4 x the maintenance diet (1.4M), and 3) 2.15 x the maintenance diet (2.15M). Before slaughtering the animals, blood samples were collected to measure plasma urea, growth hormone, and insulin concentrations. After slaughtering, the longissimus dorsi, semitendinosus, semimembranosus, gastrocnemius, soleus, and chest muscle were removed to record various parameters, including the mRNA expression levels of MSTN and IGFs, in addition to skeletal muscle fiber diameter and cross-sectional area. The result showed that as dietary intake improved, the mRNA expression levels of MSTN and IGF-II decreased, whereas IGF-Іexpression increased. The mRNA expression levels of MSTN and IGFs were significantly different in the same skeletal muscle under different dietary intake. The skeletal muscle fiber diameter and cross-sectional area increased with greater dietary intake, as observed for the mRNA expression of IGF-І; however, it contrasted to that observed for the mRNA expression of MSTN and IGF-II. In conclusion, dietary intake levels have a certain influence on MSTN and IGFs mRNA expression levels, in addition to skeletal muscle fiber diameter and cross-sectional area. This study contributes valuable information for enhancing the molecular-based breeding of sheep. PMID:25078581

  18. Hypothalamic-pituitary-adrenal activity and pro-opiomelanocortin mRNA levels in the hypothalamus and pituitary of the rat are differentially modulated by acute intermittent morphine with or without water restriction stress.

    PubMed

    Zhou, Y; Spangler, R; Maggos, C E; Wang, X M; Han, J S; Ho, A; Kreek, M J

    1999-11-01

    Acute administration of morphine stimulates the secretion of hypothalamic-pituitary-adrenal (HPA) hormones, ACTH, beta-endorphin and corticosterone in the rat. In this study we investigated the effects of repeated multiple-dose morphine on HPA activity under two different conditions: without or with water restriction stress. Rats received six intermittent injections of morphine (6.25 mg/kg per injection, s.c.) every 2 h and were killed 30 min after the last injection. The results were as follows. (1) Morphine significantly elevated plasma ACTH and corticosterone levels; water restriction also significantly increased ACTH secretion, but with no significant increase of plasma corticosterone levels. In contrast, rats treated with morphine under the water restriction condition failed to show any increases of either ACTH or corticosterone levels. (2) Morphine did not change pro-opiomelanocortin (POMC) mRNA levels in the anterior pituitary; whereas water restriction significantly increased the POMC mRNA levels. The water restriction-induced increases of POMC mRNA in the anterior pituitary were absent in the rats which received morphine. (3) Morphine significantly increased POMC mRNA levels in the hypothalamus; water restriction had no effect. The morphine-induced increases in POMC mRNA in the hypothalamus were absent in the rat under the water restriction condition. These findings, that the effects of morphine on HPA activation or POMC mRNA expression depend on the presence of stress, suggest a counter-regulatory role of opiates on a stress response and opioid gene expression. PMID:10556776

  19. MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells.

    PubMed

    Anwar-Mohamed, Anwar; Elbekai, Reem H; El-Kadi, Ayman O S

    2008-11-10

    Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 (Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 microM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content. PMID:18835339

  20. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  1. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

    PubMed

    Ohde, Daniela; Moeller, Mark; Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  2. Effects of hepatitis C virus on suppressor of cytokine signaling mRNA levels: comparison between different genotypes and core protein sequence analysis.

    PubMed

    Pascarella, Stéphanie; Clément, Sophie; Guilloux, Kévin; Conzelmann, Stéphanie; Penin, François; Negro, Francesco

    2011-06-01

    Glucose metabolism disturbances, including insulin resistance and type 2 diabetes, are frequent and important cofactors of hepatitis C. Increasing epidemiological and experimental data suggest that all major genotypes of hepatitis C virus (HCV), albeit to a different extent, cause insulin resistance. The HCV core protein has been shown to be sufficient to impair insulin signaling in vitro through several post-receptorial mechanisms, mostly via the activation of suppressor of cytokine signaling (SOCS) family members and the consequent decrease of insulin receptor substrate-1 (IRS-1). The levels of IRS-1 and SOCS were investigated upon expression of the core protein of HCV genotypes 1-4. Furthermore, the core protein sequences were analyzed to identify the amino acid residues responsible for IRS-1 decrease, with particular regard to SOCS mRNA deregulation. The results suggest that the activation of SOCS family members is a general mechanism associated with the common HCV genotypes. A rare genotype 1b variant, however, failed to activate any of the SOCS tested: this allowed to analyze in detail the distinct amino acid sequences responsible for SOCS deregulation. By combining approaches using intergenotypic chimeras and site-directed mutagenesis, genetic evidence was provided in favor of a role of amino acids 49 and 131 of the HCV core-encoding sequence in mediating SOCS transactivation. PMID:21503913

  3. Glucocorticoids regulate mRNA levels for subunits of the 19 S regulatory complex of the 26 S proteasome in fast-twitch skeletal muscles.

    PubMed Central

    Combaret, Lydie; Taillandier, Daniel; Dardevet, Dominique; Béchet, Daniel; Rallière, Cécile; Claustre, Agnès; Grizard, Jean; Attaix, Didier

    2004-01-01

    Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome. PMID:14636157

  4. Effect of α-Hederin on IL-2 and IL-17 mRNA and miRNA-133a Levels in Lungs of Ovalbumin-Sensitized Male Rats.

    PubMed

    Ebrahimi, Hadi; Fallahi, Maryam; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Keyhanmanesh, Rana

    2016-03-01

    α-hederin, a saponin that is a major constituent of English Ivy (Hedera helix) is effective in the treatment of asthma. In the present study, the effect of α-hederin on lung tissue pathology and the levels of the inflammatory mediators; IL-2 mRNA, IL-17 mRNA, and MicroRNAs (miRNA)-133a was evaluated in a rat ovalbumin (OVA)-sensitized model of asthma. Rats were divided randomly into control (C), OVA-sensitized (S), OVA-sensitized pretreated with the antioxidant, thymoquinone (3 mg/kg, S + TQ) or OVA-sensitized pretreated with α-hederin (0.02 mg/kg, S + AH) groups. Levels of IL-2 and IL-17 mRNA were higher in the OVA-sensitized group than controls while the level of miRNA-133a gene expression was lower. IL-2 mRNA and miRNA-133a gene expression in the S + TQ group was higher than in the control and OVA-sensitized groups while the level of IL-17 mRNA in the S + TQ group was lower than in the OVA-sensitized group. Pretreatment with α-hederin decreased IL-17 mRNA levels and increased miRNA-133a gene expression compared with OVA-sensitized animals. All pathological changes in pretreated groups were lower than the OVA-sensitized group. These results showed a beneficial effect of α-hederin in OVA-sensitized rats, suggesting that α-hederin affects the IL-2 and IL-17 secretion pathways, altering miRNA-133a expression. PMID:26865286

  5. Fructose containing sugars modulate mRNA of lipogenic genes ACC and FAS and protein levels of transcription factors ChREBP and SREBP1c with no effect on body weight or liver fat.

    PubMed

    Janevski, Mile; Ratnayake, Sunil; Siljanovski, Svetlana; McGlynn, Maree A; Cameron-Smith, David; Lewandowski, Paul

    2012-02-01

    The aim of this study was to determine the effects of high-glucose, high-fructose and high-sucrose diets on weight gain, liver lipid metabolism and gene expression of proteins involved with hepatic fat metabolism. Rats were fed a diet containing either 60% glucose, 60% fructose, 60% sucrose, or a standard chow for 28 days. Results indicated that high-fructose and high-sucrose diets were associated with higher mRNA levels of gene transcripts involved with fat synthesis; ACC, FAS and ChREBP, with no change in SREBP-1C mRNA. The protein level of ChREBP and SREBP1c was similar in liver homogenates from all groups, but were higher in nuclear fractions from the liver of high-fructose and high-sucrose fed rats. The mRNA level of gene transcripts involved with fat oxidation was the same in all three diets, whilst a high-fructose diet was associated with greater amount of mRNA of the fat transporter CD36. Despite the changes in mRNA of lipogenic proteins, the body weight of animals from each group was the same and the livers from rats fed high-fructose and high-sucrose diets did not contain more fat than control diet livers. In conclusion, changing the composition of the principal monosaccharide in the diet to a fructose containing sugar elicits changes in the level of hepatic mRNA of lipogenic and fat transport proteins and protein levels of their transcriptional regulators; however this is not associated with any changes in body weight or liver fat content. PMID:22159273

  6. In vitro Therapeutic Effects of Low Level Laser at mRNA Level on the Release of Skin Growth Factors from Fibroblasts in Diabetic Mice

    PubMed Central

    Khoo, Nooshafarin Kazemi; Shokrgozar, Mohammad Ali; Kashani, Iraj Ragerdi; Amanzadeh, Amir; Mostafavi, Ehsan; Sanati, Hassan; Habibi, Laleh; Talebi, Saeid; Abouzaripour, Morteza; Akrami, Seyed Mohammad

    2014-01-01

    Background Numerous in vitro reports suggest that Low Level Laser Therapy (LLLT) affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light (which is more useful in clinic because of its higher penetration) on secretion of Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF), as important growth factors in wound healing. Methods Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT (energy density of 1 J/cm 2) using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time poly-merase chain reaction. Results Diabetic irradiated group showed significant increase in FGF (p = 0.017) expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. Conclusion These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts. PMID:24834313

  7. Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles.

    PubMed

    Lovell, Tristan M; Al-Musawi, Sara L; Gladwell, Richard T; Knight, Philip G

    2007-06-01

    Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins. PMID:17636170

  8. Intake of branched-chain or essential amino acids attenuates the elevation in muscle levels of PGC-1α4 mRNA caused by resistance exercise.

    PubMed

    Samuelsson, Hedvig; Moberg, Marcus; Apró, William; Ekblom, Björn; Blomstrand, Eva

    2016-07-01

    The transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is recognized as the master regulator of mitochondrial biogenesis. However, recently a novel isoform, PGC-1α4, that specifically regulates muscle hypertrophy was discovered. Because stimulation of mechanistic target of rapamycin complex 1 (mTORC1) activity is tightly coupled to hypertrophy, we hypothesized that activation of this pathway would upregulate PGC-1α4. Eight male subjects performed heavy resistance exercise (10 × 8-12 repetitions at ∼75% of 1 repetition maximum in leg press) on four different occasions, ingesting in random order a solution containing essential amino acids (EAA), branched-chain amino acids (BCAA), leucine, or flavored water (placebo) during and after the exercise. Biopsies were taken from the vastus lateralis muscle before and immediately after exercise, as well as following 90 and 180 min of recovery. Signaling through mTORC1, as reflected in p70S6 kinase phosphorylation, was stimulated to a greater extent by the EAA and BCAA than the leucine or placebo supplements. Unexpectedly, intake of EAA or BCAA attenuated the stimulatory effect of exercise on PGC-1α4 expression by ∼50% (from a 10- to 5-fold increase with BCAA and EAA, P < 0.05) 3 h after exercise, whereas intake of leucine alone did not reduce this response. The 60% increase (P < 0.05) in the level of PGC-1α1 mRNA 90 min after exercise was uninfluenced by amino acid intake. Muscle glycogen levels were reduced and AMP-activated protein kinase α2 activity and phosphorylation of p38 mitogen-activated protein kinase enhanced to the same extent with all four supplements. In conclusion, induction of PGC-1α4 does not appear to regulate the nutritional (BCAA or EAA)-mediated activation of mTORC1 in human muscle. PMID:27245337

  9. Macrophage Migration Inhibitory Factor Promoter Polymorphisms (−794 CATT5–8 and −173 G>C): Relationship with mRNA Expression and Soluble MIF Levels in Young Obese Subjects

    PubMed Central

    Matia-García, Inés; Salgado-Goytia, Lorenzo; Muñoz-Valle, José F.; García-Arellano, Samuel; Hernández-Bello, Jorge; Salgado-Bernabé, Aralia B.; Parra-Rojas, Isela

    2015-01-01

    We analyzed the relationship of −794 CATT5–8 and −173 G>C MIF polymorphisms with mRNA and soluble MIF in young obese subjects. A total of 250 young subjects, 150 normal-weight and 100 obese subjects, were recruited in the study. Genotyping of −794 CATT5–8 and −173 G>C MIF polymorphisms was performed by PCR and PCR-RFLP, respectively. MIF mRNA expression was determined by real-time PCR and serum MIF levels were measured using an ELISA kit. For both MIF promoter polymorphisms, no significant differences in the genotype and allele frequencies between groups were observed. MIF mRNA expression was slightly higher in obese subjects than in normal-weight subjects (1.38-fold), while soluble MIF levels did not show differences between groups. In addition, we found an increase in MIF mRNA expression in carriers of the 6,6 and C/C genotypes and the 6G haplotype of the −794 CATT5–8 and −173 G>C MIF polymorphisms, although it was not significant. In conclusion, this study found no relationship between obesity and MIF gene promoter polymorphisms with MIF mRNA expression in young obese subjects. PMID:25972622

  10. Coordinate Changes in Histone Modifications, mRNA Levels, and Metabolite Profiles in Clonal INS-1 832/13 β-Cells Accompany Functional Adaptations to Lipotoxicity*

    PubMed Central

    Malmgren, Siri; Spégel, Peter; Danielsson, Anders P.H.; Nagorny, Cecilia L.; Andersson, Lotta E.; Nitert, Marloes Dekker; Ridderstråle, Martin; Mulder, Hindrik; Ling, Charlotte

    2013-01-01

    Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic β-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 β-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 β-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal β-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion. PMID:23476019

  11. Coordinate changes in histone modifications, mRNA levels, and metabolite profiles in clonal INS-1 832/13 β-cells accompany functional adaptations to lipotoxicity.

    PubMed

    Malmgren, Siri; Spégel, Peter; Danielsson, Anders P H; Nagorny, Cecilia L; Andersson, Lotta E; Nitert, Marloes Dekker; Ridderstråle, Martin; Mulder, Hindrik; Ling, Charlotte

    2013-04-26

    Lipotoxicity is a presumed pathogenetic process whereby elevated circulating and stored lipids in type 2 diabetes cause pancreatic β-cell failure. To resolve the underlying molecular mechanisms, we exposed clonal INS-1 832/13 β-cells to palmitate for 48 h. We observed elevated basal insulin secretion but impaired glucose-stimulated insulin secretion in palmitate-exposed cells. Glucose utilization was unchanged, palmitate oxidation was increased, and oxygen consumption was impaired. Halting exposure of the clonal INS-1 832/13 β-cells to palmitate largely recovered all of the lipid-induced functional changes. Metabolite profiling revealed profound but reversible increases in cellular lipids. Glucose-induced increases in tricarboxylic acid cycle intermediates were attenuated by exposure to palmitate. Analysis of gene expression by microarray showed increased expression of 982 genes and decreased expression of 1032 genes after exposure to palmitate. Increases were seen in pathways for steroid biosynthesis, cell cycle, fatty acid metabolism, DNA replication, and biosynthesis of unsaturated fatty acids; decreases occurred in the aminoacyl-tRNA synthesis pathway. The activity of histone-modifying enzymes and histone modifications of differentially expressed genes were reversibly altered upon exposure to palmitate. Thus, Insig1, Lss, Peci, Idi1, Hmgcs1, and Casr were subject to epigenetic regulation. Our analyses demonstrate that coordinate changes in histone modifications, mRNA levels, and metabolite profiles accompanied functional adaptations of clonal β-cells to lipotoxicity. It is highly likely that these changes are pathogenetic, accounting for loss of glucose responsiveness and perturbed insulin secretion. PMID:23476019

  12. Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

    PubMed

    Alves, Agnelo Neves; Ribeiro, Beatriz Guimarães; Fernandes, Kristianne Porta Santos; Souza, Nadhia Helena Costa; Rocha, Lília Alves; Nunes, Fabio Daumas; Bussadori, Sandra Kalil; Mesquita-Ferrari, Raquel Agnelli

    2016-05-01

    This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process. PMID:26914683

  13. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

    PubMed Central

    Kyöstiö, S R; Owens, R A; Weitzman, M D; Antoni, B A; Chejanovsky, N; Carter, B J

    1994-01-01

    The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence

  14. Disturbance effects of PM₁₀ on iNOS and eNOS mRNA expression levels and antioxidant activity induced by ischemia-reperfusion injury in isolated rat heart: protective role of vanillic acid.

    PubMed

    Dianat, Mahin; Radmanesh, Esmat; Badavi, Mohammad; Mard, Seyed Ali; Goudarzi, Gholamraza

    2016-03-01

    Myocardial infarction is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demand. Air pollution increases the risk of death from cardiovascular diseases (CVDs). The aim of this study was to investigate the effects of particulate matter (PM) on oxidative stress, the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) messenger RNA (mRNA) level induced by ischemia-reperfusion injury, and the protective effects of vanillic acid (VA) in the isolated rat heart. Male Wistar rats were randomly divided into eight groups (n = 10), namely control, VAc, sham, VA, PMa (0.5 mg/kg), PMb (2.5 mg/kg), PMc (5 mg/kg), and PMc + VA groups. Particles with an aerodynamic diameter <10 μm (PM10) was instilled into the trachea through a fine intubation tube. Two days following the PM10 instillation, the animal's hearts were isolated and transferred to a Langendorff apparatus. The hearts were subjected to 30 min of global ischemia followed by 60 min of reperfusion. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), xanthine oxidase (XOX), and lactate dehydrogenase (LDH) were measured using special kits. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine levels of iNOS and eNOS mRNA. An increase in left ventricular end-diastolic pressure (LVEDP), S-T elevation, and oxidative stress in PM10 groups was observed. Ischemia-reperfusion (I/R) induction showed a significant augment in the expression of iNOS mRNA level and a significant decrease in the expression eNOS mRNA level. This effect was more pronounced in the PM groups than in the control and sham groups. Vanillic acid caused a significant decrease in LVEDP, S-T elevation, and also a significant difference in eNOS mRNA expression level, antioxidant enzymes, iNOS mRNA expression level, and oxidative stress occurred on myocardial dysfunction

  15. The effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles

    PubMed Central

    DeWitt, Marsha; Johnson, Robert L.; Snyder, Paul; Fleet, James C.

    2015-01-01

    In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions ofmice with colon-specific deletion of both APC gene alleles (CAC; APCΔ580/Δ580). At four weeks of age, CAC; APCΔ580/Δ580 and control mice were injected with vehicle or 1,25(OH)2D (1 μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APCΔ580/Δ580 mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APCΔ580/Δ580 and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APCΔ580/Δ580 or control mice. VDR mRNA and protein levels were lower (−65% and −90%) in the colon of CAC; APCΔ580/Δ580 mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of Transient Receptor Potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APCΔ580/Δ580 mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. PMID:25597951

  16. Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens, and their mRNA expression levels in the liver, kidney, brain and skeletal muscle during aestivation.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2014-10-01

    Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy. PMID:25034132

  17. Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

    PubMed

    Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

    2009-05-01

    Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins. PMID:18612846

  18. IGF and GH mRNA levels are suppressed upon exposure to micromolar concentrations of cobalt and zinc in rainbow trout white muscle.

    PubMed

    Ekinci, Deniz; Ceyhun, Saltuk Buğrahan; Aksakal, Ercüment; Erdoğan, Orhan

    2011-04-01

    The objective of this study was to assess the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on insulin like growth factors (IGF) and growth hormone (GH). Mature rainbow trouts were exposed to 0.42, 2.1, 4.2, 21 and 42μmol/L Co(2+) (added as CoCl(2)·6H(2)O) and 0.34, 1.7, 3.4, 17 and 34μmol/L Zn(2+) (added as ZnSO(4)i·7H(2)O). After 6, 12, 24 and 48h of treatment, expressions of white muscle IGF-I, IGF-II and GH mRNAs were measured by means of quantitative Real Time PCR. During the exposure experiments, no mortalities occurred. The most effective metal concentrations, which caused significant alterations, were determined to be 42μmol/L Co(2+) (10mg CoCl(2)·6H(2)O/L) and 3.4μmol/L Zn(+2) (1mg ZnSO(4)·7H(2)O/L). The following results were obtained for these concentrations. Expression of IGF-I did not change at 6h in zinc treatment while the decrease (p<0.05) was observed at 12h and 24h, and this decrease became stronger at 48h. Cobalt exposure caused a decrease in IGF-I mRNA level at 6h, 12h, 24h and 48h (p<0.05). Both zinc and cobalt exposure resulted in significant decreases in GH expression at 6h. Exposure of trout to Zn resulted in a decrease in expression of IGF-II starting from 6h whereas the significant decrease started at 6h in cobalt exposure and this decrease elevated at 24h. The results indicate that micromolar cobalt and zinc exposure causes significant attenuation in the expressions of these three genes' time dependently. Our findings show that IGF-I is the most resistant and GH is the most sensitive component against cobalt and zinc exposure. We conclude that IGF/GH axis might be strongly affected by the short term exposure to low micromolar concentrations of zinc and cobalt due to alterations of these genes. PMID:21167956

  19. A quantitative analysis of the reduction in oxygen levels required to induce up-regulation of vascular endothelial growth factor (VEGF) mRNA in cervical cancer cell lines

    PubMed Central

    Chiarotto, J A; Hill, R P

    1999-01-01

    The presence of hypoxia (low oxygen concentrations) in solid tumours correlates with poor prognosis, increased metastasis, and resistance to radiotherapy and some forms of chemotherapy. Malignant cells produce an angiogenesis factor, vascular endothelial growth factor (VEGF), which may increase metastatic ability and is up-regulated in the presence of hypoxia. Clinical data for cancers of the cervix and head and neck relate oxygen levels in the tumour to treatment outcome. This suggests the possibility that the presence of VEGF mRNA might be used as a marker for relevant levels of hypoxia. Suspension cultures of three human cervical cancer cell lines, SiHa, ME-180 and HeLa, were used to investigate up-regulation of VEGF mRNA levels following exposure to precisely defined oxygen concentrations for 2 or 4 h. An oxygen sensor was used to confirm the actual levels of dissolved oxygen present. The oxygen concentrations which caused half-maximal upregulation (the Km value) of VEGF mRNA level in the three cell lines were similar except for one instance (Km at 4 h: SiHa 27.0 ± 5.7 μM, ME-180 16.8 ± 3.3 μM, HeLa 13.0 ± 1.8 μM, SiHa and HeLa P = 0.01). The Km values for the HeLa cell line as measured at 2 h (24.9 ± 0.8 μM) and 4 h (13.0 ± 1.8 μM) were significantly different (P < 0.0001). VEGF mRNA half-lives measured in air were consistent with values in the literature (SiHa 59.8 ± 5.8 min, ME-180 44.4 ± 7.2 min, HeLa 44.5 ± 6.3 min). Differences in oxygen consumption at low oxygen concentrations were noted between the different cell lines. Stirring in suspension culture was found to induce VEGF mRNA in SiHa cells. The presence of VEGF mRNA may be a marker for radiobiologic hypoxia. © 1999 Cancer Research Campaign PMID:10408392

  20. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  1. Sex- and region-specific alterations of progesterone receptor mRNA levels and estrogen sensitivity in rat brain following developmental exposure to the estrogenic UV filter 4-methylbenzylidene camphor.

    PubMed

    Maerkel, Kirsten; Lichtensteiger, Walter; Durrer, Stefan; Conscience, Marianne; Schlumpf, Margret

    2005-05-01

    Recently, we reported on in vitro and in vivo estrogenic activity of UV filters and on developmental toxicity of 4-methylbenzylidene (4-MBC) camphor [Schlumpf, M., Cotton, B., Conscience, M., Haller, V., Steinmann, B., Lichtensteiger, W., 2001a. In vitro and in vivo estrogenicity of UV screens. Environ. Health Perspect. 109, 239; Schlumpf, M., Berger, L., Cotton, B., Conscience-Egli, M., Durrer, S., Fleischmann, I., Haller, V., Maerkel, K., Lichtensteiger, W., 2001b. Estrogen active UV screens. SÖFW-J. 7, 10]. 4-MBC (7, 24, 47mg/(kgday)) was administered in chow to long Evans rats from 10 weeks before mating of the parent (F0) generation until adulthood of the F1 generation. Peripheral reproductive organs and central nervous system were studied in adult offspring. mRNA expression of progesterone receptor (PR), an estrogen-regulated gene, was investigated in medial preoptic area (MPO) and ventromedial hypothalamic nucleus (VMH) by real-time RT-PCR. We analyzed intact 12-week-old male and female offspring under steady state conditions and adult gonadectomized offspring 6h after a single s.c. injection of estradiol-17β (E2) (10 or 50μg/kg) in order to assess estrogen sensitivity. At steady state conditions we observed significantly higher PR mRNA expression in VMH of control females versus control males. 4-MBC exposed females exhibited a decrease in PR mRNA to levels of control males. The increase in PR mRNA in response to E2 was higher in VMH of males of both 4-MBC groups as compared to control males. PR mRNA levels were similar in MPO of control males and females. Developmental 4-MBC exposure increased PR mRNA levels in male MPO, but did not significantly change female levels. The acute response to the lower E2 dose was decreased in MPO of 4-MBC-exposed males, whereas females of the 7mg/kg dose group exhibited an increased reaction to 50μg/kg of E2. Our data indicate that developmental exposure to endocrine active chemicals such as the UV filter 4-MBC can

  2. Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis.

    PubMed

    Nusgens, B V; Humbert, P; Rougier, A; Colige, A C; Haftek, M; Lambert, C A; Richard, A; Creidi, P; Lapière, C M

    2001-06-01

    Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription-polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post-translational enzymes, the carboxy- and amino-procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage

  3. Sulforaphane induces CYP1A1 mRNA, protein, and catalytic activity levels via an AhR-dependent pathway in murine hepatoma Hepa 1c1c7 and human HepG2 cells.

    PubMed

    Anwar-Mohamed, Anwar; El-Kadi, Ayman O S

    2009-03-01

    Recent reports have proposed that some naturally occurring phytochemicals can function as anticancer agents mainly through inducing phase II drug detoxification enzymes. Of these phytochemicals, isothiocyanates sulforaphane (SUL), present in broccoli, is by far the most extensively studied. In spite of its positive effect on phase II drug metabolizing enzymes, its effect on the phase I bioactivating enzyme cytochrome P450 1a1 (Cyp1a1) is still a matter of debate. As a first step to investigate this effect, Hepa 1c1c7 and HepG2 cells were treated with various concentration of SUL. Our results showed that SUL-induced CYP1A1 mRNA in a dose- and time-dependent manner. Furthermore, this induction was further reflected on the protein and catalytic activity levels. Investigating the effect of SUL at the transcriptional level revealed that SUL increases the Cyp1a1 mRNA as early as 1h. The RNA polymerase inhibitor actinomycin D (Act-D) completely abolished the SUL-induced Cyp1a1 mRNA. Furthermore, SUL successfully activated AhR transformation and its subsequent binding to the XRE. At the post-transcriptional level, SUL did not affect the levels of existing Cyp1a1 mRNA transcripts. This is the first demonstration that the broccoli-derived SUL can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SUL induces this enzyme. PMID:19013013

  4. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  5. The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA.

    PubMed

    Xu, Fenghao; Morin, Charles; Mitchell, Grant; Ackerley, Cameron; Robinson, Brian H

    2004-08-15

    Leigh syndrome French Canadian (LSFC) is a variant of cytochrome oxidase deficiency found in Québec and caused by mutations in the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene. Northern blots showed that the LRPPRC mRNA levels seen in skeletal muscle>heart>placenta>kidney>liver>lung=brain were proportionally almost opposite in strength to the severity of the enzymic cytochrome oxidase defect. The levels of COX (cytochrome c oxidase) I and COX III mRNA visible on Northern blots were reduced in LSFC patients due to the common (A354V, Ala354-->Val) founder mutation. The amount of LRPPRC protein found in both fibroblast and liver mitochondria from LSFC patients was consistently reduced to <30% of control levels. Import of [(35)S]methionine LRPPRC into rat liver mitochondria was slower for the mutant (A354V) protein. A titre of LRPPRC protein was also found in nuclear fractions that could not be easily accounted for by mitochondrial contamination. [35S]Methionine labelling of mitochondrial translation products showed that the translation of COX I, and perhaps COX III, was specifically reduced in the presence of the mutation. These results suggest that the gene product of LRPPRC, like PET 309p, has a role in the translation or stability of the mRNA for mitochondrially encoded COX subunits. A more diffuse distribution of LRPPRC in LSFC cells compared with controls was evident when viewed by immunofluorescence microscopy, with less LRPPRC present in peripheral mitochondria. PMID:15139850

  6. Epithelial remodeling and claudin mRNA abundance in the gill and kidney of puffer fish (Tetraodon biocellatus) acclimated to altered environmental ion levels.

    PubMed

    Duffy, Nicole M; Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2011-02-01

    In water of varying ion content, the gills and kidney of fishes contribute significantly to the maintenance of salt and water balance. However, little is known about the molecular architecture of the tight junction (TJ) complex and the regulation of paracellular permeability characteristics in these tissues. In the current studies, puffer fish (Tetraodon biocellatus) were acclimated to freshwater (FW), seawater (SW) or ion-poor freshwater (IPW) conditions. Following acclimation, alterations in systemic endpoints of hydromineral status were examined in conjunction with changes in gill and kidney epithelia morphology/morphometrics, as well as claudin TJ protein mRNA abundance. T. biocellatus were able to maintain endpoints of hydromineral status within relatively tight limits across the broad range of water ion content examined. Both gill and kidney tissue exhibited substantial alterations in morphology as well as claudin TJ protein mRNA abundance. These responses were particularly pronounced when comparing fish acclimated to SW versus those acclimated to IPW. TEM observations of IPW-acclimated fish gills revealed the presence of cells that exhibited the typical characteristics of gill mitochondria-rich cells (e.g. voluminous, Na(+)-K(+)-ATPase-immunoreactive, exposed to the external environment at the apical surface), but were not mitochondria-rich. To our knowledge, this type of cell has not previously been described in hyperosmoregulating fish gills. Furthermore, modifications in the morphometrics and claudin mRNA abundance of kidney tissue support the notion that spatial alterations in claudin TJ proteins along the nephron of fishes will likely play an important role in the regulation of salt and water balance in these organisms. PMID:20976602

  7. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    SciTech Connect

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  8. mRNA Levels of ACh-Related Enzymes in the Hippocampus of THY-Tau22 Mouse: A Model of Human Tauopathy with No Signs of Motor Disturbance.

    PubMed

    García-Gómez, Beatriz E; Fernández-Gómez, Francisco J; Muñoz-Delgado, Encarnación; Buée, Luc; Blum, David; Vidal, Cecilio J

    2016-04-01

    The microtubule-associated protein Tau tends to form aggregates in neurodegenerative disorders referred to as tauopathies. The tauopathy model transgenic (Tg) THY-Tau22 (Tau22) mouse shows disturbed septo-hippocampal transmission, memory deficits and no signs of motor dysfunction. The reports showing a hippocampal downregulation of choline acetyltransferase (ChAT) in SAMP8 mice, a model of aging, and an upregulation of acetylcholinesterase (AChE) in Tg-VLW mice, a model of FTDP17 tauopathy, may lead to think that the supply of ACh to the hippocampus can be threatened as aging or Tau pathology progress. The above was tested by comparing the mRNA levels for ACh-related enzymes in hippocampi of wild-type (wt) and Tau22 mice at ages when the neuropathological signs are debuting (3-4 months), moderate (6-7 months) and extensive (>9 months). Age-matched Tau22 and wt mice hippocampi displayed similar ChAT, AChE-T, butyrylcholinesterase (BChE) and a proline-rich membrane anchor (PRiMA) mRNA levels, any change most likely arising from ACh homeostasis. The unchanged hippocampal levels of AChE-T mRNA and enzyme activity observed in Tau22 mice, expressing G272V-P301S hTau, differed from the increase in AChE-T mRNA and activity observed in Tg-VLW mice, expressing G272V-P301L-R406W hTau. The difference supports the idea that AChE upregulation may proceed or not depending on the particular Tau mutation, which would dictate Tau folding, the accessibility/affinity to kinases and phosphatases, and P-Tau aggregation with itself and protein partners, transcription factors included. PMID:26697857

  9. OGG1 mRNA expression and incision activity in rats are higher in foetal tissue than in adult liver tissue while 8-oxo-2'-deoxyguanosine levels are unchanged.

    PubMed

    Riis, Bente; Risom, Lotte; Loft, Steffen; Poulsen, Henrik Enghusen

    2002-09-01

    This study was set up to investigate the relationships between the formation and removal of DNA damage in form of 8-oxodeoxyguanosine (8-oxodG) in neonatal (day 16 of gestation) as compared to adult rats. The hypothesis addressed was whether the rapidly dividing foetal tissue has an enhanced requirement of DNA repair providing protection against potentially mutagenic DNA damages such as 8-oxodG. The activity of the primary 8-oxodG-repair protein OGG1 was measured by a DNA incision assay and the expression of OGG1 mRNA was measured by Real-Time PCR normalised to 18S rRNA. The tissue level of 8-oxodG was measured by HPLC-ECD. We found a 2-3-fold increased incision activity in the foetal control tissue, together with a 3-15-fold increase in mRNA of OGG1 as compared to liver tissue from adult rats. The levels of 8-oxodG in the foetal tissue were unaltered as compared to the adult groups. To increase the levels of 8-oxodG, the rats received an injection (i.p.) of the hepatotoxin 2-nitropropane. The compound induced significant levels of 8-oxodG in male rat livers 5h after the injection and in the foetuses 24h after the injection, while the female rats showed no increase in 8-oxodG. The incision activity was slightly depressed in both male and female liver tissue and in the foetal tissue 5h after the injection, but significantly increased from 5 to 24h after the injection. However, it did not reach levels significantly above the control levels. In conclusion, this study confirms that foetal tissue has increased levels of OGG1 mRNA and correspondingly an enhanced incision activity on an 8-oxodG substrate in a crude tissue extract. PMID:12509275

  10. Novel rice OsSIPK is a multiple stress responsive MAPK family member showing rhythmic expression at mRNA level.

    PubMed

    Lee, Mi-Ok; Cho, Kyoungwon; Kim, So-Hee; Jeong, Seung-Hee; Kim, Jung-A; Jung, Young-Ho; Shim, Jaekyung; Shibato, Junko; Rakwal, Randeep; Tamogami, Shigeru; Kubo, Akihiro; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

    2008-04-01

    We report isolation and transcriptional profiling of rice (Oryza sativa L.) mitogen-activated protein kinase (MAPK), OsSIPK (salicylic acid-induced protein kinase). OsSIPK gene is located on chromosome 6 most probably existing as a single copy in the rice genome, and encodes 398 amino acid polypeptide having the MAPK family signature and phosphorylation activation motif TEY. Steady state mRNA analyses of OsSIPK showed weak constitutive expression in leaves of 2-week-old rice seedlings. A time course (30-120 min) experiment using a variety of elicitors and stresses revealed that the OsSIPK mRNA is strongly induced by jasmonic acid (JA), salicylic acid (SA), ethephon, abscisic acid, cycloheximide (CHX), JA/SA + CHX, cantharidin, okadaic acid, hydrogen peroxide, chitosan, sodium chloride, and cold stress (12 degrees C), but not with wounding by cut, gaseous pollutants ozone, and sulfur dioxide, high temperature, ultraviolet C irradiation, sucrose, and drought. Its transcription was also found to be tissue-specifically regulated, and followed a rhythmic dark induction in leaves. Finally, we showed that the OsSIPK protein is localized to the nucleus. From these results, OsSIPK can be implicated in diverse stimuli-responsive signaling cascades and transcription of certain genes. PMID:18066586

  11. Dietary gamma-linolenic acid in the form of borage oil causes less body fat accumulation accompanying an increase in uncoupling protein 1 mRNA level in brown adipose tissue.

    PubMed

    Takahashi, Y; Ide, T; Fujita, H

    2000-10-01

    Rats were fed a low-fat diet containing 2% safflower oil or 20% fat diets containing either safflower oil rich in linoleic acid, borage oil containing 25% gamma (gamma)-linolenic acid or enzymatically prepared gamma-linolenic acid enriched borage oil containing 47% gamma-linolenic acid for 14 days. Energy intake and growth of animals were the same among groups. A high safflower oil diet compared with a low-fat diet caused significant increases in both epididymal and perirenal white adipose tissue weights. However, high-fat diets rich in gamma-linolenic acid failed to do so. Compared with a low-fat diet, all the high-fat diets increased mRNA levels of uncoupling protein 1 and lipoprotein lipase in brown adipose tissue. The extents of the increase were greater with high-fat diets rich in gamma-linolenic acid. Various high-fat diets, compared with a low-fat diet, decreased glucose transporter 4 mRNA in white adipose tissue to the same levels. The amount and types of dietary fat did not affect the leptin mRNA level in epididymal white adipose tissue. However, a high safflower oil diet, but not high-fat diets rich in gamma-linolenic acid relative to a low-fat diet, increased perirenal white adipose tissue leptin mRNA levels. All high-fat diets, relative to a low-fat diet, increased the hepatic mitochondrial fatty acid oxidation rate and fatty acid oxidation enzyme mRNA abundances to the same levels. High-fat diets also increased these parameters in the peroxisomal pathway, and the increases were greater with high-fat diets rich in gamma-linolenic acid. The physiological activity in increasing brown adipose tissue gene expression and peroxisomal fatty acid oxidation was similar between the two types of borage oil differing in gamma-linolenic acid content. It was suggested that dietary gamma-linolenic acid attenuates body fat accumulation through the increase in gene expressions of uncoupling protein 1 in brown adipose tissue. An increase in hepatic peroxisomal fatty acid

  12. Effect of nutrition on plasma lipid profile and mRNA levels of ovarian genes involved in steroid hormone synthesis in Hu sheep during luteal phase.

    PubMed

    Ying, S J; Xiao, S H; Wang, C L; Zhong, B S; Zhang, G M; Wang, Z Y; He, D Y; Ding, X L; Xing, H J; Wang, F

    2013-11-01

    Ovarian steroid hormones regulate follicular growth and atresia. This study aims to determine whether key ovarian sterol-regulatory genes are differentially expressed in Hu sheep under different short-term nutritional regimens. Estrus was synchronized using intravaginal progestagen sponges. The ewes were assigned randomly to 3 groups. On d 6 to 12 of their estrous cycle, the control (CON) group received a maintenance diet (1.0×M), the supplemented (SUP) group received 1.5×M, and the restricted (R) group received 0.5×M. On d 7 to 12, blood samples were taken. The sheep were slaughtered at the end of the treatment, and their organs and ovaries were collected. The plasma concentrations of urea (P<0.01), total cholesterol (P<0.01), low-density lipoprotein cholesterol (P<0.01), NEFA (P<0.01), FSH (P<0.05), and estradiol (P<0.05) increased with decreasing dietary intake, whereas plasma triglyceride (P<0.01) and triiodothyronine (T3) concentrations decreased (P<0.05). The ewes in the R group had higher spleen weight and percentage of spleen to BW and lower liver and small intestine weights and percentage of liver/stomach to BW than the SUP group ewes (P<0.05). Nutritional restriction decreased the cytochrome p450 (CYP17A1) and estrogen receptor 1 (ESR1) mRNA expression (P<0.05) and increased the cytochrome p450 aromatase (CYP19A1) mRNA expression (P<0.05) in follicles>2.5 mm. Follicle size affected the mRNA expression of very low density lipoprotein receptor (VLDLR), estrogen receptor 2 (ESR2), FSH receptor (FSHR), CYP17A1, and CYP19A1 (P<0.05). In conclusion, we suggest that a potential mechanism by which short-term negative energy balance inhibits follicular growth may involve responses to disrupted reproductive hormone concentrations and influenced the intrafollicular expression of CYP17A1, CYP19A1, and ESR1. This result may be due to increased plasma urea and lipid concentrations. PMID:24045481

  13. mRNA imprinting

    PubMed Central

    2011-01-01

    Following its synthesis in the nucleus, mRNA undergoes various stages that are critical for the proper synthesis, localization and possibly functionality of its encoded protein. Recently, we have shown that two RNA polymerase II (Pol II) subunits, Rpb4p and Rpb7p, associate with the nascent transcript co-transcriptionally. This “mRNA imprinting” lasts throughout the mRNA lifetime and is required for proper regulation of all major stages that the mRNA undergoes. Other possible cases of co-transcriptional imprinting are discussed. Since mRNAs can be transported from the synthesizing cell to other cells, we propose that mRNA imprinting can also affect the phenotype of the recipient cells. This can be viewed as “mRNA-based epigenetics.” PMID:21686103

  14. Delineation of biochemical, molecular, and physiological changes accompanying bile acid pool size restoration in Cyp7a1−/− mice fed low levels of cholic acid

    PubMed Central

    Jones, Ryan D.; Repa, Joyce J.; Russell, David W.; Dietschy, John M.

    2012-01-01

    Cholesterol 7α-hydroxylase (CYP7A1) is the initiating and rate-limiting enzyme in the neutral pathway that coverts cholesterol to primary bile acids (BA). CYP7A1-deficient (Cyp7a1−/−) mice have a depleted BA pool, diminished intestinal cholesterol absorption, accelerated fecal sterol loss, and increased intestinal cholesterol synthesis. To determine the molecular and physiological effects of restoring the BA pool in this model, adult female Cyp7a1−/− mice and matching Cyp7a1+/+ controls were fed diets containing cholic acid (CA) at modest levels [0.015, 0.030, and 0.060% (wt/wt)] for 15–18 days. A level of just 0.03% provided a CA intake of ∼12 μmol (4.8 mg) per day per 100 g body wt and was sufficient in the Cyp7a1−/− mice to normalize BA pool size, fecal BA excretion, fractional cholesterol absorption, and fecal sterol excretion but caused a significant rise in the cholesterol concentration in the small intestine and liver, as well as a marked inhibition of cholesterol synthesis in these organs. In parallel with these metabolic changes, there were marked shifts in intestinal and hepatic expression levels for many target genes of the BA sensor farnesoid X receptor, as well as genes involved in cholesterol transport, especially ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG8. In Cyp7a1+/+ mice, this level of CA supplementation did not significantly disrupt BA or cholesterol metabolism, except for an increase in fecal BA excretion and marginal changes in mRNA expression for some BA synthetic enzymes. These findings underscore the importance of using moderate dietary BA levels in studies with animal models. PMID:22628034

  15. Are we missing a mineralocorticoid in teleost fish? Effects of cortisol, deoxycorticosterone and aldosterone on osmoregulation, gill Na+,K+-ATPase activity and isoform mRNA levels in Atlantic salmon

    USGS Publications Warehouse

    McCormick, S.D.; Regish, A.; O'Dea, M. F.; Shrimpton, J.M.

    2008-01-01

    It has long been held that cortisol, acting through a single receptor, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. The recent finding that fish express a gene with high sequence similarity to the mammalian mineralocorticoid receptor (MR) suggests the possibility that a hormone other than cortisol carries out some mineralocorticoid functions in fish. To test for this possibility, we examined the effect of in vivo cortisol, 11-deoxycorticosterone (DOC) and aldosterone on salinity tolerance, gill Na+,K+-ATPase (NKA) activity and mRNA levels of NKA α1a and α1b in Atlantic salmon. Cortisol treatment for 6–14 days resulted in increased, physiological levels of cortisol, increased gill NKA activity and improved salinity tolerance (lower plasma chloride after a 24 h seawater challenge), whereas DOC and aldosterone had no effect on either NKA activity or salinity tolerance. NKA α1a and α1b mRNA levels, which increase in response to fresh water and seawater acclimation, respectively, were both upregulated by cortisol, whereas DOC and aldosterone were without effect. Cortisol, DOC and aldosterone had no effect on gill glucocorticoid receptor GR1, GR2 and MR mRNA levels, although there was some indication of possible upregulation of GR1 by cortisol (p = 0.07). The putative GR blocker RU486 inhibited cortisol-induced increases in salinity tolerance, NKA activity and NKA α1a and α1b transcription, whereas the putative MR blocker spironolactone had no effect. The results provide support that cortisol, and not DOC or aldosterone, is involved in regulating the mineralocorticoid functions of ion uptake and salt secretion in teleost fish.

  16. Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line

    PubMed Central

    Fraser, Scott E.; Trinh, Le A.

    2015-01-01

    Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ct90a). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle. PMID:26083378

  17. Distinctive expression of extracellular matrix molecules at mRNA and protein levels during formation of cellular and acellular cementum in the rat.

    PubMed

    Sasano, Y; Maruya, Y; Sato, H; Zhu, J X; Takahashi, I; Mizoguchi, I; Kagayama, M

    2001-02-01

    Little is known about differential expression of extracellular matrices secreted by cementoblasts between cellular and acellular cementum. We hypothesize that cementoblasts lining acellular cementum express extracellular matrix genes differently from those lining cellular cementum, thereby forming two distinct types of extracellular matrices. To test this hypothesis, we investigated spatial and temporal gene expression of selected extracellular matrix molecules, that is type I collagen, bone sialoprotein, osteocalcin and osteopontin, during formation of both cellular and acellular cementum using in situ hybridization. In addition, their extracellularly deposited and accumulated proteins were examined immunohistochemically. The mRNA transcripts of pro-alpha1 (I) collagen were primarily localized in cementoblasts of cellular cementum and cementocytes, while those of bone sialoprotein were predominantly seen in cementoblasts lining acellular cementum. In contrast, osteocalcin was expressed by both types of cementoblasts and cementocytes and so was osteopontin but only transiently. Our immunohistochemical examination revealed that translated proteins were localized extracellularly where the genes had been expressed intracellularly. The present study demonstrated the distinctive expression of genes and proteins of the extracellular matrix molecules between cellular and acellular cementum. PMID:11432645

  18. Putative Pacemakers in the Eyestalk and Brain of the Crayfish Procambarus clarkii Show Circadian Oscillations in Levels of mRNA for Crustacean Hyperglycemic Hormone

    PubMed Central

    Nelson-Mora, Janikua; Prieto-Sagredo, Julio; Loredo-Ranjel, Rosaura; Fanjul-Moles, María Luisa

    2013-01-01

    Crustacean hyperglycemic hormone (CHH) synthesizing cells in the optic lobe, one of the pacemakers of the circadian system, have been shown to be present in crayfish. However, the presence of CHH in the central brain, another putative pacemaker of the multi-oscillatory circadian system, of this decapod and its circadian transcription in the optic lobe and brain have yet to be explored. Therefore, using qualitative and quantitative PCR, we isolated and cloned a CHH mRNA fragment from two putative pacemakers of the multi-oscillatory circadian system of Procambarus clarkii, the optic lobe and the central brain. This CHH transcript synchronized to daily light-dark cycles and oscillated under dark, constant conditions demonstrating statistically significant daily and circadian rhythms in both structures. Furthermore, to investigate the presence of the peptide in the central brain of this decapod, we used immunohistochemical methods. Confocal microscopy revealed the presence of CHH-IR in fibers and cells of the protocerebral and tritocerebal clusters and neuropiles, particularly in some neurons located in clusters 6, 14, 15 and 17. The presence of CHH positive neurons in structures of P. clarkii where clock proteins have been reported suggests a relationship between the circadian clockwork and CHH. This work provides new insights into the circadian regulation of CHH, a pleiotropic hormone that regulates many physiological processes such as glucose metabolism and osmoregulatory responses to stress. PMID:24391849

  19. The Antagonistic Effect of Selenium on Lead-Induced Inflammatory Factors and Heat Shock Protein mRNA Level in Chicken Cartilage Tissue.

    PubMed

    Zheng, Shufang; Song, Huanyu; Gao, Han; Liu, Chunpeng; Zhang, Ziwei; Fu, Jing

    2016-09-01

    Selenium (Se) is recognized as a necessary trace mineral in animal diets, including those of birds. Lead (Pb) is a toxic heavy metal and can damage organs in humans and animals. Complex antagonistic interactions between Se and heavy metals have been reported in previous studies. However, little is known regarding the effects of Se on Pb-induced toxicity and the expression of inflammatory factors and heat shock proteins (HSPs) in the cartilage of chickens. In this present study, we fed chickens either with Se or Pb or both Se and Pb supplement and later analyzed the mRNA expressions of inflammatory factors (inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and HSPs (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90). The results showed that Se and Pb influenced the expression of inflammatory factors and HSP genes in the chicken cartilage tissues. Additionally, we also found that antagonistic interaction existed between Se and Pb supplementation. Our findings suggested that Se could exert a antagonistic effect on Pb in chicken cartilage tissues. PMID:26831653

  20. Differential mRNA Accumulation upon Early Arabidopsis thaliana Infection with ORMV and TMV-Cg Is Associated with Distinct Endogenous Small RNAs Level

    PubMed Central

    Zavallo, Diego; Manacorda, Carlos Augusto; Rodriguez, Maria Cecilia; Asurmendi, Sebastian

    2015-01-01

    Small RNAs (sRNAs) play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV) or crucifer-infecting Tobacco mosaic virus (TMV-Cg) with slightly different symptomatology at two early stages of infection (2 and 4dpi) was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions. PMID:26237414

  1. Effect of Alpha-Hederin, the active constituent of Nigella sativa, on miRNA-126, IL-13 mRNA levels and inflammation of lungs in ovalbumin-sensitized male rats

    PubMed Central

    Fallahi, Maryam; Keyhanmanesh, Rana; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Ebrahimi, Hadi

    2016-01-01

    Objective: In previous studies the therapeutic effects of Nigella sativa have been demonstrated on asthmatic animals. In the present study, the preventive effect of single dose of alpha-hederin, its active constituent, has been evaluated on lung inflammation and some inflammatory mediators in lungs of ovalbumin sensitized rat in order to elicit its mechanism. Materials and Methods: Forty rats were randomly grouped in 4 groups; control (C), sensitized (S), sensitized pretreated groups with thymoquinone (3 mg/kg i.p., S+TQ) and alpha-hederin (0.02 mg/kg i.p., S+AH). Levels of IL-13 mRNA and miRNA-126 in lung tissue and its pathological changes in each group were assessed. Results: Elevated levels of miRNA-126, IL-13 mRNA and pathological changes were observed in the sensitized group compared to the control group (p<0.001 to p<0.05). All of these factors were significantly reduced in S+TQ and S+AH groups in comparison to S group (p<0.001 to p<0.05). Although alpha-hederin decreased the levels of miRNA-126, IL-13 mRNA and pathological changes in comparison with thymoquinone, the results were statistically not significant. Conclusion: The results suggested that alpha-hederin had preventive effect on sensitized rats like thymoquinone. It may intervene in miRNA-126 expression, which consequently could interfere with IL-13 secretion pathway leading to a reduction in inflammatory responses. PMID:27247924

  2. mRNA expression levels and genetic status of genes involved in the EGFR and NF-κB pathways in metastatic non-small-cell lung cancer patients

    PubMed Central

    2011-01-01

    Background Metastatic non-small-cell lung cancer (NSCLC) has a dismal prognosis. EGFR is overexpressed or mutated in a large proportion of cases. Downstream components of the EGFR pathway and crosstalk with the NF-κB pathway have not been examined at the clinical level. We explored the prognostic significance of the mRNA expression of nine genes in the EGFR and NF-κB pathways and of BRCA1 and RAP80 in patients in whom EGFR and K-ras gene status had previously been determined. In addition, NFKBIA and DUSP22 gene status was also determined. Methods mRNA expression of the eleven genes was determined by QPCR in 60 metastatic NSCLC patients and in nine lung cancer cell lines. Exon 3 of NFKBIA and exon 6 of DUSP22 were analyzed by direct sequencing. Results were correlated with outcome to platinum-based chemotherapy in patients with wild-type EGFR and to erlotinib in those with EGFR mutations. Results BRCA1 mRNA expression was correlated with EZH2, AEG-1, Musashi-2, CYLD and TRAF6 expression. In patients with low levels of both BRCA1 and AEG-1, PFS was 13.02 months, compared to 5.4 months in those with high levels of both genes and 7.7 months for those with other combinations (P = 0.025). The multivariate analysis for PFS confirmed the prognostic role of high BRCA1/AEG-1 expression (HR, 3.1; P = 0.01). Neither NFKBIA nor DUSP22 mutations were found in any of the tumour samples or cell lines. Conclusions The present study provides a better understanding of the behaviour of metastatic NSCLC and identifies the combination of BRCA1 and AEG-1 expression as a potential prognostic model. PMID:21951562

  3. Severe von Willebrand disease due to a defect at the level of von Willebrand factor mRNA expression: Detection by exonic PCR-restriction fragment length polymorphism analysis

    SciTech Connect

    Nichols, W.C.; Lyons, S.E.; Harrison, J.S.; Cody, R.L.; Ginsburg, D. )

    1991-05-01

    von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, results from abnormalities in the plasma clotting protein von Willebrand factor (vWF). Severe (type III) vWD is autosomal recessive in inheritance and is associated with extremely low or undetectable vWF levels. The authors report a method designed to distinguish mRNA expression from the two vWF alleles by PCR analysis of peripheral blood platelet RNA using DNA sequence polymorphisms located within exons of the vWF gene. This approach was applied to a severe-vWD pedigree in which three of eight siblings are affected and the parents and additional siblings are clinically normal. Each parent was shown to carry a vWF allele that is silent at the mRNA level. Family members inheriting both abnormal alleles are affected with severe vWD, whereas individuals with only one abnormal allele are asymptomatic. Given the frequencies of the two exon polymorphisms reported here, this analysis should be applicable to {approx}70% of type I and type III vWD patients. This comparative DNA and RNA PCR-restriction fragment length polymorphism approach may also prove useful in identifying defects at the level of gene expression associated with other genetic disorders.

  4. Acute hypoxia differentially affects the NMDA receptor NR1, NR2A and NR2B subunit mRNA levels in the developing chick optic tectum: stage-dependent plasticity in the 2B-2A ratio.

    PubMed

    Vacotto, Marina; Rapacioli, Melina; Flores, Vladimir; de Plazas, Sara Fiszer

    2010-10-01

    It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1-NR2A and NR1-NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis. PMID:20596770

  5. The C-terminal domain of FUSCA3 negatively regulates mRNA and protein levels, and mediates sensitivity to the hormones abscisic acid and gibberellic acid in Arabidopsis.

    PubMed

    Lu, Qing Shi; Paz, Joelle Dela; Pathmanathan, Aathi; Chiu, Rex Shun; Tsai, Allen Yi-Lun; Gazzarrini, Sonia

    2010-10-01

    The transcription factor FUSCA3 (FUS3) controls the transition from the embryonic to the vegetative phase of development by regulating abscisic acid (ABA) and gibberellic acid (GA) levels in Arabidopsis thaliana. In a feedback loop, FUS3 accumulation is negatively and positively regulated by GA and ABA, respectively, by an uncharacterized mechanism. Here, we use a FUS3-GFP construct to show that the level of the FUS3 protein decreases dramatically during mid to late embryogenesis, whereas its mRNA is present at a high level. Deletion studies identify a C-terminal domain (CTD) that negatively regulates mRNA and protein levels, and mediates sensitivity to ABA and GA. Indeed, a CTD-truncated FUS3 variant accumulates at high level, and is insensitive to the destabilizing and stabilizing effects of GA and ABA, respectively. In contrast, fusion of various fragments of the CTD with GFP is sufficient to greatly reduce GFP fluorescence. The GFP-CTD fluorescence can be increased by ABA and paclobutrazol, an inhibitor of GA biosynthesis. Cell-free degradation assays show that FUS3 is a short-lived protein. FUS3 degradation follows the 26S proteasome in vitro and in vivo, and the CTD affects its degradation rate. In contrast to the native form, the CTD-truncated FUS3 is unable to fully rescue the fus3-3 mutant, and is thus required for FUS3 function. In conclusion, this study identifies a CTD that maintains low levels of FUS3 during embryogenesis and early germination, and is required for normal FUS3 function and sensitivity to ABA and GA. PMID:20663088

  6. Single nucleotide polymorphisms in CETP, SLC46A1, SLC19A1, CD36, BCOM1, APOA5, and ABCA1 are significant predictors of plasma HDL in healthy adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a marker-trait association study we estimated the statistical significance of 65 single nucleotide polymorphisms (SNP) in 23 candidate genes on HDL levels of two independent Caucasian populations. Each population consisted of men and women and their HDL levels were adjusted for gender and body we...

  7. Elevated expression of tryptophan hydroxylase-2 mRNA at the neuronal level in the dorsal and median raphe nuclei of depressed suicides

    PubMed Central

    Bach-Mizrachi, H; Underwood, MD; Tin, A; Ellis, SP; Mann, JJ; Arango, V

    2008-01-01

    Deficient levels of serotonin are associated with suicide and depression. Paradoxically, in the dorsal raphe nucleus (DRN) there are more serotonin neurons and more neuronal tryptophan hydroxylase-2 (TPH2) expression postmortem in depressed suicides. In this study, we sought to determine whether greater TPH2 expression in depressed suicides was the result of more TPH2 expression per neuron. In situ hybridization and computer-assisted image analysis were performed on tissue sections throughout the extent of the raphe nuclei at the level of silver grains per neuron to systematically quantify TPH2 neuronal expression. Depressed suicides have 26.5% more TPH2 grain density per neuron in the DRN compared with matched controls (P= 0.04). The difference in grain density is greater at mid- and caudal anatomical levels across the rostrocaudal axis of the DRN. Densitometric analysis of TPH2 expression in the DRN subnuclei showed that higher expression levels were observed at posterior anatomical levels of depressed suicides (121% of control in the caudal subnucleus). Higher TPH2 expression in depressed suicides may explain more TPH2 protein and reflect a homeostatic response to deficient serotonin levels in the brains of depressed suicides. Localized changes in TPH2 expression in specific subnuclei of the DRN suggest that the serotonergic compensatory mechanism in depression and suicide is specifically regulated within the DRN and has implications for regions innervated by this subnucleus. PMID:18180753

  8. Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats

    PubMed Central

    Shojaei, Shahla; Ghavami, Saeid; Panjehshahin, Mohammad Reza; Owji, Ali Akbar

    2015-01-01

    We aimed to compare the effects of oral ethanol (Eth) alone or combined with the phytoestrogen resveratrol (Rsv) on the expression of various brain-derived neurotrophic factor (BDNF) transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW)/day) dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day) dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats. PMID:26703578

  9. Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats.

    PubMed

    Shojaei, Shahla; Ghavami, Saeid; Panjehshahin, Mohammad Reza; Owji, Ali Akbar

    2015-01-01

    We aimed to compare the effects of oral ethanol (Eth) alone or combined with the phytoestrogen resveratrol (Rsv) on the expression of various brain-derived neurotrophic factor (BDNF) transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW)/day) dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day) dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats. PMID:26703578

  10. Correlation between mRNA levels and functional role of alpha1-adrenoceptor subtypes in arteries: evidence of alpha1L as a functional isoform of the alpha1A-adrenoceptor.

    PubMed

    Martí, Daniel; Miquel, Raquel; Ziani, Khalid; Gisbert, Regina; Ivorra, M Dolores; Anselmi, Elsa; Moreno, Lucrecia; Villagrasa, Victoria; Barettino, Domingo; D'Ocon, Pilar

    2005-11-01

    The mRNA levels for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The alpha1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), alpha1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both alpha1A- and alpha1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the alpha1B-subtype were a minority in all vessels (1.7-11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to alpha1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 micromol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro (4,5) decane-7-dionedihydrochloride} (BMY-7378, an alpha1D-adrenoceptor antagonist) confirm the relevant role of alpha1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an alpha1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of alpha1A- and alpha1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of alpha1A/alpha1L-adrenoceptors with minor participation of the alpha1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the alpha1L-adrenoceptor could be a functional isoform of the alpha1A-subtype. PMID:15951348

  11. Decreased Npas4 and Arc mRNA Levels in the Hippocampus of Aged Memory-Impaired Wild-Type But Not Memory Preserved 11β-HSD1 Deficient Mice.

    PubMed

    Qiu, J; Dunbar, D R; Noble, J; Cairns, C; Carter, R; Kelly, V; Chapman, K E; Seckl, J R; Yau, J L W

    2016-01-01

    Mice deficient in the glucocorticoid-regenerating enzyme 11β-HSD1 resist age-related spatial memory impairment. To investigate the mechanisms and pathways involved, we used microarrays to identify differentially expressed hippocampal genes that associate with cognitive ageing and 11β-HSD1. Aged wild-type mice were separated into memory-impaired and unimpaired relative to young controls according to their performance in the Y-maze. All individual aged 11β-HSD1-deficient mice showed intact spatial memory. The majority of differentially expressed hippocampal genes were increased with ageing (e.g. immune/inflammatory response genes) with no genotype differences. However, the neuronal-specific transcription factor, Npas4, and immediate early gene, Arc, were reduced (relative to young) in the hippocampus of memory-impaired but not unimpaired aged wild-type or aged 11β-HSD1-deficient mice. A quantitative reverse transcriptase-polymerase chain reaction and in situ hybridisation confirmed reduced Npas4 and Arc mRNA expression in memory-impaired aged wild-type mice. These findings suggest that 11β-HSD1 may contribute to the decline in Npas4 and Arc mRNA levels associated with memory impairment during ageing, and that decreased activity of synaptic plasticity pathways involving Npas4 and Arc may, in part, underlie the memory deficits seen in cognitively-impaired aged wild-type mice. PMID:26563879

  12. Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.

    PubMed

    Ahlemeyer, Barbara; Vogt, Julia-Franziska; Michel, Vera; Hahn-Kohlberger, Petra; Baumgart-Vogt, Eveline

    2014-11-01

    The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(®) 2000, FuGENE 6, HiPerFect(®), INTERFERin™, RiboJuice™) as well as microporation using the Neon™ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70%), to the reduction of PEX5 mRNA (by 90 vs. 0-50%) and PEX5 protein levels (by 70 vs. 0-50%). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15%) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded

  13. Antisense inhibition of myoD expression in regenerating rat soleus muscle is followed by an increase in the mRNA levels of myoD, myf-5 and myogenin and by a retarded regeneration.

    PubMed

    Zádor, Erno; Bottka, Sándor; Wuytack, Frank

    2002-06-12

    It has been reported that muscles of myoD-/- mice present a lower potential to regenerate, but there are no reports on the effect of acute interference with myoD expression limited in space and time to only a particular regenerating muscle. Here we relied on antisense inhibition of this factor. Four different oligos were tested. The suppression of regeneration indices (the expression of desmin, the formation of myotubes and the initiation of endplates) was the most pronounced, with the oligomer targeting a region encompassing the translation start site of myoD. A mixed backbone phosphorothioate-phosphate diester oligo (200 microl at 20 microM) was still detectable in the muscles 1 h after its administration and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the level of the targeted 5' end of the myoD mRNA was selectively decreased. The level of myoD protein was also lowered. Four hours after the antisense treatment, when the oligos were no longer detectable, the myoD mRNA level was restored and 24 h later it exceeded controls together with that of myf-5 and myogenin. After 4 weeks, the antisense-treated soleus muscles were similar to the control-treated and the untreated regenerated soleus with respect to fiber types and motor endplates, however, they contained smaller fibers which reflected the asynchronity of regeneration. This shows that successfully targeted simple antisense oligonucleotides can be used as selective tools for inhibition of individual factors in studying the process of muscle regeneration. PMID:12063168

  14. cDNA sequences and mRNA levels of two hexamerin storage proteins PinSP1 and PinSP2 from the Indianmeal moth, Plodia interpunctella.

    PubMed

    Zhu, Yu Cheng; Muthukrishnan, Subbaratnam; Kramer, Karl J

    2002-05-01

    In insects, storage proteins or hexamerins accumulate apparently to serve as sources of amino acids during metamorphosis and reproduction. Two storage protein-like cDNAs obtained from a cDNA library prepared from fourth instar larvae of the Indianmeal moth (Plodia interpunctella) were cloned and sequenced. The first clone, PinSP1, contained 2431 nucleotides with a 2295 nucleotide open reading frame (ORF) encoding a protein with 765 amino acid residues. The second cDNA, PinSP2, consisted of 2336 nucleotides with a 2250-nucleotide ORF encoding a protein with 750 amino acid residues. PinSP1 and PinSP2 shared 59% nucleotide sequence identity and 44% deduced amino acid sequence identity. A 17-amino acid signal peptide and a molecular mass of 90.4 kDa were predicted for the PinSP1 protein, whereas a 15-amino acid signal peptide and a mass of 88 kDa were predicted for PinSP2. Both proteins contained conserved insect larval storage protein signature sequence patterns and were 60-70% identical to other lepidopteran larval storage proteins. Expression of mRNA for both larval storage proteins was determined using the quantitative reverse transcription polymerase chain reaction method. Only very low levels were present in the second instar, but both mRNAs dramatically increased during the third instar, peaked in the fourth instar, decreased dramatically late in the same instar and pupal stages, and were undetectable during the adult stage. Males and females exhibited similar mRNA expression levels for both storage proteins during the pupal and adult stages. The results support the hypothesis that P. interpunctella, a species that does not feed after the larval stage, accumulates these two storage proteins as reserves during larval development for subsequent use in the pupal and adult stages. PMID:11891129

  15. Comparison of the effect of lipopolysaccharide on tumor necrosis factor α (TNF-α) secretion and TNF and TNFR1 mRNA levels in feline endometrium throughout the estrous cycle during pyometra and after medroxyprogesterone acetate treatment

    PubMed Central

    JURSZA-PIOTROWSKA, Ewelina; SIEMIENIUCH, Marta J.

    2016-01-01

    Endotoxins released by Gram-negative bacteria are potent stimulators of tumor necrosis factor α (TNF-α) production. The objectives of this study were to evaluate plasma levels of TNF-α, TNF-α secretion, and mRNA levels of TNF and TNF-α receptor type 1 (TNFR1) following exposure to lipopolysaccharide (LPS). For this, we used cultured endometrial cells or organ cultures, throughout the estrous cycle, after hormone treatment with medroxyprogesterone acetate (MPA), and during pyometra. Plasma TNF-α concentrations were increased in animals at estrus (P < 0.05) compared to other groups. In the LPS-challenged endometrium, secretion of TNF-α by tissues collected during estrus increased (P < 0.001) compared to that of other groups. LPS, alone or combined with TNF-α, upregulated TNF gene expression in the feline endometrium at diestrus (P < 0.001 for both treatments), in queens treated short-term with MPA (P < 0.01 and P < 0.05, respectively) and in queens treated long-term with MPA (P < 0.01 and P < 0.001, respectively). During pyometra, TNF and TNFR1 mRNA were increased only after tissues were challenged with TNF-α and LPS (P < 0.001 and P < 0.01, respectively). When cultured endometrial cells were challenged with LPS, the concentration of TNF-α increased only in epithelial cells after 4 h and 12 h (P < 0.05 and P < 0.01, respectively). Since LPS did not affect stromal cells, but TNF-α increased its own transcript after 2 h (P < 0.01), 4 h (P < 0.05) and 12 h (P < 0.001), we assume that stromal cells are not directly involved in pathogen recognition, as was the case for epithelial cells. PMID:27097764

  16. Comparison of the effect of lipopolysaccharide on tumor necrosis factor α (TNF-α) secretion and TNF and TNFR1 mRNA levels in feline endometrium throughout the estrous cycle during pyometra and after medroxyprogesterone acetate treatment.

    PubMed

    Jursza-Piotrowska, Ewelina; Siemieniuch, Marta J

    2016-08-25

    Endotoxins released by Gram-negative bacteria are potent stimulators of tumor necrosis factor α (TNF-α) production. The objectives of this study were to evaluate plasma levels of TNF-α, TNF-α secretion, and mRNA levels of TNF and TNF-α receptor type 1 (TNFR1) following exposure to lipopolysaccharide (LPS). For this, we used cultured endometrial cells or organ cultures, throughout the estrous cycle, after hormone treatment with medroxyprogesterone acetate (MPA), and during pyometra. Plasma TNF-α concentrations were increased in animals at estrus (P < 0.05) compared to other groups. In the LPS-challenged endometrium, secretion of TNF-α by tissues collected during estrus increased (P < 0.001) compared to that of other groups. LPS, alone or combined with TNF-α, upregulated TNF gene expression in the feline endometrium at diestrus (P < 0.001 for both treatments), in queens treated short-term with MPA (P < 0.01 and P < 0.05, respectively) and in queens treated long-term with MPA (P < 0.01 and P < 0.001, respectively). During pyometra, TNF and TNFR1 mRNA were increased only after tissues were challenged with TNF-α and LPS (P < 0.001 and P < 0.01, respectively). When cultured endometrial cells were challenged with LPS, the concentration of TNF-α increased only in epithelial cells after 4 h and 12 h (P < 0.05 and P < 0.01, respectively). Since LPS did not affect stromal cells, but TNF-α increased its own transcript after 2 h (P < 0.01), 4 h (P < 0.05) and 12 h (P < 0.001), we assume that stromal cells are not directly involved in pathogen recognition, as was the case for epithelial cells. PMID:27097764

  17. Impact of deltamethrin exposure on mRNA expression levels of metallothionein A, B and cytochrome P450 1A in rainbow trout muscles.

    PubMed

    Erdoğan, Orhan; Ceyhun, Saltuk Buğrahan; Ekinci, Deniz; Aksakal, Ercüment

    2011-09-15

    Metallothioneins (MT) are widely utilized to identify specific responses to heavy metal pollution. In addition, there is evidence demonstrating that in vertebrates MT synthesis is stimulated by different endogenous and exogenous agents in particular compounds leading to production of ROS. Also, cytochrome P450 1A can enhance the generation of ROS. On this basis, MT and CYP 1A induction can be considered as biomarkers of oxidative stress. In the current study, we examined the influences of pesticide administration on the expression of MT-A, MT-B and CYP 1A. For this purpose, we produced muscle metallothionein-A, metallothionein-B and cytochrome P450 1A cDNAs and used quantitative RT-PCR to assay mRNAs in rainbow trout exposed to acute and long-term deltamethrin administration. We observed that deltamethrin exposure significantly (p<0.05) increased the expression levels of Cyp1A, MT-A and MT-B in a time dependent manner. Results of our study contributes to the identification of inducers of such biomarkers in addition to well known agents. PMID:21658436

  18. Effects of dietary lysozyme levels on growth performance, intestinal morphology, non-specific immunity and mRNA expression in weanling piglets.

    PubMed

    Long, Yanrong; Lin, Sen; Zhu, Jiatao; Pang, Xiaoxue; Fang, Zhengfeng; Lin, Yan; Che, Lianqiang; Xu, Shengyu; Li, Jian; Huang, Yiming; Su, Xiang; Wu, De

    2016-03-01

    The aim of the present study was to determine the effect of dietary lysozyme levels on growth performance, gut health and non-specific immunity of weanling piglets. A total of 150 weanling piglets were allocated to six treatments. The piglets were fed the same basel diet supplemented with 0, 30, 60, 90 and 120 mg/kg lysozyme as well as antibiotics for 28 days. From day 14 to day 28 of dietary treatment, piglets fed 90 mg/kg lysozyme had greater average daily gain than piglets fed control diet. During the whole experimental period, piglets fed 120 mg/kg lysozyme tended to have greater average daily gain than piglets fed control diet. Compared with piglets fed control diet, piglets fed diets containing antibiotics and 90 mg/kg lysozyme had greater villus height to crypt depth ratio in duodenum and jejunum. Additionally, dietary supplementation of 60 and 90 mg/kg lysozyme as well as antibiotics enhanced the phagocytic activity of peritoneal macrophages in piglets. In conclusion, dietary lysozyme can accelerate the growth of weanling piglets by improving gut health and non-specific immunity and supplementing 90 mg/kg lysozyme is as effective as antibiotics (20 mg/kg colistin sulphate + 50 mg/kg kitasamycin) in improving the growth performance of weanling piglets. PMID:26419503

  19. Analysis of activation-induced cytidine deaminase mRNA levels in patients with chronic lymphocytic leukemia with different cytogenetic status.

    PubMed

    Gelmez, Metin Y; Teker, Aysegul B A; Aday, Aynur D; Yavuz, Akif S; Soysal, Teoman; Deniz, Gunnur; Aktan, Melih

    2014-02-01

    Activation induced cytidine deaminase (AID) enzyme, which converts cytosine into uracil and is expressed only by activated B lymphocytes, plays a role in B cells in both the mechanisms of somatic hypermutation (SHM) and class switch recombination (CSR). There are studies showing that AID can cause numerous translocations in different lymphoproliferative diseases. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in bone marrow and peripheral blood. The predictability and clinical status of B-CLL are difficult to determine. About 30-50% of patients have chromosomal abnormalities. AID, which is thought to create fraction segments for translocations, might also cause deletions in DNA regions of 17p13, 11q22.3, 13q14 and 13q34 that are associated with prognostic implications in patients with CLL. In this study, the AID gene expression in patients with CLL with and without deletions was investigated. When compared to healthy subjects and patients without deletions, increased levels of AID expression in patients with deletions of 17p13, 11q22.3 or 13q14 were found, but not for the 13q34 region. Our results show that AID expression may be associated with deletions in patients with CLL. PMID:23662991

  20. Ex Vivo Cytokine mRNA Levels Correlate with Changing Clinical Status of Ethiopian TB Patients and their Contacts Over Time

    PubMed Central

    Wassie, Liya; Demissie, Abebech; Aseffa, Abraham; Abebe, Markos; Yamuah, Lawrence; Tilahun, Hiwot; Petros, Beyene; Rook, Graham; Zumla, Alimuddin; Andersen, Peter; Doherty, T. Mark

    2008-01-01

    There is an increasing body of evidence which suggests that IL-4 plays a role in the pathogenesis of TB, but a general consensus on its role remains elusive. We have previously published data from a cohort of Ethiopian TB patients, their contacts, and community controls suggesting that enhanced IL-4 production is associated with infection with M. tuberculosis, rather than overt disease and that long-term protection in infected community controls is associated with co-production of the IL-4 antagonist IL-4d2, alongside elevated IL-4. Here, for the first time, we compare data on expression of IFN-γ, IL-4 and IL-4δ2 over time in TB patients and their household contacts. During the follow-up period, the TB patients completed therapy and ceased to display TB-like symptoms. This correlated with a decrease in the relative amount of IL-4 expressed. Over the same period, the clinical status of some of their contacts also changed, with a number developing TB-like symptoms or clinically apparent TB. IL-4 expression was disproportionately increased in this group. The findings support the hypothesis that elevated IL-4 production is generally associated with infection, but that TB disease is associated with a relatively increased expression of IL-4 compared to IFN-γ and IL-4δ2. However, the data also suggest that there are no clear-cut differences between groups: the immune response over time appears to include changes in the expression of IFN-γ, IL-4 and IL-4δ2, and it is the relative, not absolute levels of cytokine expression that are characteristic of clinical status. PMID:18231607

  1. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  2. A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

    PubMed

    Cipolla, Gabriel A; Park, Jong Kook; de Oliveira, Liana A; Lobo-Alves, Sara Cristina; de Almeida, Rodrigo C; Farias, Ticiana D J; Lemos, Débora de S; Malheiros, Danielle; Lavker, Robert M; Petzl-Erler, Maria Luiza

    2016-10-01

    Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus. PMID:27424220

  3. Expression of RXR, EcR, E75 and VtG mRNA levels in the hepatopancreas and ovary of the freshwater edible crab, Oziothelphusa senex senex (Fabricius, 1798) during different vitellogenic stages

    NASA Astrophysics Data System (ADS)

    Girish, B. P.; Swetha, CH.; Reddy, P. Sreenivasula

    2015-04-01

    The objective of the present study was to investigate the expression profile of retinoid X receptor ( RXR), ecdysone receptor ( EcR) and ecdysone inducible gene ( E75) in the hepatopancreas and ovary of Oziothelphusa senex senex during different vitellogenic stages. RXR, EcR and E75 complementary DNAs (cDNAs) were isolated from the ovaries, while vitellogenin ( VtG) cDNA was isolated from the hepatopancreas of vitellogenic female crab. Deduced amino acid sequence of the messenger RNAs (mRNAs) of RXR, EcR and E75 showed more than 80 % identity with their respective mRNAs of other brachyurans. VtG mRNA was not detected in the ovary throughout vitellogenic stages. RXR and EcR were significantly increased in the ovaries during vitellogenic stage I. The levels of EcR, E75 and VtG in the hepatopancreas elevated significantly during vitellogenic stages I and II, whereas the levels of RXR elevated only in vitellogenic stage I. During vitellogenic stage III, the levels of RXR, EcR and VtG in the hepatopancreas were significantly decreased. Immunoprecipitation analysis revealed the presence of VtG in the haemolymph, hepatopancreas and ovary extracts from the females but absent in haemolymph and hepatopancreas extract of males. It can be inferred that RXR, EcR and E75 are involved in the regulation of synthesis of VtG in hepatopancreas, whereas in ovary, it is hypothesized that they play an important role in the uptake of VtG from the haemolymph, probably by regulating the levels of vitellogenin receptor. These are the first data showing an association between the expression levels of RXR, EcR and E75 and vitellogenesis and provide an alternative molecular intervention mechanism to the traditional eyestalk ablation to induce vitellogenesis and ovarian maturation in crustaceans.

  4. Effects of pectin pentaoligosaccharide from Hawthorn ( Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet.

    PubMed

    Li, Tuo-Ping; Zhu, Ru-Gang; Dong, Yin-Ping; Liu, Yong-Hui; Li, Su-Hong; Chen, Gang

    2013-08-01

    The regulatory effects of haw pectin pentaoligosaccharide (HPPS) on fatty acid oxidation-related enzyme activities and mRNA levels were investigated in the liver of high fat diet induced hyperlipidemic mice. Results showed that HPPS (150 mg/kg for 10 weeks) significantly suppresses weight gain (32.3 ± 0.26 and 21.1 ± 0.14 g for high-fat diet and HPPS groups, respectively), decreases serum triacylglycerol levels (1.64 ± 0.09 and 0.91 ± 0.02 mmol/L, respectively), and increases lipid excretion in feces (55.7 ± 0.38 and 106.4 ± 0.57 mg/g for total lipid, respectively), compared to high-fat diet as control. HPPS significantly increased the hepatic fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, carnitine palmitoyltransferase I, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase by 53.8, 74.2, 47.1, and 24.2%, respectively. Meanwhile, the corresponding mRNAs were up-regulated by 89.6, 85.8, 82.9, and 30.9%, respectively. Moreover, HPPS was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α. Results suggest that continuous HPPS ingestion may be used as dietary therapy to prevent obesity and cardiovascular diseases. PMID:23855516

  5. RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes.

    PubMed

    Daffu, Gurdip; Shen, Xiaoping; Senatus, Laura; Thiagarajan, Devi; Abedini, Andisheh; Hurtado Del Pozo, Carmen; Rosario, Rosa; Song, Fei; Friedman, Richard A; Ramasamy, Ravichandran; Schmidt, Ann Marie

    2015-12-01

    Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator-activated receptor-γ (PPARG)-responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68(+) macrophages from atherosclerotic plaques of Ldlr(-/-) mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr(-/-) mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications. PMID:26253613

  6. Sex differences in constitutive mRNA levels of CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46 in the pig liver: Comparison between Meishan and Landrace pigs.

    PubMed

    Kojima, Misaki; Degawa, Masakuni

    2016-06-01

    Breed and sex differences in hepatic mRNA levels of cytochrome P450 (CYP) isoforms (CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46) were examined in 5-month-old Meishan, Landrace, and their crossbred F1 (LM and ML) pigs. Serum testosterone levels in male Meishan, LM, and ML pigs were 2.5-3.5-fold higher than in Landrace pigs. CYP3A46 mRNA was breed-specifically detected only in Landrace, LM, and ML pigs. In Meishan, LM, and ML pigs only, male-predominant expressions of CYP2B22, CYP2C33, CYP2C49 and CYP3A29 mRNAs were observed; CYP3A22 mRNA expression showed the opposite pattern. Male-dominant mRNA expression was also observed in LM and ML pigs for CYP3A46. The sex differences in CYP mRNA levels in Meishan pigs disappeared when males were castrated and were restored by testosterone propionate (TP) administration to the castrated males. In Landrace pigs, TP administration to castrated males and intact females significantly increased the levels of CYP2B22, CYP2C33, and CYP3A46 mRNAs. Immature (1-month-old) pigs showed no breed or sex differences in CYP mRNA expressions. The results demonstrated that androgen is an important determinant of sex-associated expression of several CYPs and suggested that breed differences in sex-associated expression could be caused by differences in serum androgen level and by other genetic traits. PMID:27080814

  7. Effect of excess levels of lysine and leucine in wheat-based, amino acid-fortified diets on the mRNA expression of two selected cationic amino acid transporters in pigs.

    PubMed

    Morales, A; Barrera, M A; Araiza, A B; Zijlstra, R T; Bernal, H; Cervantes, M

    2013-04-01

    An experiment was conducted to evaluate the effect of excess levels of Leu and Lys on the expression of b(0,+) and CAT-1 mRNA in jejunum, liver and the muscles Longissimus dorsi (LDM) and Semitendinosus (STM). Twenty pigs with an average initial BW of 16.4 ± 1.7 kg were used in a Randomized Complete Block. Dietary treatments (T) were as follows: T1, basal diet; T2, basal plus 3.5 g l-Lys/kg diet; T3, basal plus 1.5 g l-Leu/kg diet; T4, basal plus 3.5 g l-Lys plus 1.5 g l-Leu/kg diet. Diets in T1 and T3 met 100% the requirement of Lys for pigs within the 10 to 20 kg body weight range; diets in T2 and T4 contained 35% excess of Lys. Also, diets in T1 and T2 supplied 104%, whereas diets in T3 and T4 supplied 116% the requirement of Leu. The expression of b(0,+) in jejunum was reduced (p = 0.002) because of the supplementation of l-Leu, but l-Lys supplementation had no effect (p = 0.738). In contrast, the expression of b(0,+) in STM (p = 0.012) and liver (p = 0.095) was reduced by the high level of Lys, but Leu had no effect (p > 0.100). CAT-1 expression in STM increased by high Lys (p = 0.023) and Leu (p = 0.007) levels. In liver, the expression of CAT-1 substantially increased (p = 0.001) because of Lys. In conclusion, excess levels of dietary Lys and Leu affect the expression of cationic amino acid transporters, and this effect varies depending on the studied tissue. PMID:22211733

  8. mRNA transcript therapy.

    PubMed

    Weissman, Drew

    2015-02-01

    mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery. PMID:25359562

  9. Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins

    PubMed Central

    Tseng, Pei-Chi; Lee, Tzong-Shyuan; Lee, Wen-Jane; Chang, Pey-Jium; Chiang, An-Na

    2016-01-01

    Metabolic syndrome (MetS) is a complicated health problem that encompasses a variety of metabolic disorders. In this study, we analyzed the relationship between the major biochemical parameters associated with MetS and circulating levels of microRNA (miR)-33, miR-103, and miR-155. We found that miRNA-33 levels were positively correlated with levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, LDL-cholesterol, and triacylglycerol, but negatively correlated with HDL-cholesterol levels. In the cellular study, miR-33 levels were increased in macrophages treated with high glucose and cholesterol-lowering drugs atorvastatin and pitavastatin. miR-33 has been reported to play an essential role in cholesterol homeostasis through ATP-binding cassette transporter A1 (ABCA1) regulation and reverse cholesterol transport. However, the molecular mechanism underlying the linkage between miR-33 and statin treatment remains unclear. In the present study, we investigated whether atorvastatin and pitavastatin exert their functions through the modulation of miR-33 and ABCA1-mediated cholesterol efflux from macrophages. The results showed that treatment of the statins up-regulated miR-33 expression, but down-regulated ABCA1 mRNA levels in RAW264.7 cells and bone marrow-derived macrophages. Statin-mediated ABCA1 regulation occurs at the post-transcriptional level through targeting of the 3′-UTR of the ABCA1 transcript by miR-33. Additionally, we found significant down-regulation of ABCA1 protein expression in macrophages treated with statins. Finally, we showed that high glucose and statin treatment significantly suppressed cholesterol efflux from macrophages. These findings have highlighted the complexity of statins, which may exert detrimental effects on metabolic abnormalities through regulation of miR-33 target genes. PMID:27139226

  10. Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins.

    PubMed

    Chen, Wei-Ming; Sheu, Wayne H-H; Tseng, Pei-Chi; Lee, Tzong-Shyuan; Lee, Wen-Jane; Chang, Pey-Jium; Chiang, An-Na

    2016-01-01

    Metabolic syndrome (MetS) is a complicated health problem that encompasses a variety of metabolic disorders. In this study, we analyzed the relationship between the major biochemical parameters associated with MetS and circulating levels of microRNA (miR)-33, miR-103, and miR-155. We found that miRNA-33 levels were positively correlated with levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, LDL-cholesterol, and triacylglycerol, but negatively correlated with HDL-cholesterol levels. In the cellular study, miR-33 levels were increased in macrophages treated with high glucose and cholesterol-lowering drugs atorvastatin and pitavastatin. miR-33 has been reported to play an essential role in cholesterol homeostasis through ATP-binding cassette transporter A1 (ABCA1) regulation and reverse cholesterol transport. However, the molecular mechanism underlying the linkage between miR-33 and statin treatment remains unclear. In the present study, we investigated whether atorvastatin and pitavastatin exert their functions through the modulation of miR-33 and ABCA1-mediated cholesterol efflux from macrophages. The results showed that treatment of the statins up-regulated miR-33 expression, but down-regulated ABCA1 mRNA levels in RAW264.7 cells and bone marrow-derived macrophages. Statin-mediated ABCA1 regulation occurs at the post-transcriptional level through targeting of the 3'-UTR of the ABCA1 transcript by miR-33. Additionally, we found significant down-regulation of ABCA1 protein expression in macrophages treated with statins. Finally, we showed that high glucose and statin treatment significantly suppressed cholesterol efflux from macrophages. These findings have highlighted the complexity of statins, which may exert detrimental effects on metabolic abnormalities through regulation of miR-33 target genes. PMID:27139226

  11. Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.

    PubMed

    Su, Wei; Slevin, Michael K; Marzluff, William F; Rhoads, Robert E

    2016-01-01

    Since DNA and histone levels must be closely balanced for cell survival, histone expressions are highly regulated. The regulation of replication-dependent histone expression is mainly achieved at the mRNA level, as the mRNAs are rapidly removed when DNA replication is inhibited during S-phase. Histone mRNA degradation initiates with addition of multiple uridines (oligouridylation) following the 3' stem-loop (SL) catalyzed by terminal uridyltransferase (TUTase). Previous studies showed that histone mRNA degradation occurs through both 5' → 3' and 3' → 5' processes, but the relative contributions are difficult to dissect due to lack of established protocols. The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by structural modifications at the both 5' and 3' termini. In this chapter, we present methods of utilizing modified cap dinucleotide analogs to block 5' → 3' degradation of a reporter mRNA containing canonical histone mRNA 3' SL and monitoring how oligouridylation and 3' → 5' degradation occur. Protocols are presented for synthesis of reporter mRNA containing the histone 3' SL and modified cap analogs, monitoring mRNA stability and unidirectional degradation either from 5' or 3' termini, and detection of oligo(U) tracts from degradation products by either traditional or deep sequencing. PMID:27236794

  12. Adaptive capability as indicated by behavioral and physiological responses, plasma HSP70 level, and PBMC HSP70 mRNA expression in Osmanabadi goats subjected to combined (heat and nutritional) stressors

    NASA Astrophysics Data System (ADS)

    Shilja, Shaji; Sejian, V.; Bagath, M.; Mech, A.; David, C. G.; Kurien, E. K.; Varma, Girish; Bhatta, Raghavendra

    2015-12-01

    A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0 kg) were used in the present study. The bucks were divided into four groups viz., C (n = 6; control), HS (n = 6; heat stress), NS (n = 6; nutritional stress), and CS (n = 6; combined stress). The study was conducted for a period of 45 days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30 % intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6 h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly (P < 0.01) between ad libitum fed groups (C and HS) and restricted feeding groups (NS and CS). The highest (P < 0.01) lying time was recorded in the CS group while the lowest in the C and HS groups. The highest (P < 0.01) drinking frequency was also recorded in the CS group. Water intake recorded was significantly (P < 0.01) higher in both the HS and CS groups. The highest respiration rate (RR), pulse rate (PR), and rectal temperature (RT) during the afternoon were also recorded in the CS group. Further, skin temperature of the head, flank, and scrotum during the afternoon was also higher (P < 0.01) in the CS group. In addition, both plasma HSP70 concentration and PBMC HSP70 messenger RNA (mRNA) transcript expression were also significantly (P < 0.01) higher in the CS group. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on adaptive capabilities of Osmanabadi bucks as compared to that would occur individually. Further, the study

  13. Adaptive capability as indicated by behavioral and physiological responses, plasma HSP70 level, and PBMC HSP70 mRNA expression in Osmanabadi goats subjected to combined (heat and nutritional) stressors.

    PubMed

    Shilja, Shaji; Sejian, V; Bagath, M; Mech, A; David, C G; Kurien, E K; Varma, Girish; Bhatta, Raghavendra

    2016-09-01

    A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0 kg) were used in the present study. The bucks were divided into four groups viz., C (n = 6; control), HS (n = 6; heat stress), NS (n = 6; nutritional stress), and CS (n = 6; combined stress). The study was conducted for a period of 45 days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30 % intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6 h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly (P < 0.01) between ad libitum fed groups (C and HS) and restricted feeding groups (NS and CS). The highest (P < 0.01) lying time was recorded in the CS group while the lowest in the C and HS groups. The highest (P < 0.01) drinking frequency was also recorded in the CS group. Water intake recorded was significantly (P < 0.01) higher in both the HS and CS groups. The highest respiration rate (RR), pulse rate (PR), and rectal temperature (RT) during the afternoon were also recorded in the CS group. Further, skin temperature of the head, flank, and scrotum during the afternoon was also higher (P < 0.01) in the CS group. In addition, both plasma HSP70 concentration and PBMC HSP70 messenger RNA (mRNA) transcript expression were also significantly (P < 0.01) higher in the CS group. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on adaptive capabilities of Osmanabadi bucks as compared to that would occur individually

  14. Probing dimensionality beyond the linear sequence of mRNA.

    PubMed

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions. PMID:26650615

  15. Sodium Channel Inhibitors Reduce DMPK mRNA and Protein.

    PubMed

    Witherspoon, Luke; O'Reilly, Sean; Hadwen, Jeremiah; Tasnim, Nafisa; MacKenzie, Alex; Farooq, Faraz

    2015-08-01

    Myotonic dystrophy type 1 (DM1) is caused by an expanded trinucleotide (CTG)n tract in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. This results in the aggregation of an expanded mRNA forming toxic intranuclear foci which sequester splicing factors. We believe down-regulation of DMPK mRNA represents a potential, and as yet unexplored, DM1 therapeutic avenue. Consequently, a computational screen for agents which down-regulate DMPK mRNA was undertaken, unexpectedly identifying the sodium channel blockers mexiletine, prilocaine, procainamide, and sparteine as effective suppressors of DMPK mRNA. Analysis of DMPK mRNA in C2C12 myoblasts following treatment with these agents revealed a reduction in the mRNA levels. In vivo analysis of CD1 mice also showed DMPK mRNA and protein down-regulation. The role of DMPK mRNA suppression in the documented efficacy of this class of compounds in DM1 is worthy of further investigation. PMID:26011798

  16. Signaling Pathways That Control mRNA Turnover

    PubMed Central

    Thapar, Roopa; Denmon, Andria P.

    2013-01-01

    Cells regulate their genomes mainly at the level of transcription and at the level of mRNA decay. While regulation at the level of transcription is clearly important, the regulation of mRNA turnover by signaling networks is essential for a rapid response to external stimuli. Signaling pathways result in posttranslational modification of RNA binding proteins by phosphorylation, ubiquitination, methylation, acetylation etc. These modifications are important for rapid remodeling of dynamic ribonucleoprotein complexes and triggering mRNA decay. Understanding how these posttranslational modifications alter gene expression is therefore a fundamental question in biology. In this review we highlight recent findings on how signaling pathways and cell cycle checkpoints involving phosphorylation, ubiquitination, and arginine methylation affect mRNA turnover. PMID:23602935

  17. Changes in Chloroplast mRNA Stability during Leaf Development.

    PubMed Central

    Klaff, P; Gruissem, W

    1991-01-01

    During spinach leaf development, chloroplast-encoded mRNAs accumulate to different steady-state levels. Their relative transcription rates alone, however, cannot account for the changes in mRNA amount. In this study, we examined the importance of mRNA stability for the regulation of plastid mRNA accumulation using an in vivo system to measure mRNA decay in intact leaves by inhibiting transcription with actinomycin D. Decay of psbA and rbcL mRNAs was assayed in young and mature leaves. The psbA mRNA half-life was increased more than twofold in mature leaves compared with young leaves, whereas rbcL mRNA decayed with a similar relative half-life at both leaf developmental stages. The direct in vivo measurements demonstrated that differential mRNA stability in higher plant plastids can account for differences in mRNA accumulation during leaf development. The role of polysome association in mRNA decay was also investigated. Using organelle-specific translation inhibitors that force mRNAs into a polysome-bound state or deplete mRNAs of ribosomes, we measured mRNA decay in vivo in either state. The results showed that rbcL and psbA mRNAs are less stable when bound to polysomes relative to the polysome-depleted mRNAs and that their stabilities are differentially affected by binding to polysomes. The results suggested that ribosome binding and/or translation of the psbA and rbcL mRNAs may function to modulate the rate of their decay in chloroplasts. PMID:12324602

  18. The LXR ligand GW3965 inhibits Newcastle disease virus infection by affecting cholesterol homeostasis.

    PubMed

    Sheng, Xiang-Xiang; Sun, Ying-Jie; Zhan, Yuan; Qu, Yu-Rong; Wang, Hua-Xia; Luo, Miao; Liao, Ying; Qiu, Xu-Sheng; Ding, Chan; Fan, Hong-Jie; Mao, Xiang

    2016-09-01

    Newcastle disease (ND) is a contagious disease that affects most species of birds. Its causative pathogen, Newcastle disease virus (NDV), also exhibits considerable oncolytic activity against mammalian cancers. A better understanding of the pathogenesis of NDV will help us design efficient vaccines and novel anticancer strategies. GW3965, a widely used synthetic ligand of liver X receptor (LXR), induces the expression of LXRs and its downstream genes, including ATP-binding cassette transporter A1 (ABCA1). ABCA1 regulates cellular cholesterol homeostasis. Here, we found that GW3965 inhibited NDV infection in DF-1 cells. It also inhibited NF-κB activation and reduced the upregulation of proinflammatory cytokines induced by the infection. Further studies showed that GW3965 exerted its inhibitory effects on virus entry and replication. NDV infection increased the mRNA levels of several lipogenic genes but decreased the ABCA1 mRNA level. Overexpression of ABCA1 inhibited NDV infection and reduced the cholesterol content in DF-1 cells, but when the cholesterol was replenished, NDV infection was restored. GW3965 treatment prevented cholesterol accumulation in the perinuclear area of the infected cells. In summary, our studies suggest that GW3965 inhibits NDV infection, probably by affecting cholesterol homeostasis. PMID:27357231

  19. Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme.

    PubMed Central

    Schwer, B; Mao, X; Shuman, S

    1998-01-01

    Current models of mRNA decay in yeast posit that 3' deadenylation precedes enzymatic removal of the 5' cap, which then exposes the naked end to 5' exonuclease action. Here, we analyzed gene expression in Saccharomyces cerevisiae cells bearing conditional mutations of Ceg1 (capping enzyme), a 52 kDa protein that transfers GMP from GTP to the 5' end of mRNA to form the GpppN cap structure. Shift of ceg1 mutants to restrictive temperature elicited a rapid decline in the rate of protein synthesis, which correlated with a sharp reduction in the steady-state levels of multiple individual mRNAs. ceg1 mutations prevented the accumulation of SSA1 and SSA4 mRNAs that were newly synthesized at the restrictive temperature. Uncapped poly(A)+ SSA4 mRNA accumulated in cells lacking the 5' exoribonuclease Xrn1. These findings provide genetic evidence for the long-held idea that the cap guanylate is critical for mRNA stability. The deadenylation-decapping-degradation pathway appears to be short-circuited when Ceg1 is inactivated. PMID:9547258

  20. Effects of DNA replication on mRNA noise.

    PubMed

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  1. Effects of DNA replication on mRNA noise

    PubMed Central

    Peterson, Joseph R.; Cole, John A.; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A.

    2015-01-01

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript’s half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  2. mRNA Composition and Control of Bacterial Gene Expression

    PubMed Central

    Liang, S.-T.; Xu, Y.-C.; Dennis, P.; Bremer, H.

    2000-01-01

    The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of β-galactosidase expressed from three different promoter-lacZ fusions (Pspc-lacZ, PRNAI-lacZ, and PRNAII-lacZ) was measured (i) in a relA+ background during exponential growth at different rates and (ii) in relA+ and ΔrelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp. PMID:10809680

  3. Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells.

    PubMed

    Benteyn, Daphné; Heirman, Carlo; Thielemans, Kris; Bonehill, Aude

    2016-01-01

    Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs. PMID:27236795

  4. Several genetic polymorphisms interact with overweight/obesity to influence serum lipid levels

    PubMed Central

    2012-01-01

    Background Information about the interactions of single nucleotide polymorphisms (SNPs) and overweight/obesity on serum lipid profiles is still scarce. The present study was undertaken to detect ten SNPs and their interactions with overweight/obesity on serum lipid levels. Methods A total of 978 normal weight and 751 overweight/obese subjects of Bai Ku Yao were randomly selected from our previous stratified randomized cluster samples. Normal weight, overweight and obesity were defined as a body mass index (BMI) < 24, 24–28, and > 28 kg/m2; respectively. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein (Apo) A1 and ApoB levels were measured. Genotyping of ATP-binding cassette transporter A1 (ABCA-1) V825I, acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) rs1044925, low density lipoprotein receptor (LDL-R) AvaII, hepatic lipase gene (LIPC) -250G>A, endothelial lipase gene (LIPG) 584C>T, methylenetetrahydrofolate reductase (MTHFR) 677C>T, the E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP) rs3757354, proprotein convertase subtilisin-like kexin type 9 (PCSK9) E670G, peroxisome proliferator-activated receptor delta (PPARD) +294T>C, and Scavenger receptor class B type 1 (SCARB1) rs5888 was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. The interactions were detected by factorial design covariance analysis. Results The genotypic and allelic frequencies of LIPC and PCSK9 were different between normal weight and overweight/obese subjects, the genotypic frequency of LIPG and allelic frequency of MYLIP were also different between normal weight and overweight/obese subjects (P < 0.05-0.001). The levels of TC, ApoA1 (ABCA-1); TC, LDL-C, ApoA1, ApoB and ApoA1/ApoB (LIPC); TG, HDL-C, and ApoA1 (LIPG); TC, HDL-C, LDL-C, ApoA1 and Apo

  5. 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

  6. Nucleolin mediates microRNA-directed CSF-1 mRNA deadenylation but increases translation of CSF-1 mRNA.

    PubMed

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K

    2013-06-01

    CSF-1 mRNA 3'UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3'UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3'UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  7. Nuclear Retention of mRNA in Mammalian Tissues

    PubMed Central

    Bahar Halpern, Keren; Caspi, Inbal; Lemze, Doron; Levy, Maayan; Landen, Shanie; Elinav, Eran; Ulitsky, Igor; Itzkovitz, Shalev

    2015-01-01

    Summary mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise. PMID:26711333

  8. Interactions of several genetic polymorphisms and alcohol consumption on blood pressure levels.

    PubMed

    Yin, Rui-Xing; Aung, Lynn Htet Htet; Long, Xing-Jiang; Yan, Ting-Ting; Cao, Xiao-Li; Huang, Feng; Wu, Jin-Zhen; Yang, De-Zhai; Lin, Wei-Xiong; Pan, Shang-Ling

    2015-01-01

    This study aimed to detect the interactions of several single nucleotide polymorphisms (SNPs) and alcohol consumption on blood pressure levels. Genotypes of 10 SNPs in the ATP-binding cassette transporter A1 (ABCA-1), acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), low density lipoprotein receptor (LDLR), hepatic lipase gene (LIPC), endothelial lipase gene (LIPG), methylenetetrahydrofolate reductase (MTHFR), the E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP), proprotein convertase subtilisin-like kexin type 9 (PCSK9), peroxisome proliferator-activated receptor delta (PPARD), and Scavenger receptor class B type 1 (SCARB1) genes were determined in 616 nondrinkers and 608 drinkers. The genotypic frequencies of LDLR rs5925, LIPC rs2070895, MTHFR rs1801133, and MYLIP rs3757354 SNPs were significantly different between nondrinkers and drinkers. The levels of systolic blood pressure (ABCA-1 rs2066715 and rs2070895), diastolic blood pressure (rs2070895), and pulse pressure (PP) (rs2066715, ACAT-1 rs1044925, and rs1801133) in nondrinkers, and systolic blood pressure (rs1044925 and SCARB1 rs5888), diastolic blood pressure (rs1044925 and LIPG rs2000813), and PP (PCSK9 rs505151 and rs5888) in drinkers were different among the genotypes (P < 0.005-0.001). The interactions of several SNPs and alcohol consumption on systolic blood pressure (rs2066715, rs1044925, rs5925, rs2070895, rs1801133, rs3757354, PPARD rs2016520, and rs5888), diastolic blood pressure (rs2066715, rs1044925, rs5925, rs2000813, rs3757354, and rs2016520), and PP (rs1044925, rs2070895, rs1801133, rs3757354, rs505151, and rs5888) were observed (P < 0.005-0.001). The differences in blood pressure levels between the nondrinkers and drinkers might be partially attributed to the interactions of these SNPs and alcohol consumption. PMID:26354227

  9. mRNA quality control goes transcriptional

    PubMed Central

    Kilchert, Cornelia; Vasiljeva, Lidia

    2013-01-01

    Eukaryotic mRNAs are extensively processed to generate functional transcripts, which are 5′ capped, spliced and 3′ polyadenylated. Accumulation of unprocessed (aberrant) mRNAs can be deleterious for the cell, hence processing fidelity is closely monitored by QC (quality control) mechanisms that identify erroneous transcripts and initiate their selective removal. Nucleases including Xrn2/Rat1 and the nuclear exosome have been shown to play an important role in the turnover of aberrant mRNAs. Recently, with the growing appreciation that mRNA processing occurs concomitantly with polII (RNA polymerase II) transcription, it has become evident that QC acts at the transcriptional level in addition to degrading aberrant RNAs. In the present review, we discuss mechanisms that allow cells to co-transcriptionally initiate the removal of RNAs as well as down-regulate transcription of transcripts where processing repeatedly fails. PMID:24256272

  10. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  11. Dimethyl sulphoxide and haemin induce ferrochelatase mRNA by different mechanisms in murine erythroleukaemia cells.

    PubMed

    Fukuda, Y; Fujita, H; Taketani, S; Sassa, S

    1993-03-01

    The level of mRNA encoding ferrochelatase (FeC), the terminal enzyme of the haem biosynthetic pathway, was examined in murine erythroleukaemia (MEL) cells when they were induced to undergo erythroid cell differentiation by treatment with dimethyl sulphoxide (DMSO), or haemin. FeC mRNA increased within 12 h after DMSO or haemin treatment of MEL cells, and its level continued to increase for 48 h. Treatment of cells with succinylacetone (SA), a potent inhibitor of haem synthesis, suppressed a DMSO-mediated increase in FeC mRNA, and haemin treatment reversed a SA-mediated decrease in FeC mRNA. Nuclear runoff analyses showed that, while DMSO increased the rate of transcription of FeC mRNA, haemin did not. These results indicate that the induction of FeC mRNA by DMSO is largely transcriptional, while that by haemin is post-transcriptional. PMID:8485055

  12. Plasma visfatin levels and mRNA expression of visfatin in peripheral blood mononuclear cells and peripheral blood monocyte-derived macrophages from normal weight females with polycystic ovary syndrome

    PubMed Central

    ZHANG, JING; ZHOU, LINGLING; TANG, LIULIN; XU, LIANGZHI

    2014-01-01

    Polycystic ovary syndrome (PCOS) is a common reproductive endocrinology disease, however, an explicit etiology is not known. Insulin resistance (IR) appears to be central to the pathogenesis of PCOS and inflammation may be significant in the pathogenesis of IR in PCOS. The aims of the present study were to investigate the plasma visfatin level and the gene expression of visfatin in peripheral blood mononuclear cells (PBMCs) and peripheral blood monocyte-derived macrophages (PBMMs) from PCOS patients, in addition to investigating the association between PCOS and IR. A total of 21 PCOS patients and 21 control subjects were enrolled in the study; the homeostasis model assessment of insulin resistance (HOMA-IR) was considered to be a stratified method for establishing the subgroups. Fasting blood samples were collected and the levels of sex hormones, insulin, glucose, blood lipids and visfatin were measured. In addition, visfatin gene expression levels in PBMCs and PBMMs were assessed using quantitative polymerase chain reaction. The plasma visfatin and gene expression levels of visfatin in PBMCs and PBMMs were not observed to increase in the normal weight PCOS and normal weight IR patients. Furthermore, plasma visfatin levels did not correlate with the normal weight PCOS patients or the normal weight IR patients per se. Further investigation into the role of visfatin in the pathogenesis of PCOS or IR should examine macrophages in the tissues, rather than macrophages in the peripheral blood. PMID:24940414

  13. Feline Calicivirus Can Tolerate Gross Changes of Its Minor Capsid Protein Expression Levels Induced by Changing Translation Reinitiation Frequency or Use of a Separate VP2-Coding mRNA

    PubMed Central

    Meyers, Gregor

    2014-01-01

    Caliciviruses use reinitiation of translation governed by a ‘termination upstream ribosomal binding site’ (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level. PMID:25007260

  14. Localization of histidine decarboxylase mRNA in rat brain.

    PubMed

    Bayliss, D A; Wang, Y M; Zahnow, C A; Joseph, D R; Millhorn, D E

    1990-08-01

    The recent cloning of a cDNA encoding fetal rat liver histidine decarboxylase (HDC), the synthesizing enzyme for histamine, allows the study of the central histaminergic system at the molecular level. To this end, Northern blot and in situ hybridization analyses were used to determine the regional and cellular distribution of neurons which express HDC mRNA in rat brain. Three hybridizing species which migrate as 1.6-, 2.6-, and 3.5-kb RNA were identified with Northern blots. The major (2.6 kb) and minor (3.5 kb) species, characteristic of HDC mRNA in fetal liver, were expressed at high levels in diencephalon and at just detectable levels in hippocampus, but not in other brain regions. In contrast, the 1.6-kb species was present in all brain regions examined except the olfactory bulb. Cells which contain HDC mRNA were found by in situ hybridization in the hypothalamus; HDC mRNA-containing cells were not detected in other areas, including the hippocampus. Hypothalamic neurons which express HDC mRNA were localized to all aspects of the tuberomammillary nucleus, a result consistent with previous immunohistochemical findings. PMID:19912749

  15. Regulation of neuronal oxytocin mRNA by ovarian steroids in the mature and developing hypothalamus.

    PubMed Central

    Miller, F D; Ozimek, G; Milner, R J; Bloom, F E

    1989-01-01

    We have examined the changes in neuronal expression of oxytocin mRNA in the perinatal and mature female rat as a function of endogenous gonadal steroids. Northern blot analysis demonstrated a significant developmental increase in the abundance of oxytocin mRNA in the female brain concomitant with puberty. Ovariectomy of adult females decreased total brain oxytocin mRNA to significantly lower levels. In contrast, lactating mothers had increased levels of neuronal oxytocin mRNA. In situ hybridization analysis of neuronal oxytocin mRNA in adolescent, mature virgin, and ovariectomized virgin female brains demonstrated that the location and number of neurons expressing oxytocin mRNA was unchanged and that total brain oxytocin mRNA differences were attributable to amounts expressed per neuron. Differences in mRNA abundance were noted in oxytocin neurons throughout the hypothalamus, including those known to project as magnocellular neurons to the neurohypophysis and those of parvocellular origin thought to make wholly intracerebral connections. This developmental and dynamic regulation of oxytocin mRNA levels during gonadal maturation may coordinate the peripheral and central effects of this peptide on the reproductive biology of the female rat. Images PMID:2928343

  16. Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli

    PubMed Central

    Geyer, Chelsie N.; Fowler, Randal C.; Johnson, James R.; Johnston, Brian; Weissman, Scott J.; Hawkey, Peter; Hanson, Nancy D.

    2016-01-01

    Objectives High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam. Methods Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT–PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives. Results mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2–48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5–15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2–3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm. Conclusions CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels. PMID:26612874

  17. Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish.

    PubMed

    Canosa, Luis Fabián; Stacey, Norm; Peter, Richard Ector

    2008-12-01

    In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain. PMID:18815210

  18. Gene regulation by structured mRNA elements.

    PubMed

    Wachter, Andreas

    2014-05-01

    The precise temporal and spatial coordination of gene activity, based on the integration of internal and external signals, is crucial for the accurate functioning of all biological processes. Although the basic principles of gene expression were established some 60 years ago, recent research has revealed a surprising complexity in the control of gene activity. Many of these gene regulatory mechanisms occur at the level of the mRNA, including sophisticated gene control tasks mediated by structured mRNA elements. We now know that mRNA folds can serve as highly specific receptors for various types of molecules, as exemplified by metabolite-binding riboswitches, and interfere with pro- and eukaryotic gene expression at the level of transcription, translation, and RNA processing. Gene regulation by structured mRNA elements comprises versatile strategies including self-cleaving ribozymes, RNA-folding-mediated occlusion or presentation of cis-regulatory sequences, and sequestration of trans-acting factors including other RNAs and proteins. PMID:24780087

  19. Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

    PubMed Central

    Gong, Z Y; Brandhorst, B P

    1988-01-01

    An increased level of unpolymerized tubulin caused by depolymerization of microtubules in sea urchin larvae resulted in a rapid loss of tubulin mRNA, which was prevented by nearly complete inhibition of protein synthesis. Results of an RNA run-on assay indicated that inhibition of protein synthesis does not alter tubulin gene transcription. Analysis of the decay of tubulin mRNA in embryos in which RNA synthesis was inhibited by actinomycin D indicated that inhibition of protein synthesis prevents the destabilization of tubulin mRNA. The effect was similar whether mRNA was maintained on polysomes in the presence of emetine or anisomycin or displaced from the polysomes in the presence of puromycin or pactamycin; thus, the stabilization of tubulin mRNA is not dependent on the state of the polysomes after inhibition of protein synthesis. Even after tubulin mRNA declined to a low level after depolymerization of microtubules, it could be rescued by treatment of embryos with inhibitors of protein synthesis. Tubulin mRNA could be induced to accumulate prematurely in gastrulae but not in plutei if protein synthesis was inhibited, an observation that is indicative of the importance of the autogenous regulation of tubulin mRNA stability during embryogenesis. Possible explanations for the role of protein synthesis in the control of mRNA stability are discussed. Images PMID:3211150

  20. Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood.

    PubMed

    Rönn, Tina; Volkov, Petr; Gillberg, Linn; Kokosar, Milana; Perfilyev, Alexander; Jacobsen, Anna Louisa; Jørgensen, Sine W; Brøns, Charlotte; Jansson, Per-Anders; Eriksson, Karl-Fredrik; Pedersen, Oluf; Hansen, Torben; Groop, Leif; Stener-Victorin, Elisabet; Vaag, Allan; Nilsson, Emma; Ling, Charlotte

    2015-07-01

    Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed the DNA methylation of ∼480 000 sites in human adipose tissue from 96 males and 94 females and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic biomarkers of aging in blood, including ELOVL2, FHL2, KLF14 and GLRA1, also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2. We identified 2825 genes (e.g. FTO, ITIH5, CCL18, MTCH2, IRS1 and SPP1) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28-46 mmol/mol) correlated significantly with the methylation of 711 sites, annotated to, for example, RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for obesity, type 2 diabetes and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue. PMID:25861810

  1. Biomaterials for mRNA delivery.

    PubMed

    Islam, Mohammad Ariful; Reesor, Emma K G; Xu, Yingjie; Zope, Harshal R; Zetter, Bruce R; Shi, Jinjun

    2015-12-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  2. Biomaterials for mRNA Delivery

    PubMed Central

    Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  3. Regulation of the mRNA half-life in breast cancer.

    PubMed

    Griseri, Paola; Pagès, Gilles

    2014-08-10

    The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3'UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

  4. Regulation of the mRNA half-life in breast cancer

    PubMed Central

    Griseri, Paola; Pagès, Gilles

    2014-01-01

    The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3’UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

  5. Regulation of luteinizing hormone receptor mRNA expression by a specific RNA binding protein in the ovary*

    PubMed Central

    Menon, K.M.J.; Nair, Anil K.; Wang, Lei; Peegel, Helle

    2009-01-01

    Summary The expression of LH receptor mRNA shows significant changes during different physiological states of the ovary. Previous studies from our laboratory have identified a post-transcriptional mechanism by which LH receptor mRNA is regulated following preovulatory LH surge or in response to hCG administration. A specific binding protein, identified as mevalonate kinase, binds to the open reading frame of LH receptor mRNA. The protein binding site is localized to nucleotides 203–220 of the LH receptor mRNA and exhibits a high degree of specificity. The expression levels of the protein show an inverse relationship to the LH receptor mRNA levels. The hCG-induced down-regulation of LH receptor mRNA can be mimicked by increasing the intracellular levels of cyclic AMP by a phosphodiesterase inhibitor. An in vitro mRNA decay assay showed that addition of the binding protein to the decay system caused accelerated LH receptor mRNA decay. Our results therefore show that LH receptor mRNA expression in the ovary is regulated post-transcriptionally by altering the rate of mRNA degradation by a specific mRNA binding protein. PMID:17055149

  6. Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

    SciTech Connect

    Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

    2013-11-15

    Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.

  7. Regulation and deregulation of mRNA translation during myeloid maturation.

    PubMed

    Khanna-Gupta, Arati

    2011-02-01

    Gene expression in the eukaryotic cell is regulated at a number of levels, including transcription of genomic DNA into messenger RNA (mRNA), nucleocytoplasmic export of mRNA, and translation of the exported mRNA into proteins in the cytoplasm by ribosomes. The role played by epigenetics and transcription factors associated with the control of gene expression in the developing neutrophil has been well documented and appreciated over the years. A wealth of information on the role played by transcription factors in myeloid biology has contributed to our understanding of both normal and abnormal neutrophil development. However, regulation of mRNA translation in myeloid cell maturation is much less well-studied. A better understanding of the translational control of myeloid gene expression may provide important insights into both normal and abnormal myeloid maturation. This review summarizes our current understanding of the regulation of myeloid gene expression at the mRNA translational level. PMID:21093533

  8. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    PubMed Central

    Bonnet, Amandine; Palancade, Benoit

    2014-01-01

    Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

  9. Endurance training enhances LXRα gene expression in Wistar male rats.

    PubMed

    Kazeminasab, Fatemeh; Marandi, Mohammad; Ghaedi, Kamran; Esfarjani, Fahimeh; Moshtaghian, Jamal

    2013-09-01

    Liver X receptor α (LXRα) is a member of the ligand-activated transcription factor of nuclear hormonal receptor superfamily, whose activation leads to modulation in the expression of genes involved in cholesterol homeostasis including ATP-binding cassette transporter A1 (ABCA1), which plays a crucial role in plasma high-density lipoprotein cholesterol (HDL-C) remodeling. The purpose of this study was to investigate whether endurance training enhanced the expression level of liver LXRα gene. Twelve adult male Wistar rats (200-220 g) were divided into control and training groups. Training group received exercise on a motor-driven treadmill at 28 m/min (0 % grade) for 60 min/day, 5 days/week for 8 weeks. Twenty-four hours after the last exercise session, the rats were killed and blood was taken from the right ventricle of each rat. Plasma was collected for HDL-C, low-density lipoprotein cholesterol (LDL-C), TC and TG measurements. Furthermore, a portion of the liver of each rat was excised and washed in ice-cold saline and frozen in liquid nitrogen for assessment of LXRα and ABCA1 mRNA levels. Data indicated significant increase in both LXRα and ABCA1 mRNA levels in trained rats, compared to control rats. Plasma HDL-C concentration was significantly higher (P < 0.001) in trained rats at the end of treadmill exercise. However, there was a significant decrease in LDL-C (P < 0.003), TG, TC concentration, TC/HDL-C and LDL/HDL-C ratios in trained rats compared with those in the control group (P < 0.001). In conclusion, we found that endurance training induced significant elevation in LXRα gene expression, which correlated with enhanced levels of ABCA1 mRNA and plasma HDL-C concentration. PMID:23674092

  10. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  11. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  12. Stochastic mRNA synthesis in mammalian cells.

    PubMed

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  13. All-in-one detector of circulating mRNA based on a smartphone

    NASA Astrophysics Data System (ADS)

    Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

    2016-03-01

    Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

  14. Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion.

    PubMed

    Ernfors, Patrik; Persson, Håkan

    1991-01-01

    Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia. PMID:12106253

  15. Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

    PubMed

    Blair, E D; Blair, C C; Wagner, E K

    1987-08-01

    To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation. PMID:3037112

  16. Genome-wide analysis of mRNA decay patterns during early Drosophila development

    PubMed Central

    2010-01-01

    Background The modulation of mRNA levels across tissues and time is key for the establishment and operation of the developmental programs that transform the fertilized egg into a fully formed embryo. Although the developmental mechanisms leading to differential mRNA synthesis are heavily investigated, comparatively little attention is given to the processes of mRNA degradation and how these relate to the molecular programs controlling development. Results Here we combine timed collection of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally and zygotically encoded factors to mRNA degradation, and the ways in which mRNA decay profiles relate to gene function, mRNA localization patterns, translation rates and protein turnover. We also detect cis-regulatory sequences enriched in transcripts with common degradation patterns and propose several proteins and microRNAs as developmental regulators of mRNA decay during early fruit fly development. Finally, we experimentally validate the effects of a subset of cis-regulatory sequences and trans-regulators in vivo. Conclusions Our work advances the current understanding of the processes controlling mRNA degradation during early Drosophila development, taking us one step closer to the understanding of mRNA decay processes in all animals. Our data also provide a valuable resource for further experimental and computational studies investigating the process of mRNA decay. PMID:20858238

  17. Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells.

    PubMed

    Shi, Jing; Zhou, Ming; Gong, Aihua; Li, Qijun; Wu, Qian; Cheng, Gary J; Yang, Mingyang; Sun, Yaocheng

    2016-02-16

    The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biological and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepared nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomolecules. PMID:26813157

  18. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons

    PubMed Central

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A.; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E.

    2016-01-01

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay. The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. PMID:26717982

  19. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

    PubMed

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

    2016-05-01

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. PMID:26717982

  20. Variations in cytokine mRNA expression during normal human pregnancy

    PubMed Central

    Kruse, N; Greif, M; Moriabadi, N F; Marx, L; Toyka, K V; Rieckmann, P

    2000-01-01

    Epidemiological data provide evidence that disease activity of T cell-mediated, organ-specific autoimmune diseases is reduced during pregnancy. Although there are several experimental animal studies on the effect of pregnancy on the immune system, the situation in humans is less clear. We therefore performed a prospective analysis of cytokine mRNA expression in whole blood by a new on-line reverse transcriptase-polymerase chain reaction technique and of serum hormone levels during pregnancy in healthy women. The control group included age-matched non-pregnant healthy women. Quantitativecytokine mRNA expression revealed significantly reduced IL-18, interferon-gamma (IFN-γ), and IL-2 mRNA levels in the first and second trimester in pregnancy compared with non-pregnant women. No difference between groups was detected for tumour necrosis factor-alpha (TNF-α) mRNA. IL-4 and IL-10 mRNA were detected at low levels in only 20% of pregnant women and were reduced to a statistically significant extent in the second and third trimester compared with the control group. Changes in IL-18 mRNA expression correlated inversely with serum values for human choriogonadotropin (HCG) and IL-10 serum levels correlated with increases in serum 17β-oestradiol levels. These data indicate immunomodulatory effects of pregnancy at the cytokine level which may be related to the variations in the clinical course of organ-specific, T cell-mediated autoimmune diseases during pregnancy. PMID:10632669

  1. Cholesterol efflux is LXRα isoform-dependent in human macrophages

    PubMed Central

    2014-01-01

    Background The nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. Methods We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques. Results Here we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 su