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Sample records for abca1 protein levels

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  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. 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

  12. 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.

  13. 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

  14. 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

  15. 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...

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  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. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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...

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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 ...

  8. 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 ...

  9. 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

  10. 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

  11. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    PubMed Central

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Clinical significance of high-density lipoproteins and the development of atherosclerosis: focus on the role of the adenosine triphosphate-binding cassette protein A1 transporter.

    PubMed

    Brewer, H Bryan; Santamarina-Fojo, Silvia

    2003-08-21

    Low levels of high-density lipoprotein (HDL) cholesterol constitute a risk factor for coronary artery disease, and there is evidence that increasing HDL cholesterol levels reduces cardiovascular risk. The phenotype of low HDL cholesterol with or without elevated triglycerides is at least as common in patients hospitalized for cardiovascular disease as is hypercholesterolemia, and it is characteristic of diabetes and the metabolic syndrome, conditions associated with increased cardiovascular risk. Recent studies have elucidated mechanisms by which HDL acts to reduce cardiovascular risk, bolstering the rationale for targeting of HDL in lipid-modifying therapy. In particular, HDL (1) carries excess cholesterol from peripheral cells to the liver for removal in the process termed reverse cholesterol transport, (2) reduces oxidative modification of low-density lipoproteins (LDL), and (3) inhibits cytokine-induced expression of cellular adhesion molecules on endothelial cells. Studies of the newly described adenosine triphosphate-binding cassette protein A1 (ABCA1) transporter have established a crucial role for this transporter in modulating the levels of plasma HDL and intracellular cholesterol in the liver as well as in peripheral cells. Elevated levels of intracellular cholesterol stimulate the liver X receptor pathway, enhancing the expression of ABCA1, which increases intracellular trafficking of excess cholesterol to the cell surface for interaction with lipid-poor apolipoprotein A-I to form nascent HDL. Nascent HDL facilitates the removal of additional excess cellular cholesterol, which is esterified by lecithin-cholesterol acyltransferase with conversion of the nascent HDL to mature spherical HDL. Overexpression of ABCA1 in mice on a regular chow or Western diet results in a marked increase in plasma HDL, increased LDL, and increased transport of cholesterol to the liver. On a high cholesterol/cholate diet, transgenic mice overexpressing ABCA1 have increased HDL

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. Atomic-level analysis of membrane-protein structure.

    PubMed

    Hendrickson, Wayne A

    2016-06-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. PMID:27273628

  4. Yeast prions: Paramutation at the protein level?

    PubMed

    Tuite, Mick F

    2015-08-01

    Prions are proteins that have the potential to refold into a novel conformation that templates the conversion of like molecules to the altered infectious form. In the yeast Saccharomyces cerevisiae, trans-generational epigenetic inheritance can be mediated by a number of structurally and functionally diverse prions. Prionogenesis can confer both loss-of-function and gain-of-function properties to the prion protein and this in turn can have a major impact on host phenotype, short-term adaptation and evolution of new traits. Prionogenesis shares a number of properties in common with paramutation and can be considered as a mitotically and meiotically heritable change in protein conformation induced by trans-interactions between homologous proteins. PMID:26386407

  5. High levels of plasma protein C in nephrotic syndrome.

    PubMed

    Pabinger-Fasching, I; Lechner, K; Niessner, H; Schmidt, P; Balzar, E; Mannhalter, C

    1985-02-18

    In patients with severe nephrotic syndrome determinations of plasma protein C: Ag levels (8 patients: 5 adults, 3 children) and protein C activity (3 out of 8 patients) revealed significantly elevated plasma protein C concentrations. Furthermore we observed a significant inverse correlation of protein C: Ag to AT III: Ag levels. No protein C: Ag could be detected in the urine of two patients studied. We conclude from our data, that changes of plasma protein C do not contribute to the high thrombotic tendency in nephrotic syndrome. PMID:3838827

  6. Dietary protein source and level alters growth in neon tetras.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutritional studies for aquarium fish like the neon tetra are sparse in comparison with those for food fish. To determine the optimum dietary protein level and source for growth of neon tetras, diets were formulated to contain 25, 35, 45 and 55% dietary protein from either marine animal protein or ...

  7. Novel mechanism of transcriptional repression of the human ATP binding cassette transporter A1 gene in hepatic cells by the winged helix/forkhead box transcription factor A2.

    PubMed

    Thymiakou, Efstathia; Kardassis, Dimitris

    2014-06-01

    ATP binding cassette transporter A1 (ABCA1) plays a key role in the biogenesis of HDL by promoting the efflux of cellular cholesterol and phospholipids to lipid free apoA-I. Mutations in the ABCA1 gene cause Tangier disease which is characterized by near or complete absence of circulating plasma HDL. In the present study we show that the winged helix/forkhead box containing transcription factor A2 (FOXA2) shown previously to play a role in glucose and bile acid homeostasis in the liver and in energy utilization in adipose tissue is a negative modulator of ABCA1 gene expression in hepatic cells. We show that the ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of the sites are localized in a region of the ABCA1 promoter enriched in binding elements for transcriptional repressor proteins whereas the third site is the core of the TATA element of the ABCA1 promoter. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or FOXA2 gene expression by siRNA was associated with increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibited both the constitutive ABCA1 gene expression as well as ABCA1 gene induction by oxysterols and retinoids via nuclear receptors LXRα/RXRα. In summary, the present study identifies transcription factor FOXA2 as a negative modulator of ABCA1 gene expression in hepatic cells and reveals a novel mechanism of transcriptional repression by FOXA2 which involves the TATA element of the ABCA1 gene. PMID:24807696

  8. Synaptic protein levels altered in vascular dementia

    PubMed Central

    Sinclair, Lindsey I; Tayler, Hannah M; Love, Seth

    2015-01-01

    Introduction Cerebral ischaemia is the defining pathophysiological abnormality in most forms of vascular dementia (VAD), but the pathogenesis of the dementia remains poorly understood. In Alzheimer's disease (AD), there is early loss of synaptic proteins, but these have been little studied in VAD. Materials and Methods We measured synaptophysin, postsynaptic density protein 95 (PSD-95), drebrin, synaptosomal-associated protein 25 (SNAP-25) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assays in superior temporal cortex from 11 patients with VAD and, initially, 11 non-dementia controls. We corrected for neuronal content by measurement of neuron-specific enolase. A further 11 controls were subsequently used in a validation study. Simulation of post-mortem delay found that PSD-95 was stable at 4°C but declined slightly at RT. SNAP-25 and drebrin showed good post-mortem stability. Previous studies had shown good post-mortem preservation of synaptophysin and VEGF. Results The VAD cases had lower synaptophysin (but P > 0.05 in initial study), significantly lower SNAP-25 (P = 0.024) and significantly higher drebrin (P = 0.020). On comparison with the second control group, the reduction in synaptophysin was significant (P = 0.008), and the other results were confirmed. Conclusion There is probably a reduction in presynaptic proteins in the temporal cortex in VAD, although not as marked as in AD. In VAD, there is also an increase in drebrin, which may be a response to reduced synaptic input. PMID:25559750

  9. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  10. Dietary protein level and performance of growing Baladi kids

    PubMed Central

    Abdelrahman, M. M.; Aljumaah, R. S.

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity. PMID:27175130

  11. Protein engineering strategies with potential applications for altering clinically relevant cellular pathways at the protein level.

    PubMed

    Regan, Lynne; Hinrichsen, Michael R; Oi, Curran

    2016-05-01

    All diseases can be fundamentally viewed as the result of malfunctioning cellular pathways. Protein engineering offers the potential to develop new tools that will allow these dysfunctional pathways to be better understood, in addition to potentially providing new routes to restore proper function. Here we discuss different approaches that can be used to change the intracellular activity of a protein by intervening at the protein level: targeted protein sequestration, protein recruitment, protein degradation, and selective inhibition of binding interfaces. The potential of each of these tools to be developed into effective therapeutic treatments will also be discussed, along with any major barriers that currently block their translation into the clinic. PMID:27031866

  12. Protein expression analyses at the single cell level.

    PubMed

    Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

    2014-01-01

    The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. PMID:25197931

  13. Source and level of supplemental protein for growing lambs.

    PubMed

    Dabiri, N; Thonney, M L

    2004-11-01

    Two 3 x 2 factorial growth trials and a companion metabolism trial with 13, 15, or 17% dietary CP (DM basis), with or without 3% of the DM replaced with slowly degraded menhaden fish meal, were conducted to determine if level of dietary protein influences whether slowly degraded protein improves lamb growth and protein use. The growth trials included 32 and 34 pens of two weanling lambs initially weighing 23 to 26 kg and fed for 42 d. The metabolism trial included 12 additional lambs fed in metabolism cages with a 2-wk adjustment period, a 1-wk preliminary period, and a 7-d collection period. Plasma urea N (PUN) was measured in all lambs at the conclusion of the second growth trial and at the end of the metabolism trial. There was a protein level x protein source interaction (P = 0.05) for PUN of the 12 lambs in the metabolism trial but not for the 68 lambs in the second growth trial. Replacement of part of the soybean meal protein with protein from fish meal did not affect ADG or G:F at any protein level, but it lowered (P = 0.08) PUN in the second growth trial. Plasma urea N values were higher (P = 0.002) in lambs fed diets with 15 or 17% CP; however, ADG (P = 0.037 in Exp. 1 and P = 0.055 in Exp. 2), and G:F (P = 0.094 in Exp. 1 and P = 0.003 in Exp. 2) were lower for lambs fed the diets with 13% CP. There was little difference in ADG or G:F between lambs fed the diets with 15 or 17% CP, suggesting that a CP level of 15% with supplemental protein from soybean meal would be optimal for 25- to 40-kg growing Finnsheep x Dorset lambs. PMID:15542470

  14. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  15. A residue level protein-protein interaction model in electrolyte solutions

    NASA Astrophysics Data System (ADS)

    Song, Xueyu

    2014-03-01

    The osmotic second virial coefficients B2 are directly related to the solubility of protein molecules in electrolyte solutions and can be useful to narrow down the search parameter space of protein crystallization conditions. Using a residue level model of protein-protein interaction in electrolyte solutions B2 of bovine pancreatic trypsin inhibitor and lysozyme in various solution conditions such as salt concentration, pH and temperature are calculated using an extended Fast Multipole Methods in combination with the boundary element formulation. Overall, the calculated B2 are well correlated with the experimental observations for various solution conditions. In combination with our previous work on the binding affinity calculations of protein complexes it is demonstrated that our residue level model can be used as a reliable model to describe protein-protein interaction in solutions.

  16. Rice bran proteins and their hydrolysates modulate cholesterol metabolism in mice on hypercholesterolemic diets.

    PubMed

    Zhang, Huijuan; Wang, Jing; Liu, Yingli; Gong, Lingxiao; Sun, Baoguo

    2016-06-15

    The hypolipidemic properties of defatted rice bran protein (DRBP), fresh rice bran protein (FRBP), DRBP hydrolysates (DRBPH), and FRBP hydrolysates (FRBPH) were determined in mice on high fat diets for four weeks. Very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C) contents, and the hepatic total cholesterol content were reduced while fecal total cholesterol and total bile acid (TBA) contents were increased in the FRBPH diet group. The expression levels of hepatic genes for cholesterol biosynthesis HMG-CoAR and SREBP-2 were lowest in the FRBPH diet group. The mRNA level of HMG-CoAR was significantly positively correlated with the hepatic TG content (r = 0.82, P < 0.05). The mRNA levels of genes related to bile acid biosynthesis and cholesterol efflux, CYP7A1, ABCA1, and PPARγ were up-regulated in all test groups. The results suggest that FRBPH regulates cholesterol metabolism in mice fed the high fat and cholesterol diet by increasing fecal steroid excretion and expression levels of genes related to bile acid synthesis and cholesterol efflux, and the down-regulation of the expression levels of genes related to cholesterol biosynthesis. PMID:27216972

  17. Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

    2015-01-01

    Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent). PMID:26157620

  18. DPF2 regulates OCT4 protein level and nuclear distribution.

    PubMed

    Liu, Chao; Zhang, Dijuan; Shen, Yuxian; Tao, Xiaofang; Liu, Lihua; Zhong, Yongwang; Fang, Shengyun

    2015-12-01

    The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein. PMID:26417682

  19. Smoking, COPD and 3-Nitrotyrosine Levels of Plasma Proteins

    SciTech Connect

    Jin, Hongjun; Webb-Robertson, Bobbie-Jo M.; Peterson, Elena S.; Tan, Ruimin; Bigelow, Diana J.; Scholand, Mary Beth; Hoidal, John R.; Pounds, Joel G.; Zangar, Richard C.

    2011-09-01

    BACKGROUND: Nitric oxide is a physiologically regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of cigarette smoke, although it is not clear if this effect results from decreased nitric oxide production or oxidation of nitric oxide to reactive, nitrating, species. These processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we determine the effects of smoking and chronic obstructive pulmonary disease (COPD) on circulating levels of nitrotyrosine, and thereby gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was used to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. Plasma samples from 458 individuals were analyzed. RESULTS: Nitrotyrosine levels in circulating proteins were uniformly reduced in smokers but increased in COPD patients. We also observed a persistent suppression of nitrotyrosine in former smokers. CONCLUSIONS: Smoking broadly suppresses the levels of 3-nitrotyrosine in plasma proteins, suggesting that cigarette smoke suppresses endothelial nitric oxide production. In contrast, the increase in nitrotyrosine levels in COPD patients most likely results from inflammatory processes. This study provides the first evidence that smoking has irreversible effects on endothelial production of nitric oxide, and provides insight into how smoking could induce a loss of elasticity in the vasculature and a long-term increase in the risk of cardiovascular disease.

  20. Predicting Abdominal Aortic Aneurysm Target Genes by Level-2 Protein-Protein Interaction

    PubMed Central

    Fu, Yi; Cui, Qinghua; Kong, Wei

    2015-01-01

    Abdominal aortic aneurysm (AAA) is frequently lethal and has no effective pharmaceutical treatment, posing a great threat to human health. Previous bioinformatics studies of the mechanisms underlying AAA relied largely on the detection of direct protein-protein interactions (level-1 PPI) between the products of reported AAA-related genes. Thus, some proteins not suspected to be directly linked to previously reported genes of pivotal importance to AAA might have been missed. In this study, we constructed an indirect protein-protein interaction (level-2 PPI) network based on common interacting proteins encoded by known AAA-related genes and successfully predicted previously unreported AAA-related genes using this network. We used four methods to test and verify the performance of this level-2 PPI network: cross validation, human AAA mRNA chip array comparison, literature mining, and verification in a mouse CaPO4 AAA model. We confirmed that the new level-2 PPI network is superior to the original level-1 PPI network and proved that the top 100 candidate genes predicted by the level-2 PPI network shared similar GO functions and KEGG pathways compared with positive genes. PMID:26496478

  1. ORMDL proteins regulate ceramide levels during sterile inflammation.

    PubMed

    Cai, Lin; Oyeniran, Clement; Biswas, Debolina D; Allegood, Jeremy; Milstien, Sheldon; Kordula, Tomasz; Maceyka, Michael; Spiegel, Sarah

    2016-08-01

    The bioactive sphingolipid metabolite, ceramide, regulates physiological processes important for inflammation and elevated levels of ceramide have been implicated in IL-1-mediated events. Although much has been learned about ceramide generation by activation of sphingomyelinases in response to IL-1, the contribution of the de novo pathway is not completely understood. Because yeast ORM1 and ORM2 proteins negatively regulate ceramide levels through inhibition of serine palmitoyltransferase, the first committed step in ceramide biosynthesis, we examined the functions of individual mammalian ORM orthologs, ORM (yeast)-like (ORMDL)1-3, in regulation of ceramide levels. In HepG2 liver cells, downregulation of ORMDL3 markedly increased the ceramide precursors, dihydrosphingosine and dihydroceramide, primarily from de novo biosynthesis based on [U-(13)C]palmitate incorporation into base-labeled and dual-labeled dihydroceramides, whereas downregulation of each isoform increased dihydroceramides [(13)C]labeled in only the amide-linked fatty acid. IL-1 and the IL-6 family cytokine, oncostatin M, increased dihydroceramide and ceramide levels in HepG2 cells and concomitantly decreased ORMDL proteins. Moreover, during irritant-induced sterile inflammation in mice leading to induction of the acute-phase response, which is dependent on IL-1, expression of ORMDL proteins in the liver was strongly downregulated and accompanied by increased ceramide levels in the liver and accumulation in the blood. Together, our results suggest that ORMDLs may be involved in regulation of ceramides during IL-1-mediated sterile inflammation. PMID:27313060

  2. Predicting Protein-Protein Interactions from the Molecular to the Proteome Level.

    PubMed

    Keskin, Ozlem; Tuncbag, Nurcan; Gursoy, Attila

    2016-04-27

    Identification of protein-protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete interactions at the proteome level. This review aims to provide a background on PPIs and their types. Computational methods for PPI predictions can use a variety of biological data including sequence-, evolution-, expression-, and structure-based data. Physical and statistical modeling are commonly used to integrate these data and infer PPI predictions. We review and list the state-of-the-art methods, servers, databases, and tools for protein-protein interaction prediction. PMID:27074302

  3. Low copper and high manganese levels in prion protein plaques

    USGS Publications Warehouse

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  4. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level.

    PubMed

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-in

    2016-02-14

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces. PMID:26794230

  5. Effect of Crude Protein Levels in Concentrate and Concentrate Levels in Diet on In vitro Fermentation

    PubMed Central

    Van Dung, Dinh; Shang, Weiwei; Yao, Wen

    2014-01-01

    The effect of concentrate mixtures with crude protein (CP) levels 10%, 13%, 16%, and 19% and diets with roughage to concentrate ratios 80:20, 60:40, 40:60, and 20:80 (w/w) were determined on dry matter (DM) and organic matter (OM) digestibility, and fermentation metabolites using an in vitro fermentation technique. In vitro fermented attributes were measured after 4, 24, and 48 h of incubation respectively. The digestibility of DM and OM, and total volatile fatty acid (VFA) increased whereas pH decreased with the increased amount of concentrate in the diet (p<0.001), however CP levels of concentrate did not have any influence on these attributes. Gas production reduced with increased CP levels, while it increased with increasing concentrate levels. Ammonia nitrogen (NH3-N) concentration and microbial CP production increased significantly (p<0.05) by increasing CP levels and with increasing concentrate levels in diet as well, however, no significant difference was found between 16% and 19% CP levels. Therefore, 16% CP in concentrate and increasing proportion of concentrate up to 80% in diet all had improved digestibility of DM and organic matter, and higher microbial protein production, with improved fermentation characteristics. PMID:25050017

  6. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-In

    2016-02-01

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those

  7. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  8. Macrophage-activating lipopeptide-2 downregulates the expression of ATP-binding cassette transporter A1 by activating the TLR2/NF-кB/ZNF202 pathway in THP-1 macrophages.

    PubMed

    Peng, Liangjie; Zhang, Zizhen; Zhang, Min; Yu, Xiaohua; Yao, Feng; Tan, Yulin; Liu, Dan; Gong, Duo; Chong, Huang; Liu, Xiaoyan; Zheng, Xilong; Tian, Guoping; Tang, Chaoke

    2016-04-01

    Macrophage-activating lipopeptide-2 (MALP-2) has been shown to promote the development of atherosclerosis. ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein, plays a critical role in mediating cholesterol export from macrophages to apolipoprotein A-I (apoA-I). However, whether MALP-2 can regulate the expression of ABCA1 is still largely unknown. The aim of this study was to explore the effects of MALP-2 on ABCA1 expression in THP-1 macrophages and the underlying mechanisms. Our results showed that the treatment of cells with MALP-2 decreased ABCA1 level and suppressed cholesterol efflux in both concentration- and time-dependent manners. The contents of intracellular cholesterol were significantly increased in the presence of MALP-2. Moreover, MALP-2-mediated inhibition of ABCA1 expression was abolished by siRNA of either Toll-like receptor 2 (TLR2) or nuclear factor κB (NF-κB). A similar effect was produced by treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate. In addition, MALP-2-induced activation of NF-κB markedly increased zinc finger protein 202 (ZNF202) level, and ZNF202 siRNA impaired the effects of MALP-2 on ABCA1 expression. Taken together, these results suggest that MALP-2 can decrease ABCA1 expression and subsequent cholesterol efflux through activation of the TLR2/NF-κB/ZNF202 signaling pathway in THP-1 macrophages. PMID:26922321

  9. Effects of soy protein and calcium levels on mineral bioaccessibility and protein digestibility from enteral formulas.

    PubMed

    Galán, María Gimena; Drago, Silvina Rosa

    2014-09-01

    Enteral formulas (EF) are complex food systems which have all the nutrients in their matrix for the complete human nourishment. However, there are components in EF which can interact with minerals, reducing their absorption, and thereof the EF nutritional quality. The effect of soy protein (SP) and Ca content on Fe, Zn, and Ca bioaccessibility and protein digestibility (%DP) was assessed using a response surface design in EF. Tested SP levels were 2.5-5.0 g/100 mL of total protein. Ca levels were adjusted with Ca citrate within a range between 50 and 100 mg/100 mL. SP content negatively influenced %DP and Fe, Zn and Ca bioaccessibility. As SP content increased, mineral bioaccessibility and %DP decreased, probably due to the increased levels of phytic acid and trypsin inhibitors from SP. Ca content only affected %DCa, which had a direct relationship with Ca levels, while did not affect Fe and Zn bioaccessibility or %DP. Since Ca citrate did not impair Fe and Zn bioaccessibility, it could be an appropriate Ca source for EF fortification. PMID:25079612

  10. C-reactive protein levels in hereditary angioedema.

    PubMed

    Hofman, Z L M; Relan, A; Hack, C E

    2014-07-01

    Hereditary angioedema (HAE) patients experience recurrent episodes of angioedema attacks that can be painful, disfiguring and even life-threatening. The disorder results from a mutation in the gene that controls the synthesis of C1-inhibitor (C1INH). C1INH is a major regulator of activation of the contact system. It is often assumed that attacks results from uncontrolled local activation of the contact system with subsequent formation of bradykinin. To evaluate the involvement of inflammatory reactions in HAE, we analysed C-reactive protein (CRP) levels. HAE patients included in a clinical database of recombinant human C1-inhibitor (rhC1INH) studies were evaluated. For the current study we analysed CRP levels when patients were asymptomatic, during a clinical attack and in a follow-up period, and correlated these with the clinical manifestations of the attack. Data from 68 HAE patients were analysed and included CRP levels on 273 occasions. While asymptomatic, 20% of the patients analysed had increased CRP. At the onset of the attack (P = 0·049) and during the next 24 h CRP rose significantly (P = 0·002) in patients with an abdominal location, and post-attack levels were significantly higher in these patients than in patients with attacks at other locations (P = 0·034). In conclusion, CRP levels are elevated in a substantial proportion of asymptomatic HAE patients. Levels of CRP increase significantly during an abdominal attack. These data suggest low-grade systemic inflammatory reactions in HAE patients as well as a triggering event for attacks that starts prior to symptom onset. PMID:24588117

  11. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  12. Sputum and BAL Clara cell secretory protein and surfactant protein D levels in asthma.

    PubMed

    Emmanouil, P; Loukides, S; Kostikas, K; Papatheodorou, G; Papaporfyriou, A; Hillas, G; Vamvakaris, I; Triggidou, R; Katafigiotis, P; Kokkini, A; Papiris, S; Koulouris, N; Bakakos, P

    2015-06-01

    Clara cell secretory protein (CC16) is associated with Th2 modulation. Surfactant protein D (SPD) plays an important role in surfactant homeostasis and eosinophil chemotaxis. We measured CC16 and SPD in sputum supernatants of 84 asthmatic patients and 12 healthy controls. In 22 asthmatics, we additionally measured CC16 and SPD levels in BAL and assessed smooth muscle area (SMA), reticular basement membrane (RBM) thickness, and epithelial detachment (ED) in bronchial biopsies. Induced sputum CC16 and SPD were significantly higher in patients with severe asthma (SRA) compared to mild-moderate and healthy controls. BAL CC16 and SPD levels were also higher in SRA compared to mild-moderate asthma. CC16 BAL levels correlated with ED, while SPD BAL levels correlated with SMA and RBM. Severity represented a significant covariate for these associations. CC16 and SPD levels are upregulated in SRA and correlate with remodeling indices, suggesting a possible role of these biomarkers in the remodeling process. PMID:25728058

  13. Honey bee protein atlas at organ-level resolution.

    PubMed

    Chan, Queenie W T; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J

    2013-11-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed. PMID:23878156

  14. Honey bee protein atlas at organ-level resolution

    PubMed Central

    Chan, Queenie W.T.; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J.

    2013-01-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed. PMID:23878156

  15. Auxin acts independently of DELLA proteins in regulating gibberellin levels.

    PubMed

    Reid, James B; Davidson, Sandra E; Ross, John J

    2011-03-01

    Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation, along with environmental and endogenous factors, including other hormones such as the brassinosteroids. The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism, since auxin up-regulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and down regulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA 1. In our recent paper, we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action, since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA 1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level. However, our recent results, in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined. PMID:21358281

  16. Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages*

    PubMed Central

    Karunakaran, Denuja; Kockx, Maaike; Owen, Dylan M.; Burnett, John R.; Jessup, Wendy; Kritharides, Leonard

    2013-01-01

    Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ϵ, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages. PMID:23288845

  17. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    PubMed Central

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  18. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    PubMed

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  19. Microsecond protein dynamics observed at the single-molecule level

    PubMed Central

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-01-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

  20. Atomic-level Snapshot Catches Protein Motor in Action

    SciTech Connect

    2009-01-01

    Using a state-of-the-art protein crystallography beamline at Berkeley Labs Advanced Light Source, researchers have captured a critical action shapshot of an enzyme that is vital to the survival of all biological cells.

  1. 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...

  2. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  3. Methods to alter levels of a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  4. Effect of N-acetylcysteine administration on homocysteine level, oxidative damage to proteins, and levels of iron (Fe) and Fe-related proteins in lead-exposed workers.

    PubMed

    Kasperczyk, Sławomir; Dobrakowski, Michał; Kasperczyk, Aleksandra; Romuk, Ewa; Rykaczewska-Czerwińska, Monika; Pawlas, Natalia; Birkner, Ewa

    2016-09-01

    N-Acetylcysteine (NAC) could be included in protocols designed for the treatment of lead toxicity. Therefore, in this study, we decided to investigate the influence of NAC administration on homocysteine (Hcy) levels, oxidative damage to proteins, and the levels of iron (Fe), transferrin (TRF), and haptoglobin (HPG) in lead (Pb)-exposed workers. The examined population (n = 171) was composed of male employees who worked with Pb. They were randomized into four groups. Workers who were not administered any antioxidants, drugs, vitamins, or dietary supplements were classified as the reference group (n = 49). The remaining three groups consisted of workers who were treated orally with NAC at three different doses (1 × 200, 2 × 200, or 2 × 400 mg) for 12 weeks. After the treatment, blood Pb levels significantly decreased in the groups receiving NAC compared with the reference group. The protein concentration was not affected by NAC administration. In contrast, Hcy levels significantly decreased or showed a strong tendency toward lower values depending on the NAC dose. Levels of the protein carbonyl groups were significantly decreased in all of the groups receiving NAC. Conversely, glutamate dehydrogenase activity was significantly elevated in all of the groups receiving NAC, while the level of protein thiol groups was significantly elevated only in the group receiving 200 mg of NAC. Treatment with NAC did not significantly affect Fe and TRF levels, whereas HPG levels showed a tendency toward lower values. Treatment with NAC normalized the level of Hcy and decreased oxidative stress as measured by the protein carbonyl content; this effect occurred in a dose-dependent manner. Moreover, small doses of NAC elevated the levels of protein thiol groups. Therefore, NAC could be introduced as an alternative therapy for chronic Pb toxicity in humans. PMID:25731901

  5. Several lipid-related gene polymorphisms interact with overweight/obesity to modulate blood pressure levels.

    PubMed

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

    2012-01-01

    Little is known about the interactions of single nucleotide polymorphisms (SNPs) and overweight/obesity on blood pressure levels. The present study was undertaken to detect 10 lipid-related gene SNPs and their interactions with overweight/obesity 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, 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 in 978 normal weight and 751 overweight/obese subjects. The interactions were detected by factorial regression analysis. The genotypes of ACAT-1 AC, LIPC GA and AA, and SCARB1 TT; LDL-R A-A- and LIPC GA; and SCARB1 TT were interacted with overweight/obesity to increase systolic, diastolic blood pressure (SBP, DBP) and pulse pressure (PP) levels; respectively. The genotypes of ACAT-1 CC; ACAT-1 AA and CC were interacted with overweight/obesity to decrease SBP, PP levels (p < 0.01-0.001); respectively. The differences in blood pressure levels between normal weight and overweight/obese subjects might partly result from different interactions of several SNPs and overweight/obesity. PMID:23109900

  6. Several Lipid-Related Gene Polymorphisms Interact with Overweight/Obesity to Modulate Blood Pressure Levels

    PubMed Central

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

    2012-01-01

    Little is known about the interactions of single nucleotide polymorphisms (SNPs) and overweight/obesity on blood pressure levels. The present study was undertaken to detect 10 lipid-related gene SNPs and their interactions with overweight/obesity 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, 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 in 978 normal weight and 751 overweight/obese subjects. The interactions were detected by factorial regression analysis. The genotypes of ACAT-1 AC, LIPC GA and AA, and SCARB1 TT; LDL-R A-A- and LIPC GA; and SCARB1 TT were interacted with overweight/obesity to increase systolic, diastolic blood pressure (SBP, DBP) and pulse pressure (PP) levels; respectively. The genotypes of ACAT-1 CC; ACAT-1 AA and CC were interacted with overweight/obesity to decrease SBP, PP levels (p < 0.01–0.001); respectively. The differences in blood pressure levels between normal weight and overweight/obese subjects might partly result from different interactions of several SNPs and overweight/obesity. PMID:23109900

  7. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  8. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  9. Protein metabolism in growing pigs fed corn or cassava peel based diets containing graded protein levels.

    PubMed

    Tewe, O O

    1985-05-01

    Sixty-four Large White cross Landrace weanling pigs were randomly allotted to eight treatments in a two by four factorial arrangement. The two dietary variables were cassava peel (0 and 40 per cent) and crude protein (20, 15, 10 and 5 per cent). Total serum protein concentration was significantly (P less than 0.01) reduced by protein deficiency and by its interaction with cassava peel. The multiple coefficient of determination (R2) showed that protein intake was the primary factor determining changes in serum protein. R2 values for cyanide intake (independent variable) on serum protein (dependent variable) increased from day 30 to 90 of the trial. Serum urea was increased on the 5 per cent protein diets on days 60 and 90 of the trial. The R2 values for cyanide and protein intake on serum urea concentration increased from day 30 to day 90 of the trial. Serum creatinine increased (P less than 0.05) on the 5 per cent protein diet on day 90 of the trial. The R2 value for the effects of protein intake on serum creatinine was higher than for cyanide intake on days 30 and 90. The results confirm the progressive and pronounced effects of long term cyanide intake on serum nitrogenous metabolites in pigs consuming between 110 and 120 ppm hydrocyanic acid, especially in diets containing 10 per cent or less protein. PMID:2989987

  10. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  11. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis.

    PubMed

    Cui, Huanhuan L; Ditiatkovski, Michael; Kesani, Rajitha; Bobryshev, Yuri V; Liu, Yingying; Geyer, Matthias; Mukhamedova, Nigora; Bukrinsky, Michael; Sviridov, Dmitri

    2014-07-01

    Patients with HIV are at an increased risk of cardiovascular disease. In this study we investigated the effect of Nef, a secreted HIV protein responsible for the impairment of cholesterol efflux, on the development of atherosclerosis in two animal models. ApoE(-/-) mice fed a high-fat diet and C57BL/6 mice fed a high-fat, high-cholesterol diet were injected with recombinant Nef (40 ng/injection) or vehicle, and the effects of Nef on development of atherosclerosis, inflammation, and dyslipidemia were assessed. In apoE(-/-) mice, Nef significantly increased the size of atherosclerotic lesions and caused vessel remodeling. Nef caused elevation of total cholesterol and triglyceride levels in the plasma while reducing high-density lipoprotein cholesterol levels. These changes were accompanied by a reduction of ABCA1 abundance in the liver, but not in the vessels. In C57BL/6 mice, Nef caused a significant number of lipid-laden macrophages presented in adventitia of the vessels; these cells were absent from the vessels of control mice. Nef caused sharp elevations of plasma triglyceride levels and body weight. Taken together, our findings suggest that Nef causes dyslipidemia and accumulation of cholesterol in macrophages within the vessel wall, supporting the role of Nef in pathogenesis of atherosclerosis in HIV-infected patients.-Cui, H. L., Ditiatkovski, M., Kesani, R., Bobryshev, Y. V., Liu, Y., Geyer, M., Mukhamedova, N., Bukrinsky, M., Sviridov, D. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis. PMID:24642731

  12. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis

    PubMed Central

    Cui, Huanhuan L.; Ditiatkovski, Michael; Kesani, Rajitha; Bobryshev, Yuri V.; Liu, Yingying; Geyer, Matthias; Mukhamedova, Nigora; Bukrinsky, Michael; Sviridov, Dmitri

    2014-01-01

    Patients with HIV are at an increased risk of cardiovascular disease. In this study we investigated the effect of Nef, a secreted HIV protein responsible for the impairment of cholesterol efflux, on the development of atherosclerosis in two animal models. ApoE−/− mice fed a high-fat diet and C57BL/6 mice fed a high-fat, high-cholesterol diet were injected with recombinant Nef (40 ng/injection) or vehicle, and the effects of Nef on development of atherosclerosis, inflammation, and dyslipidemia were assessed. In apoE−/− mice, Nef significantly increased the size of atherosclerotic lesions and caused vessel remodeling. Nef caused elevation of total cholesterol and triglyceride levels in the plasma while reducing high-density lipoprotein cholesterol levels. These changes were accompanied by a reduction of ABCA1 abundance in the liver, but not in the vessels. In C57BL/6 mice, Nef caused a significant number of lipid-laden macrophages presented in adventitia of the vessels; these cells were absent from the vessels of control mice. Nef caused sharp elevations of plasma triglyceride levels and body weight. Taken together, our findings suggest that Nef causes dyslipidemia and accumulation of cholesterol in macrophages within the vessel wall, supporting the role of Nef in pathogenesis of atherosclerosis in HIV-infected patients.—Cui, H. L., Ditiatkovski, M., Kesani, R., Bobryshev, Y. V., Liu, Y., Geyer, M., Mukhamedova, N., Bukrinsky, M., Sviridov, D. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis. PMID:24642731

  13. High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

    PubMed

    Chen, W J; Moomaw, J F; Overton, L; Kost, T A; Casey, P J

    1993-05-01

    The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme. PMID:8486655

  14. Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats.

    PubMed

    Taciak, Marcin; Barszcz, Marcin; Tuśnio, Anna; Pastuszewska, Barbara

    2015-01-01

    The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon. PMID:26536028

  15. Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats

    PubMed Central

    Taciak, Marcin; Barszcz, Marcin; Tuśnio, Anna; Pastuszewska, Barbara

    2015-01-01

    The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon. PMID:26536028

  16. Progranulin protein levels are differently regulated in plasma and CSF

    PubMed Central

    Nicholson, Alexandra M.; Finch, NiCole A.; Thomas, Colleen S.; Wojtas, Aleksandra; Rutherford, Nicola J.; Mielke, Michelle M.; Roberts, Rosebud O.; Boeve, Bradley F.; Knopman, David S.; Petersen, Ronald C.

    2014-01-01

    Objective: We aimed to investigate the relationship between plasma and CSF progranulin (PGRN) levels. Methods: Plasma and CSF PGRN were measured in a cohort of 345 subjects from the Mayo Clinic Study of Aging by ELISA. Single nucleotide polymorphism genotyping was performed using TaqMan assays. Associations between PGRN and sex, age at sample collection, diagnosis, single nucleotide polymorphism genotypes (GRN, SORT1, and APOE), and Pittsburgh compound B score were explored separately in CSF and plasma using single variable linear regression models. Pearson partial correlation coefficient was used to estimate the correlation of PGRN in CSF and plasma. Results: Plasma (p = 0.0031) and CSF (p = 0.0044) PGRN significantly increased with age, whereas plasma PGRN levels were 7% lower (p = 0.0025) and CSF PGRN levels 5% higher (p = 0.0024) in male compared with female participants. Correcting for age and sex, higher plasma PGRN was associated with higher CSF PGRN (partial r = 0.17, p = 0.004). In plasma, both rs5848 (GRN; p = 0.002) and rs646776 (SORT1; p = 3.56E-7) were associated with PGRN, while only rs5848 showed highly significant association in CSF (p = 5.59E-14). Age, sex, rs5848 genotype, and plasma PGRN together accounted for only 18% of the variability observed in CSF PGRN. Conclusions: While some correlation exists between plasma and CSF PGRN, age, sex, and genetic factors differently affect PGRN levels. Therefore, caution should be taken when using plasma PGRN to predict PGRN changes in the brain. These findings further highlight that plasma PGRN levels may not accurately predict clinical features or response to future frontotemporal lobar degeneration therapies. PMID:24771538

  17. Calcium, phosphorus and protein levels as factors in the distribution of the pheasant

    USGS Publications Warehouse

    Dale, F.H.; DeWitt, J.B.

    1958-01-01

    Summary of work on pheasant nutrition conducted since 1949 at the Patuxent Research Refuge. Pheasant chicks fed experimental diets failed to develop normally on protein levels of 15 and 18%. With 22% protein they grew at a reduced rate as compared to those on 28%. Protein level of the reproductive diet was shown to be important; low production of eggs and young resulted from levels below 25%. Calcium was found to be even more critical than protein level for reproduction; birds on a winter diet that furnished 145 mg./kg. per day had poor reproductive success the following spring. About 600 mg./kg. of Ca per day was necessary in the reproduction diet. Birds on an intermediate level of Ca (about 0.5% of diet) showed evidence of cumulative deficiency. It was concluded that pheasants receiving levels of Ca no higher than 0.5% in nature might display 'straggling failure' such as has been observed in several midwestern areas.

  18. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels

    PubMed Central

    Schraivogel, Daniel; Schindler, Susann G.; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-01-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels. PMID:26170235

  19. A local average connectivity-based method for identifying essential proteins from the network level.

    PubMed

    Li, Min; Wang, Jianxin; Chen, Xiang; Wang, Huan; Pan, Yi

    2011-06-01

    Identifying essential proteins is very important for understanding the minimal requirements of cellular survival and development. Fast growth in the amount of available protein-protein interactions has produced unprecedented opportunities for detecting protein essentiality from the network level. Essential proteins have been found to be more abundant among those highly connected proteins. However, there exist a number of highly connected proteins which are not essential. By analyzing these proteins, we find that few of their neighbors interact with each other. Thus, we propose a new local method, named LAC, to determine a protein's essentiality by evaluating the relationship between a protein and its neighbors. The performance of LAC is validated based on the yeast protein interaction networks obtained from two different databases: DIP and BioGRID. The experimental results of the two networks show that the number of essential proteins predicted by LAC clearly exceeds that explored by Degree Centrality (DC). More over, LAC is also compared with other seven measures of protein centrality (Neighborhood Component (DMNC), Betweenness Centrality (BC), Closeness Centrality (CC), Bottle Neck (BN), Information Centrality (IC), Eigenvector Centrality (EC), and Subgraph Centrality (SC)) in identifying essential proteins. The comparison results based on the validations of sensitivity, specificity, F-measure, positive predictive value, negative predictive value, and accuracy consistently show that LAC outweighs these seven previous methods. PMID:21704260

  20. A Bayesian framework for cell-level protein network analysis for multivariate proteomics image data

    NASA Astrophysics Data System (ADS)

    Kovacheva, Violet N.; Sirinukunwattana, Korsuk; Rajpoot, Nasir M.

    2014-03-01

    The recent development of multivariate imaging techniques, such as the Toponome Imaging System (TIS), has facilitated the analysis of multiple co-localisation of proteins. This could hold the key to understanding complex phenomena such as protein-protein interaction in cancer. In this paper, we propose a Bayesian framework for cell level network analysis allowing the identification of several protein pairs having significantly higher co-expression levels in cancerous tissue samples when compared to normal colon tissue. It involves segmenting the DAPI-labeled image into cells and determining the cell phenotypes according to their protein-protein dependence profile. The cells are phenotyped using Gaussian Bayesian hierarchical clustering (GBHC) after feature selection is performed. The phenotypes are then analysed using Difference in Sums of Weighted cO-dependence Profiles (DiSWOP), which detects differences in the co-expression patterns of protein pairs. We demonstrate that the pairs highlighted by the proposed framework have high concordance with recent results using a different phenotyping method. This demonstrates that the results are independent of the clustering method used. In addition, the highlighted protein pairs are further analysed via protein interaction pathway databases and by considering the localization of high protein-protein dependence within individual samples. This suggests that the proposed approach could identify potentially functional protein complexes active in cancer progression and cell differentiation.

  1. Multi-level learning: improving the prediction of protein, domain and residue interactions by allowing information flow between levels

    PubMed Central

    Yip, Kevin Y; Kim, Philip M; McDermott, Drew; Gerstein, Mark

    2009-01-01

    Background Proteins interact through specific binding interfaces that contain many residues in domains. Protein interactions thus occur on three different levels of a concept hierarchy: whole-proteins, domains, and residues. Each level offers a distinct and complementary set of features for computationally predicting interactions, including functional genomic features of whole proteins, evolutionary features of domain families and physical-chemical features of individual residues. The predictions at each level could benefit from using the features at all three levels. However, it is not trivial as the features are provided at different granularity. Results To link up the predictions at the three levels, we propose a multi-level machine-learning framework that allows for explicit information flow between the levels. We demonstrate, using representative yeast interaction networks, that our algorithm is able to utilize complementary feature sets to make more accurate predictions at the three levels than when the three problems are approached independently. To facilitate application of our multi-level learning framework, we discuss three key aspects of multi-level learning and the corresponding design choices that we have made in the implementation of a concrete learning algorithm. 1) Architecture of information flow: we show the greater flexibility of bidirectional flow over independent levels and unidirectional flow; 2) Coupling mechanism of the different levels: We show how this can be accomplished via augmenting the training sets at each level, and discuss the prevention of error propagation between different levels by means of soft coupling; 3) Sparseness of data: We show that the multi-level framework compounds data sparsity issues, and discuss how this can be dealt with by building local models in information-rich parts of the data. Our proof-of-concept learning algorithm demonstrates the advantage of combining levels, and opens up opportunities for further

  2. Effects of dietary protein level on growth and utilization of protein and energy by juvenile mangrove red snapper (Lutjanus argentimaculatus)

    NASA Astrophysics Data System (ADS)

    Ghulam, Abbas; Khalid, Jamil; Rukhsana, Akhtar; Lin, Hong

    2005-01-01

    A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ-1 to 20.03 mg protein kJ-1. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m3 seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=-1.6295 + 0.1114 X 2,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein

  3. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  4. Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells[S

    PubMed Central

    Röhrl, Clemens; Eigner, Karin; Winter, Katharina; Korbelius, Melanie; Obrowsky, Sascha; Kratky, Dagmar; Kovacs, Werner J.; Stangl, Herbert

    2014-01-01

    Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function. PMID:24179149

  5. Pitavastatin Differentially Modulates MicroRNA-Associated Cholesterol Transport Proteins in Macrophages

    PubMed Central

    Moran, George; Sun, Tao; Gotto, Antonio M.; Hajjar, David P.

    2016-01-01

    There is emerging evidence identifying microRNAs (miRNAs) as mediators of statin-induced cholesterol efflux, notably through the ATP-binding cassette transporter A1 (ABCA1) in macrophages. The objective of this study was to assess the impact of an HMG-CoA reductase inhibitor, pitavastatin, on macrophage miRNAs in the presence and absence of oxidized-LDL, a hallmark of a pro-atherogenic milieu. Treatment of human THP-1 cells with pitavastatin prevented the oxLDL-mediated suppression of miR-33a, -33b and -758 mRNA in these cells, an effect which was not uniquely attributable to induction of SREBP2. Induction of ABCA1 mRNA and protein by oxLDL was inhibited (30%) by pitavastatin, while oxLDL or pitavastatin alone significantly induced and repressed ABCA1 expression, respectively. These findings are consistent with previous reports in macrophages. miRNA profiling was also performed using a miRNA array. We identified specific miRNAs which were up-regulated (122) and down-regulated (107) in THP-1 cells treated with oxLDL plus pitavastatin versus oxLDL alone, indicating distinct regulatory networks in these cells. Moreover, several of the differentially expressed miRNAs identified are functionally associated with cholesterol trafficking (six miRNAs in cells treated with oxLDL versus oxLDL plus pitavastatin). Our findings indicate that pitavastatin can differentially modulate miRNA in the presence of oxLDL; and, our results provide evidence that the net effect on cholesterol homeostasis is mediated by a network of miRNAs. PMID:27415822

  6. Pitavastatin Differentially Modulates MicroRNA-Associated Cholesterol Transport Proteins in Macrophages.

    PubMed

    Zhang, Haijun; Lamon, Brian D; Moran, George; Sun, Tao; Gotto, Antonio M; Hajjar, David P

    2016-01-01

    There is emerging evidence identifying microRNAs (miRNAs) as mediators of statin-induced cholesterol efflux, notably through the ATP-binding cassette transporter A1 (ABCA1) in macrophages. The objective of this study was to assess the impact of an HMG-CoA reductase inhibitor, pitavastatin, on macrophage miRNAs in the presence and absence of oxidized-LDL, a hallmark of a pro-atherogenic milieu. Treatment of human THP-1 cells with pitavastatin prevented the oxLDL-mediated suppression of miR-33a, -33b and -758 mRNA in these cells, an effect which was not uniquely attributable to induction of SREBP2. Induction of ABCA1 mRNA and protein by oxLDL was inhibited (30%) by pitavastatin, while oxLDL or pitavastatin alone significantly induced and repressed ABCA1 expression, respectively. These findings are consistent with previous reports in macrophages. miRNA profiling was also performed using a miRNA array. We identified specific miRNAs which were up-regulated (122) and down-regulated (107) in THP-1 cells treated with oxLDL plus pitavastatin versus oxLDL alone, indicating distinct regulatory networks in these cells. Moreover, several of the differentially expressed miRNAs identified are functionally associated with cholesterol trafficking (six miRNAs in cells treated with oxLDL versus oxLDL plus pitavastatin). Our findings indicate that pitavastatin can differentially modulate miRNA in the presence of oxLDL; and, our results provide evidence that the net effect on cholesterol homeostasis is mediated by a network of miRNAs. PMID:27415822

  7. Systematic Protein Level Regulation via Degradation Machinery Induced by Genotoxic Drugs.

    PubMed

    Kume, Kohei; Ishida, Kazushige; Ikeda, Miyuki; Takemoto, Kazuhiro; Shimura, Tsutomu; Young, Lynn; Nishizuka, Satoshi S

    2016-01-01

    In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using "reverse-phase" protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (dCor). Twenty-two percent of the pairs showed dCor > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high dCor, the average dCor of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs. PMID:26625007

  8. Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2

    PubMed Central

    Herrero, Eva; Thorpe, Peter H.

    2016-01-01

    The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore. PMID:26891228

  9. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels. PMID:27410252

  10. A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

    PubMed Central

    Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

    2013-01-01

    The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

  11. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

    PubMed Central

    Tatham, Amy L.; Crabtree, Mark J.; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J.; Channon, Keith M.

    2009-01-01

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r2 = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r2 = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  12. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  13. 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

  14. Identification and Validation of Genetic Variants that Influence Transcription Factor and Cell Signaling Protein Levels

    PubMed Central

    Hause, Ronald J.; Stark, Amy L.; Antao, Nirav N.; Gorsic, Lidija K.; Chung, Sophie H.; Brown, Christopher D.; Wong, Shan S.; Gill, Daniel F.; Myers, Jamie L.; To, Lida Anita; White, Kevin P.; Dolan, M. Eileen; Jones, Richard Baker

    2014-01-01

    Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression. PMID:25087611

  15. Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

    PubMed

    Chino, Ayako; Makanae, Koji; Moriya, Hisao

    2013-01-01

    We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the "genetic tug-of-war" (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted. PMID:24019917

  16. Optimality and evolutionary tuning of the expression level of a protein.

    PubMed

    Dekel, Erez; Alon, Uri

    2005-07-28

    Different proteins have different expression levels. It is unclear to what extent these expression levels are optimized to their environment. Evolutionary theories suggest that protein expression levels maximize fitness, but the fitness as a function of protein level has seldom been directly measured. To address this, we studied the lac system of Escherichia coli, which allows the cell to use the sugar lactose for growth. We experimentally measured the growth burden due to production and maintenance of the Lac proteins (cost), as well as the growth advantage (benefit) conferred by the Lac proteins when lactose is present. The fitness function, given by the difference between the benefit and the cost, predicts that for each lactose environment there exists an optimal Lac expression level that maximizes growth rate. We then performed serial dilution evolution experiments at different lactose concentrations. In a few hundred generations, cells evolved to reach the predicted optimal expression levels. Thus, protein expression from the lac operon seems to be a solution of a cost-benefit optimization problem, and can be rapidly tuned by evolution to function optimally in new environments. PMID:16049495

  17. High level expression of peptides and proteins using cytochrome b5 as a fusion host.

    PubMed

    Mitra, Ashima; Chakrabarti, Kalyan Sundar; Shahul Hameed, M S; Srinivas, Kalyan V; Senthil Kumar, Ganesan; Sarma, Siddhartha P

    2005-05-01

    A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa). PMID:15802225

  18. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

    PubMed Central

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

    2015-01-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

  19. Monitoring protein phosphatase 1 isoform levels as a marker for cellular stress.

    PubMed

    Amador, Fátima Camões; Henriques, Ana Gabriela; da Cruz E Silva, Odete A B; da Cruz E Silva, Edgar F

    2004-01-01

    Reversible protein phosphorylation is a central mechanism regulating many biological functions, and abnormal protein phosphorylation can have a devastating impact on cellular control mechanisms, including a contributing role in neurodegenerative processes. Hence, many promising novel drug development strategies involve targeting protein phosphorylation systems. In this study, we demonstrate that various cellular stresses relevant to neurodegeneration can specifically affect the protein expression levels of protein phosphatase 1 (PP1). PP1 levels were altered upon exposure of PC12 and COS-1 cells to aluminium, Abeta peptides, sodium azide, and even heat shock. Particularly interesting, given PP1's involvement in aging and neurodegeneration, was the consistent decrease in PP1gamma(1) levels in response to stress agents. In fact, alterations in the expression levels of PP1 appear to correspond to an early response of stress induction, that is, before alterations in heat shock proteins can be detected. Our data suggest that monitoring PP1 isoform expression could constitute a useful diagnostic tool for cellular stress, possibly even neurodegeneration. Additionally, our results strengthen the rationale for signal transduction therapeutics and indicate that altering the specific activity of PP1 either directly or by targeting its regulatory proteins may be a useful therapeutic development strategy for the future. PMID:15113600

  20. Increased levels of hyper-stable protein aggregates in plasma of older adults.

    PubMed

    Xia, Ke; Trasatti, Hannah; Wymer, James P; Colón, Wilfredo

    2016-06-01

    Proteins that misfold into hyper-stable/degradation-resistant species during aging may accumulate and disrupt protein homeostasis (i.e., proteostasis), thereby posing a survival risk to any organism. Using the method diagonal two-dimensional (D2D) SDS-PAGE, which separates hyper-stable SDS-resistant proteins at a proteomics level, we analyzed the plasma of healthy young (<30 years) and older (60-80 years) adults. We discovered the presence of soluble SDS-resistant protein aggregates in the plasma of older adults, but found significantly lower levels in the plasma of young adults. We identified the inflammation-related chaperone protein haptoglobin as the main component of the hyper-stable aggregates. This observation is consistent with the growing link between accumulations of protein aggregates and aging across many organisms. It is plausible higher amounts of SDS-resistant protein aggregates in the plasma of older adults may reflect a compromise in proteostasis that may potentially indicate cellular aging and/or disease risk. The results of this study have implications for further understanding the link between aging and the accumulation of protein aggregates, as well as potential for the development of aging-related biomarkers. More broadly, this novel application of D2D SDS-PAGE may be used to identify, quantify, and characterize the degradation-resistant protein aggregates in human plasma or any biological system. PMID:27179971

  1. System-level comparison of protein-protein interactions between viruses and the human type I interferon system network.

    PubMed

    Navratil, V; de Chassey, B; Meyniel, L; Pradezynski, F; André, P; Rabourdin-Combe, C; Lotteau, V

    2010-07-01

    Innate immunity has evolved complex molecular pathways to protect organisms from viral infections. One pivotal line of cellular defense is the induction of the antiviral effect of interferon. To circumvent this primary response and achieve their own replication, viruses have developed complex molecular strategies. Here, we provide a systems-level study of the human type I interferon system subversion by the viral proteome, by reconstructing the underlying protein-protein interaction network. At this network level, viruses establish a massive and a gradual attack, from receptors to transcription factors, by interacting preferentially with highly connected and central proteins as well as interferon-induced proteins. We also demonstrate that viruses significantly target 22% of the proteins directly interacting with the type I interferon system network, suggesting the relevance of our network-based method to identify new candidates involved in the regulation of the antiviral response. Finally, based on the comparative analysis of interactome profiles across four viral families, we provide evidence of common and differential targeting strategies. PMID:20459142

  2. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry.

    PubMed

    Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

    2009-07-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg 0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weight 0.78 g) reared at 15 degrees C for 31 feeding d. The Lys requirements were estimated based on the relationships between weight, protein, and Lys gains (g/kg 0.75 per d) and Lys concentration (g/kg DM or g/100 g protein) or Lys intake (g/kg 0.75 per d), using the broken-line model (BLM) and the non-linear four-parameter saturation kinetics model (SKM-4). Both the model and the response criterion chosen markedly impacted the relative Lys requirement. The relative Lys requirement for Lys gain of rainbow trout estimated with the BLM (and SKM-4 at 90 % of the maximum response) increased from 16.8 (19.6) g/kg DM at a low protein level to 23.4 (24.5) g/kg DM at a high protein level. However, the dietary protein content affected neither the absolute Lys requirement nor the relative Lys requirement expressed as g Lys/100 g protein nor the Lys requirement for maintenance (21 mg Lys/kg 0.75 per d). PMID:19138439

  3. C-reactive protein gene variants: independent association with late-life depression and circulating protein levels.

    PubMed

    Ancelin, M-L; Farré, A; Carrière, I; Ritchie, K; Chaudieu, I; Ryan, J

    2015-01-01

    C-reactive protein (CRP) is a heritable biomarker of systemic inflammation that is commonly elevated in depressed patients. Variants in the CRP gene that influence protein levels could thus be associated with depression but this has seldom been examined, especially in the elderly. Depression was assessed in 990 people aged at least 65 years as part of the ESPRIT study. A clinical level of depression (DEP) was defined as having a score of ⩾16 on The Center for Epidemiologic Studies Depression scale or a diagnosis of current major depression based on the Mini-International Neuropsychiatric Interview and according to Diagnostic and Statistical Manual of Mental Disorders-IV criteria. Five single-nucleotide polymorphisms spanning the CRP gene were genotyped, and circulating levels of high-sensitivity CRP were determined. Multivariable analyses adjusted for socio-demographic characteristics, smoking, ischemic pathologies, cognitive impairment and inflammation-related chronic pathologies. The minor alleles of rs1130864 and rs1417938 were associated with a decreased risk of depression in women at Bonferroni-corrected significance levels (P=0.002). CRP gene variants were associated with serum levels in a gender-specific manner, but only rs1205 was found to be nominally associated with both an increased risk of DEP and lower circulating CRP levels in women. Variants of the CRP gene thus influence circulating CRP levels and appear as independent susceptibility factors for late-life depression. PMID:25603415

  4. EFFECTS OF VARYING DIETARY PROTEIN AND ENERGY LEVELS ON THE PRODUCTION OF LACTATING DAIRY COWS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty-five multiparous and 18 primiparous Holstein cows averaging 41 kg/d of milk were fed three levels of crude protein (CP), each at three levels of neutral detergent fiber (NDF), to identify optimal dietary CP and energy. Cows were blocked by lactation and days in milk into seven groups of nine a...

  5. Burial Level Change Defines a High Energetic Relevance for Protein Binding Interfaces.

    PubMed

    Li, Zhenhua; He, Ying; Wong, Limsoon; Li, Jinyan

    2015-01-01

    Protein-protein interfaces defined through atomic contact or solvent accessibility change are widely adopted in structural biology studies. But, these definitions cannot precisely capture energetically important regions at protein interfaces. The burial depth of an atom in a protein is related to the atom's energy. This work investigates how closely the change in burial level of an atom/residue upon complexation is related to the binding. Burial level change is different from burial level itself. An atom deeply buried in a monomer with a high burial level may not change its burial level after an interaction and it may have little burial level change. We hypothesize that an interface is a region of residues all undergoing burial level changes after interaction. By this definition, an interface can be decomposed into an onion-like structure according to the burial level change extent. We found that our defined interfaces cover energetically important residues more precisely, and that the binding free energy of an interface is distributed progressively from the outermost layer to the core. These observations are used to predict binding hot spots. Our approach's F-measure performance on a benchmark dataset of alanine mutagenesis residues is much superior or similar to those by complicated energy modeling or machine learning approaches. PMID:26357227

  6. Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility.

    PubMed

    Kratz, Ewa Maria; Piwowar, Agnieszka; Zeman, Michal; Stebelová, Katarína; Thalhammer, Theresia

    2016-03-01

    Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n=37), theratozoospermia (n=29) and fertile controls (normozoospermia, n=37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P<0.0001) and theratozoospermic (P<0.0001) patients versus fertile men, and correlated negatively (r=-0.33, P=0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P=0.0022) and the control group (P=0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P=0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative-antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential. PMID:25218686

  7. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently. PMID:15607223

  8. Exceptionally high heterologous protein levels in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Angenon, Geert; Depicker, Ann

    2003-01-01

    Seeds are concentrated sources of protein and thus may be ideal 'bioreactors' for the production of heterologous proteins. For this application, strong seed-specific expression signals are required. A set of expression cassettes were designed using 5' and 3' regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) from Phaseolus vulgaris, and evaluated for the production of heterologous proteins in dicotyledonous plant species. A murine single-chain variable fragment (scFv) was chosen as model protein because of the current industrial interest to produce antibodies and derived fragments in crops. Because the highest scFv accumulation in seed had previously been achieved in the endoplasmic reticulum (ER), the scFv-encoding sequence was provided with signal sequences for accumulation in the ER. Transgenic Arabidopsis seed stocks, expressing the scFv under control of the 35S promoter, contained scFv accumulation levels in the range of 1% of total soluble protein (TSP). However, the seed storage promoter constructs boosted the scFv to exceptionally high levels. Maximum scFv levels were obtained in homozygous seed stocks, being 12.5% of TSP under control of the arc5-I regulatory sequences and even up to 36.5% of TSP upon replacing the arc5-I promoter by the beta-phaseolin promoter of Phaseolus vulgaris. Even at such very high levels, the scFv proteins retain their full antigen-binding activity. Moreover, the presence of very high scFv levels has only minory effects on seed germination and no effect on seed production. These results demonstrate that the expression levels of arcelin 5-I and beta-phaseolin seed storage protein genes can be transferred to heterologous proteins, giving exceptionally high levels of heterologous proteins, which can be of great value for the molecular farming industry by raising production yield and lowering bio-mass production and purification costs. Finally, the feasibility of heterologous protein production using the

  9. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    PubMed Central

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  10. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    PubMed

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai

    2016-08-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  11. Controlled levels of protein modification through a chromatography-mediated bioconjugation

    DOE PAGESBeta

    Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; Francis, Matthew B.

    2015-02-27

    Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety usingmore » an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.« less

  12. Controlled levels of protein modification through a chromatography-mediated bioconjugation

    SciTech Connect

    Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; Francis, Matthew B.

    2015-02-27

    Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.

  13. A two level hierarchical model of protein retention in ion exchange chromatography.

    PubMed

    Salvalaglio, Matteo; Paloni, Matteo; Guelat, Bertrand; Morbidelli, Massimo; Cavallotti, Carlo

    2015-09-11

    Predicting protein retention in ion exchange chromatography (IEX) from first principles is a fascinating perspective. In this work a two level hierarchical modeling strategy is proposed in order to calculate protein retention factors. Model predictions are tested against experimental data measured for Lysozyme and Chymotrypsinogen A in IEX columns as a function of ionic strength and pH. At the highest level of accuracy Molecular Dynamics (MD) simulations in explicit water are used to determine the interaction free energy between each of the two proteins and the IEX stationary phase for a reference pH and ionic strength. At a lower level of accuracy a linear response model based on an implicit treatment of solvation and adopting a static protein structure is used to calculate interaction free energies for the full range of pHs and ionic strengths considered. A scaling coefficient, determined comparing MD and implicit solvent simulations, is then introduced in order to correct the linear response model for errors induced by the adoption of a static protein structure. The calculated free energies are then used to compute protein retention factors, which can be directly compared with experimental data. The possibility to introduce a third level of accuracy is explored testing the predictions of a semiempirical model. A quantitative agreement between the predicted and measured protein retention factors is obtained using the coupled MD-linear response models, supporting the reliability of the proposed approach. The model allows quantifying the electrostatic, van der Waals, and conformational contributions to the interaction free energies. A good agreement between experiments and model is obtained also using the semiempirical model that, although requiring parameterization over higher level models or experimental data, proves to be useful in order to rapidly determine protein retention factors across wide pH and ionic strength ranges as it is computationally inexpensive

  14. BCRP protein levels do not differ regionally in adult human livers, but decline in the elderly.

    PubMed

    Riches, Zoe; Abanda, Ngu; Collier, Abby C

    2015-12-01

    The aim of this study was to characterize the ontogeny and variability of the BCRP (ABCG2) transporter in healthy human liver. Levels of BCRP mRNA and protein were determined with q-RT-PCR and western blot in a cohort of 87 human livers aged from 7 days to 87 years. A study of the regional expression of BCRP within adult livers was also performed in a nested cohort of 14 individuals with multiple samples per person collected from pre-selected sites. Levels of BCRP mRNA were not significantly different at any age, but protein levels for BCRP were lower in the elderly compared with adults (p < 0.001) and children (p < 0.05). The intra-liver levels of BCRP protein ranged approximately 6.5-fold and inter-liver BCRP protein varied 8.5-fold in the cohort. No differences in BCRP mRNA or protein were observed with sex or ethnicity, although higher levels of BCRP mRNA were observed in livers from overweight individuals (Body Mass Index ≥ 25-29.9) as compared to underweight or ideal weight individuals. There were no differences in the levels of BCRP mRNA or protein in different regions of the large lobe (n = 3 regions), small lobe (n = 3 regions), directly adjacent to the portal vein or directly adjacent to the common bile duct. This indicates that BCRP researchers can source tissue from all parts of the adult liver without artificial bias in their results. Lower BCRP protein expression in the elderly may be associated with compromised xeno- and endobiotic transport. PMID:26462791

  15. Reduced activity-dependent protein levels in a mouse model of the fragile X premutation.

    PubMed

    von Leden, Ramona E; Curley, Lindsey C; Greenberg, Gian D; Hunsaker, Michael R; Willemsen, Rob; Berman, Robert F

    2014-03-01

    Environmental enrichment results in increased levels of Fmrp in brain and increased dendritic complexity. The present experiment evaluated activity-dependent increases in Fmrp levels in the motor cortex in response to training on a skilled forelimb reaching task in the CGG KI mouse model of the fragile X premutation. Fmrp, Arc, and c-Fos protein levels were quantified by Western blot in the contralateral motor cortex of mice following training to reach for sucrose pellets with a non-preferred paw and compared to levels in the ipsilateral motor cortex. After training, all mice showed increases in Fmrp, Arc, and c-Fos protein levels in the contralateral compared to the ipsilateral hemisphere; however, the increase in CGG KI mice was less than wildtype mice. Increases in Fmrp and Arc proteins scaled with learning, whereas this relationship was not observed with the c-Fos levels. These data suggest the possibility that reduced levels of activity-dependent proteins associated with synaptic plasticity such as Fmrp and Arc may contribute to the neurocognitive phenotype reported in the CGG KI mice and the fragile X premutation. PMID:24462720

  16. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    PubMed Central

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  17. AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

    PubMed

    Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V

    2016-10-01

    Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins. PMID:27451365

  18. Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

    PubMed Central

    Reuter, Christopher J.; Uthandi, Sivakumar; Puentes, Jose A.; Maupin-Furlow, Julie A.

    2010-01-01

    Proteolysis is important not only to cell physiology but also to the successful development of biocatalysts. While a wide-variety of signals are known to trigger protein degradation in bacteria and eukaryotes, these mechanisms are poorly understood in archaea, known for their ability to withstand harsh conditions. Here we present a systematic study in which single C-terminal amino acid residues were added to a reporter protein and shown to influence its levels in an archaeal cell. All 20 amino acid residues were examined for their impact on protein levels, using the reporter protein soluble modified red-shifted GFP (smRS-GFP) expressed in the haloarchaeon Haloferax volcanii as a model system. Addition of hydrophobic residues, including Leu, Cys, Met, Phe, Ala, Tyr, Ile and Val, gave the most pronounced reduction in smRS-GFP levels compared with the addition of either neutral or charged hydrophilic residues. In contrast to the altered protein levels, the C-terminal alterations had no influence on smRS-GFP-specific transcript levels, thus revealing that the effect is post-transcriptional. PMID:19850616

  19. Protein Consumption and the Elderly: What Is the Optimal Level of Intake?

    PubMed Central

    Baum, Jamie I.; Kim, Il-Young; Wolfe, Robert R.

    2016-01-01

    Maintaining independence, quality of life, and health is crucial for elderly adults. One of the major threats to living independently is the loss of muscle mass, strength, and function that progressively occurs with aging, known as sarcopenia. Several studies have identified protein (especially the essential amino acids) as a key nutrient for muscle health in elderly adults. Elderly adults are less responsive to the anabolic stimulus of low doses of amino acid intake compared to younger individuals. However, this lack of responsiveness in elderly adults can be overcome with higher levels of protein (or essential amino acid) consumption. The requirement for a larger dose of protein to generate responses in elderly adults similar to the responses in younger adults provides the support for a beneficial effect of increased protein in older populations. The purpose of this review is to present the current evidence related to dietary protein intake and muscle health in elderly adults. PMID:27338461

  20. Effect of dietary protein levels on sex hormones in growing male rats kept under constant darkness.

    PubMed

    Hanai, Miho; Esashi, Takatoshi

    2012-01-01

    The purpose of this experiment was to clarify the effects of dietary protein levels on the gonadal development and sex hormones in male rats kept under constant darkness as a model of disturbed daily rhythm. Four-week-old male rats (Fischer 344 strain) were kept under constant darkness or normal lighting (12-h light/dark cycle). Two kinds of experimental diet were prepared, one with low dietary protein levels (9% casein) and one with normal levels (18% casein). Harper mineral mixture and Panvitan were used as mineral and vitamin sources, respectively. After 4 weeks, gonadal weight, serum testosterone, and other hormone contents were evaluated. The gonadal weight in the constant darkness groups (D-groups) was lower than that in the normal lighting groups (N-groups). Although the low-protein diet in the D-groups significantly reduced gonadal weight, the normal-protein diet mitigated the reduction of gonadal weight in rats kept under constant darkness. Serum testosterone and androstenedione concentrations were lower in D-group rats fed the low-protein diet. There were no effects of lighting condition or protein levels on serum luteinizing hormone (LH), follicle- stimulating hormone (FSH), or progesterone concentrations. These results indicate that the suppression of gonadal development in D-group rats fed the low-protein diet was caused by low testosterone, which we attribute to the inhibition of synthesized androstenedione, a precursor of testosterone. The present study showed that constant darkness and the low- protein diet inhibited the synthetic pathway from progesterone to androstenedione. PMID:23095819

  1. Expression of fas protein on CD4+T cells irradiated by low level He-Ne

    NASA Astrophysics Data System (ADS)

    Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

    2005-07-01

    Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2(60 minutes)and 45.84J/cm2(120minutes), the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

  2. Protein Kinase C Is Involved in the Induction of ATP-Binding Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor Agonist in Human Macrophages.

    PubMed

    Huwait, Etimad A; Singh, Nishi N; Michael, Daryn R; Davies, Thomas S; Moss, Joe W E; Ramji, Dipak P

    2015-09-01

    The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have, therefore, investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies, therefore, reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages. PMID:25752819

  3. Acute effect of protein or carbohydrate breakfasts on human cerebrospinal fluid monoamine precursor and metabolite levels.

    PubMed

    Teff, K L; Young, S N; Marchand, L; Botez, M I

    1989-01-01

    Patients with normal pressure hydrocephalus who had three lumbar punctures during 1 week ingested either water, a protein breakfast, or a carbohydrate breakfast 2.5 h before each of the lumbar punctures. The CSF was analyzed for biogenic amine precursors and metabolites. The protein meal raised CSF tyrosine levels, a finding consistent with animal data, but did not alter those of tryptophan or any of the biogenic amine metabolites. The carbohydrate meal increased CSF 3-methoxy-4-hydroxyphenylethylene glycol, an unexplained finding. The carbohydrate meal did not affect CSF tryptophan, tyrosine, 5-hydroxyindoleacetic acid, or homovanillic acid. Our results support the idea that in humans protein or carbohydrate meals do not alter plasma amino acid levels sufficiently to cause appreciable changes in CNS tryptophan levels or 5-hydroxytryptamine synthesis. PMID:2462018

  4. Differential Acute and Chronic Effects of Leptin on Hypothalamic Astrocyte Morphology and Synaptic Protein Levels

    PubMed Central

    García-Cáceres, Cristina; Fuente-Martín, Esther; Burgos-Ramos, Emma; Granado, Miriam; Frago, Laura M.; Barrios, Vicente; Horvath, Tamas

    2011-01-01

    Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance. PMID:21343257

  5. Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

    PubMed

    Lee, Irene; Berdis, Anthony J

    2016-01-01

    Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:26277093

  6. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  7. Ground level environmental protein concentrations in various ecuadorian environments: potential uses of aerosolized protein for ecological research

    USGS Publications Warehouse

    Staton, Sarah J.R.; Woodward, Andrea; Castillo, Josemar A.; Swing, Kelly; Hayes, Mark A.

    2014-01-01

    Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m3, highly altered 0.07 ± 0.05 μg/m3, regrowth transitional 0.17 ± 0.06 μg/m3, and suburban deforested 0.09 ± 0.04 μg/m3. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m3 and wet season 0.10 ± 0.04 μg/m3), environmental fragmentation (buffered 0.19 ± 0.06 μg/m3 and edge 0.15 ± 0.06 μg/m3), and sampling height (ground level 0.32 ± 0.09 μg/m3 and 10 m 0.24 ± 0.04 μg/m3) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r2 = 0.801), net primary production (NPP) (r2 = 0.827), leaf area index (LAI) (r2 = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated

  8. Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

    PubMed

    Jablonski, Joseph E; Fu, Tong-Jen; Jackson, Lauren S; Gendel, Steven M

    2010-01-01

    Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil. PMID:20334183

  9. 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

  10. 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

  11. Levels of major proteins of Escherichia coli during growth at different temperatures.

    PubMed Central

    Herendeen, S L; VanBogelen, R A; Neidhardt, F C

    1979-01-01

    The adaptation of Escherichia coli B/r to temperature was studied by measuring the levels of 133 proteins (comprising 70% of the cell's protein mass) during balanced growth in rich medium at seven temperatures from 13.5 to 46 degrees C. The growth rate of this strain in either rich or minimal medium varies as a simple function of temperature with an Arrhenius constant of approximately 13,500 cal (ca. 56,500 J) per mol from 23 to 37 degrees C, the so-called normal range; above and below this range the growth rate decreases sharply. Analysis of the detailed results indicates that (i) metabolic coordination within the normal (Arrhenius) range is largely achieved by modulation of enzyme activity rather than amount; (ii) the restricted growth that occurs outside this range is accompanied by marked changes in the levels of most of these proteins; (iii) a few proteins are thermometer-like in varying simply with temperature over the whole temperature range irrespective of the influence of temperature on cell growth; and (iv) the temperature response of half of the proteins can be predicted from current information on their metabolic role or from their variation in level in different media at 37 degrees C. PMID:156716

  12. Protein Fibrillar Nanopolymers: Molecular-Level Insights into Their Structural, Physical and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.

    2015-09-01

    Amyloid fibrils represent a generic class of mechanically strong and stable biomaterials with extremely advantageous properties. Although amyloids were initially associated only with severe neurological disorders, the role of these structures nowadays is shifting from health debilitating to highly beneficial both in biomedical and technological aspects. Intensive involvement of fibrillar assemblies into the wide range of pathogenic and functional processes strongly necessitate the molecular level characterization of the structural, physical and elastic features of protein nanofibrils. In the present contribution, we made an attempt to highlight the up-to-date progress in the understanding of amyloid properties from the polymer physics standpoint. The fundamental insights into protein fibril behavior are essential not only for development of therapeutic strategies to combat the protein misfolding disorders but also for rational and precise design of novel biodegradable protein-based nanopolymers.

  13. Insulin regulates milk protein synthesis at multiple levels in the bovine mammary gland.

    PubMed

    Menzies, Karensa K; Lefèvre, Christophe; Macmillan, Keith L; Nicholas, Kevin R

    2009-05-01

    The role of insulin in milk protein synthesis is unresolved in the bovine mammary gland. This study examined the potential role of insulin in the presence of two lactogenic hormones, hydrocortisone and prolactin, in milk protein synthesis. Insulin was shown to stimulate milk protein gene expression, casein synthesis and (14)C-lysine uptake in mammary explants from late pregnant cows. A global assessment of changes in gene expression in mammary explants in response to insulin was undertaken using Affymetrix microarray. The resulting data provided insight into the molecular mechanisms stimulated by insulin and showed that the hormone stimulated the expression of 28 genes directly involved in protein synthesis. These genes included the milk protein transcription factor, ELF5, translation factors, the folate metabolism genes, FOLR1 and MTHFR, as well as several genes encoding enzymes involved in catabolism of essential amino acids and biosynthesis of non-essential amino acids. These data show that insulin is not only essential for milk protein gene expression, but stimulates milk protein synthesis at multiple levels within bovine mammary epithelial cells. PMID:19107532

  14. 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.

  15. Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation.

    PubMed

    Hucul, J A; Henshaw, E C; Young, D A

    1985-12-15

    A sensitive assay method developed to examine the effects of subtle, physiologically relevant, changes in the levels of adenine and guanine mono-, di-, and triphosphorylated nucleotides specifically on the initiation of protein synthesis is described. Initiation rates are quantified by measuring the amount of protein synthesis resulting from the run-off of ribosomes which have initiated during defined intervals in a modified in vitro protein-synthesizing system developed from Ehrlich ascites tumor cell lysates (Henshaw, E.C., and Panniers, R. (1983) Methods Enzymol. 101, 616-629). The modifications include the attenuation of the ATP-regenerating system so that the relative nucleotide levels more nearly reflect actual intracellular conditions. With this system the rate of initiation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios, but indifferent to the absolute levels of either diphosphate. While the tight coupling of these two ratios by endogenous nucleoside diphosphate kinase activity prevents the independent manipulation of either ratio, the data do eliminate both AMP and GMP per se as inhibitory species. The close agreement of our data calculated in terms of energy charge to previously published results on overall rates of protein synthesis in rat thymocytes (Mendelsohn, S.K., Nordeen, S.K., and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79, 53-60) continues to suggest a physiologically relevant regulatory influence of subtle changes in nucleotides acting at the level of the initiation reaction. PMID:2999123

  16. Systemic Glucose Level Changes with a Carbohydrate-Restricted and Higher Protein Diet Combined with Exercise

    ERIC Educational Resources Information Center

    Bowden, Rodney G.; Lanning, Beth A.; Doyle, Eva I.; Slonaker, Becky; Johnston, Holly M.; Scanes, Georgene

    2007-01-01

    Objective: The authors' purpose in this study was to compare the effects of macronutrient intake on systemic glucose levels in previously sedentary participants who followed 1 of 4 diets that were either higher protein or high carbohydrate, while initiating an exercise program. Participants and Methods: The authors randomly assigned 94 sedentary…

  17. Low levels of aflatoxin B1, ricin and milk enhance recombinant protein production in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changing the optimal tissue culture medium by adding low levels of environmental stress such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin in transduced mammalian cells or 1% reconstituted milk enhances transcription and increases production of the foll...

  18. Effects of prebiotic, protein level, and stocking density on performance, immunity, and stress indicators of broilers.

    PubMed

    Houshmand, M; Azhar, K; Zulkifli, I; Bejo, M H; Kamyab, A

    2012-02-01

    An experiment was conducted to determine the effects of period on the performance, immunity, and some stress indicators of broilers fed 2 levels of protein and stocked at a normal or high stocking density. Experimental treatments consisted of a 2 × 2 × 2 factorial arrangement with 2 levels of prebiotic (with or without prebiotic), 2 levels of dietary CP [NRC-recommended or low CP level (85% of NRC-recommended level)], and 2 levels of stocking density (10 birds/m(2) as the normal density or 16 birds/m(2) as the high density), for a total of 8 treatments. Each treatment had 5 replicates (cages). Birds were reared in 3-tiered battery cages with wire floors in an open-sided housing system under natural tropical conditions. Housing and general management practices were similar for all treatment groups. Starter and finisher diets in mash form were fed from 1 to 21 d and 22 to 42 d of age, respectively. Supplementation with a prebiotic had no significant effect on performance, immunity, and stress indicators (blood glucose, cholesterol, corticosterone, and heterophil:lymphocyte ratio). Protein level significantly influenced broiler performance but did not affect immunity or stress indicators (except for cholesterol level). The normal stocking density resulted in better FCR and also higher antibody titer against Newcastle disease compared with the high stocking density. However, density had no significant effect on blood levels of glucose, cholesterol, corticosterone, and the heterophil:lymphocyte ratio. Significant interactions between protein level and stocking density were observed for BW gain and final BW. The results indicated that, under the conditions of this experiment, dietary addition of a prebiotic had no significant effect on the performance, immunity, and stress indicators of broilers. PMID:22252353

  19. Relationship between C-reactive protein levels and obstructive sleep apnea syndrome.

    PubMed

    Tie, Y X; Fu, Y Y; Xu, Z; Peng, Y

    2016-01-01

    This study aims to determine the relationship between C-reactive protein levels and obstructive sleep apnea syndrome (OSAS). We recruited 30 OSAS patients into the observation group (OSAS group), and subdivided them into mild, moderate and severe groups according to the apnea hypopnea index. In addition, 20 normal individuals were included in the control group. Plasma CRP levels of two groups were measured. As compared with the control group, the CRP levels in the OSAS group were significantly increased (P < 0.05). ANOVA showed that CRP levels in the three subgroups differ; statistically significant differences between the mild and severe OSA patients were observed (P < 0.05). It was hypothesized that OSAS patients show elevated serum CRP levels, and that serum CRP levels are associated with OSAS severity. PMID:27323094

  20. Influence of thyroid dysfunction on serum levels of angiopoietin-like protein 6.

    PubMed

    Lim, Jung Ah; Kim, Hyo Jeong; Ahn, Hwa Young; Park, Kyoung Un; Yi, Ka Hee; Park, Do Joon; Jang, Hak Chul; Park, Young Joo

    2015-10-01

    Angiopoietin-like protein 6 (ANGPTL6) is a novel metabolic regulator that modulates energy expenditure as well as glucose and lipid metabolism. Thyroid hormone can induce metabolic changes that are similar to those induced by ANGPTL6. Herein, we investigated whether circulating ANGPTL6 levels change according to thyroid hormone status in humans. We measured the serum levels of ANGPTL6 and metabolic parameters in 150 drug-naïve subjects with overt hyperthyroid, subclinical hyperthyroid, euthyroid, subclinical hypothyroid, or overt hypothyroid status (n=30 in each group). Serum ANGPTL6 levels were significantly higher in patients with overt hypothyroidism than in the other subjects. Women had significantly higher serum levels of ANGPTL6 than men. ANGPTL6 levels correlated positively with thyroid stimulating hormone (TSH), total cholesterol, aspartate aminotransferase, and alanine aminotransferase (ALT) and negatively with serum free thyroxine (T4) level. Multiple stepwise linear regression analysis revealed that sex, TSH, free T4, and ALT were independent predictors of serum ANGPTL6 levels. In summary, serum ANGPTL6 levels increased in patients with a hypothyroid status, and both TSH and free T4 levels are associated with ANGPLT6 levels, suggesting a possible association between thyroid function and ANGPTL6 levels. Whether the upregulated ANGPTL6 level in the hypothyroid status is primarily owing to a direct association or a compensatory mechanism remains to be determined. PMID:26189599

  1. Protein Domain-Level Landscape of Cancer-Type-Specific Somatic Mutations

    PubMed Central

    Yang, Fan; Petsalaki, Evangelia; Rolland, Thomas; Hill, David E.; Vidal, Marc; Roth, Frederick P.

    2015-01-01

    Identifying driver mutations and their functional consequences is critical to our understanding of cancer. Towards this goal, and because domains are the functional units of a protein, we explored the protein domain-level landscape of cancer-type-specific somatic mutations. Specifically, we systematically examined tumor genomes from 21 cancer types to identify domains with high mutational density in specific tissues, the positions of mutational hotspots within these domains, and the functional and structural context where possible. While hotspots corresponding to specific gain-of-function mutations are expected for oncoproteins, we found that tumor suppressor proteins also exhibit strong biases toward being mutated in particular domains. Within domains, however, we observed the expected patterns of mutation, with recurrently mutated positions for oncogenes and evenly distributed mutations for tumor suppressors. For example, we identified both known and new endometrial cancer hotspots in the tyrosine kinase domain of the FGFR2 protein, one of which is also a hotspot in breast cancer, and found new two hotspots in the Immunoglobulin I-set domain in colon cancer. Thus, to prioritize cancer mutations for further functional studies aimed at more precise cancer treatments, we have systematically correlated mutations and cancer types at the protein domain level. PMID:25794154

  2. Elevated levels of an exocrine pancreatic secretory protein in Alzheimer disease brain.

    PubMed Central

    Ozturk, M; de la Monte, S M; Gross, J; Wands, J R

    1989-01-01

    We identified by a monoclonal antibody-based immunoradiometric assay high concentrations of an exocrine pancreatic protein called pancreatic thread protein (PTP) in several areas of Alzheimer disease (AD) brain. The saline-extractable soluble immunoreactivity shares at least three epitopes in common with the native pancreatic form of the protein; the Mr varies from approximately 17,000 to 20,000. Quantitative measurements of PTP immunoreactivity in various regions of several AD brains revealed levels varying from 12 to 295 ng/g of tissue (mean, 116 ng/g) compared with 1-11 ng/g of tissue (mean, 5 ng/g) found in comparable areas of control brains. Immunocytochemistry performed with the anti-PTP monoclonal antibodies demonstrate PTP immunoreactivity within large pyramidal neurons--many of which contain neurofibrillary tangles in both AD and Down syndrome. Less accumulation was observed in astrocytes, and some PTP immunoreactivity was found extracellularly. The highest number of labeled cells in AD and Down syndrome was seen in the hippocampal formation. Fewer positive-staining cells were noted in normal and disease control brains. We conclude, therefore, that an exocrine pancreatic protein is present in the central nervous system of normal individuals at low levels; in AD brain concentrations of this protein are much higher. Images PMID:2463628

  3. Understanding epigenetic regulation: Tracking protein levels across multiple generations of cells

    NASA Astrophysics Data System (ADS)

    Rowat, A. C.; Weitz, D. A.

    2009-11-01

    Cells and organisms are remarkably robust: they alter the variety and levels of expressed genes and proteins in response to environmental stimuli, including temperature, chemicals, and the stiffness of their surroundings. Ultimately changes in gene and protein expression can result in a distinct phenotypic state, which in some cases is maintained over multiple generations; the ability to pass on a particular phenotypic state to progeny cells is critical for differentiation. Moreover, epigenetic regulation of phenotype is also thought to provide an evolutionary advantage for a population of cells adapting to a fluctuating environment on faster timescales than the occurrence of genetic mutations. However, simple methods to study patterns of gene and protein expression on multi-generational timescales are sparse. Here we describe a technique to study lineages of single cells over multiple generations using a microfluidic device; this reveals patterns of expression where protein levels are correlated across multiple generations. Such quantitative information of protein expression in the context of pedigree remains hidden when studying the population as an ensemble.

  4. SPX proteins regulate Pi homeostasis and signaling in different subcellular level

    PubMed Central

    Zhou, Zhipeng; Wang, Zhiye; Lv, Qundan; Shi, Jing; Zhong, Yongjia; Wu, Ping; Mao, Chuanzao

    2015-01-01

    To cope with low phosphate (Pi) availability, plants have to adjust its gene expression profile to facilitate Pi acquisition and remobilization. Sensing the levels of Pi is essential for reprogramming the gene expression profile to adapt to the fluctuating Pi environment. AtPHR1 in Arabidopsis and OsPHR2 in rice are central regulators of Pi signaling, which regulates the expression of phosphate starvation-induced (PSI) genes by binding to the P1BS elements in the promoter of PSI genes. However, how the Pi level affects the central regulator to regulate the PSI genes have puzzled us for a decade. Recent progress in SPX proteins indicated that the SPX proteins play important role in regulating the activity of central regulator AtPHR1/OsPHR2 in a Pi dependent manner at different subcellular levels. PMID:26224365

  5. Estimation of Levels of Salivary Mucin, Amylase and Total Protein in Gingivitis and Chronic Periodontitis Patients

    PubMed Central

    Bhandary, Rahul; Thomas, Biju; Kumari, Suchetha

    2014-01-01

    Background: Periodontal diseases are a group of inflammatory conditions resulting from interaction between a pathogenic bacterial biofilm and susceptible host’s inflammatory response eventually leading to the destruction of periodontal structures and subsequent tooth loss. Hence, investigation of salivary proteins in individuals with periodontal diseases may be useful to enhance the knowledge of their roles in these diseases. Materials and Methods: This case-control study was conducted at A.B. Shetty Memorial Institute of Dental Sciences, Mangalore. The study comprised of 90 patients of age between 25-60 years who were clinically examined and divided into three groups of 30 each: namely clinically healthy, gingivitis and chronic periodontitis. These were classified according to the values of gingival index score, clinical attachment loss and probing pocket depth. Unstimulated saliva was collected and salivary mucin, amylase and total protein levels were determined. Statistical analysis: Results obtained were tabulated and statistically analyzed using ANOVA test and Karl pearson’s correlation test. Results: The results of the study showed an increased concentration of salivary mucin, amylase and total protein in gingivitis patients and increased levels of amylase and total protein in saliva of chronic periodontitis patients compared to healthy individuals which were statistically significant. A decrease in mucin concentration was observed in the periodontitis group compared to gingivitis group. A positive correlation was present between salivary mucin, amylase and total protein levels in the three groups. Conclusion: Salivary mucin, amylase and total protein may serve as an important biochemical parameter of inflammation of the periodontium. Also, it can be hypothesized that various enzyme inhibitors might be useful as a part of host modulation therapy in the treatment of periodontal diseases. PMID:25478449

  6. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  7. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  8. Effects crude protein levels on female Nile tilapia (Oreochromis niloticus) reproductive performance parameters.

    PubMed

    de Oliveira, Marinez Moraes; Ribeiro, Tainá; Orlando, Tamira Maria; de Oliveira, Dênio Garcia Silva; Drumond, Mariana Martins; de Freitas, Rilke Tadeu Fonseca; Rosa, Priscila Vieira

    2014-11-10

    The goal of the present work was to study the reproductive performance of Nile Tilapia (Oreochromis niloticus) female broodstock fed diets containing different levels of crude protein (CP). Two hundred and forty Nile tilapia (O. niloticus) were used at an average age of 30 months, with 180 females and 60 males. The broodstock were lodged separately in masonry tanks with continuous water flow. The females were stocked in thirty tanks with dimensions of 8 m(3) in a completely randomized design consisting of five treatments and six replications. The treatments consisted of five diets with different levels of CP (32, 34, 36, 38 and 40%) and with digestible energy per gram of protein of 9.5 kg of feed. The crude protein (CP) levels positively influenced (p<0.05) reproductive parameters (female relative and absolute fecundity, egg diameter, fasting larvae survival capacity), the somatic indexes (gonadosomatic (GSI), hepatosomatic (HIS), viscerosomatic (VSI)), total plasma protein, albumin and triglycerides. There were no significant differences (p>0.05) observed with regard to spawning weight and female weight. The reproductive parameters studied in the present research indicate that diets formulated with 38% CP with digestible energy per gram of CP of 9.5 were the best diets for tilapia females during the reproductive period. PMID:25201770

  9. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture. PMID:9104035

  10. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed. PMID:23093835

  11. Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli.

    PubMed

    Retnoningrum, Debbie S; Pramesti, H T; Santika, P Y; Valerius, O; Asjarie, S; Suciati, T

    2012-08-01

    Codons in the open reading frame (ORF) encoding for human bone morphogenetic protein-2 (hBMP-2) were optimized to reach high level expression in Escherichia coli. The optimization was done by the computer programs DNA works and DNA Star according to Thermodynamically Balanced Inside Out (TBIO) approach. The ORF consisting of 342 base pairs (bp) was assembled using two-steps Polymerase Chain Reaction, cloned into a pGEM-T vector with a mutation rate of 6.38 bp per kb and transformed into E. coli JM109. After a DNA sequence confirmation, mutation-free ORF was subcloned into pET32b and transformed into E. coli BL21(DE3). The rhBMP-2 was produced as a thioredoxin-his-tag fusion protein at relatively high level, approximately 60% of total intracellular proteins as inclusion bodies (IB), with a yield of 1.39 g per liter culture. Solubilization of IB gave soluble monomer rhBMP-2 with a recovery of 13.6% and refolding of soluble rhBMP-2 produced dimeric forms with a yield of 8.7%. The size and identity of the purified rhBMP-2 was confirmed by nano-LC-MS/MS2 analysis. Our work demonstrates for the first time that by using TBIO approach, a codon-optimized ORF encoding for rhBMP-2 protein can be expressed at high level in E. coli expression system. PMID:22691543

  12. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  13. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  14. Low plasma protein nitrotyrosine levels distinguish primary Raynaud's phenomenon from scleroderma

    PubMed Central

    Kingdon, E J; Mani, A R; Frost, M T; Denton, C P; Powis, S H; Black, C M; Moore, K P

    2006-01-01

    Objective To investigate the hypothesis that increased formation of reactive nitrogen species may contribute to the vascular pathology that develops in patients with connective tissue disease such as scleroderma. Patients and methods The level of protein‐bound nitrotyrosine in plasma was measured by stable isotope dilution gas chromatography/negative ion chemical ionisation mass spectrometry in 11 patients with primary Raynaud's phenomenon, 37 with scleroderma, 13 with chronic renal impairment, and in 23 healthy controls. Results Plasma protein‐bound nitrotyrosine was markedly decreased in patients with primary Raynaud's phenomenon (mean (SEM) 0.60 (0.06) ng/mg dry protein) compared with patients with scleroderma (1.78 (0.21) ng/mg protein), chronic renal impairment (1.42 (0.17) ng/mg protein) or healthy controls (1.63±0.15 ng/mg protein, ANOVA p<0.001). Conclusion These data suggest that there is decreased nitration of plasma proteins, or increased degradation of nitrated proteins from the circulation of patients with primary but not secondary Raynaud's phenomenon. PMID:16308344

  15. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  16. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    SciTech Connect

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Weijun

    2013-07-05

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a median CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

  17. Modulation of Protein Levels in Chromosomal Dosage Series of Maize: The Biochemical Basis of Aneuploid Syndromes

    PubMed Central

    Birchler, James A.; Newton, Kathleen J.

    1981-01-01

    Genetically defined dosage series of chromosome arms 1L, 3L, 4S, 5L, 7L, 9S, 10L and combinations of 1L–3L, collectively spanning approximately one-third of the maize genome, were examined for alterations in the expression of total protein profiles in scutellar tissue. The major effects found were negative correlations of specific proteins with the dosage of particular regions in a manner similar to that previously described for enzyme activity levels (Birchler 1979). Chromosome arms 1L, 4S and 5L produced the most severe negative effects, with 3L and 7L exhibiting this phenomenon to a lesser degree. Positive correlations of certain proteins were observed with the dosage of the 1L, 3L, 5L and 7L regions. The structural locus of one of the major scutellar proteins (PRO) is present in the long arm of chromosome 1 (Schwartz 1979), but exhibits compensation in a dosage series involving whole-arm comparisons. Multiple factors in 1L affect the level of the protein. The compound TB-1La-3L4759-3 (1L 0.20–0.39) has a slight negative effect on PRO, while TB-1La-3Le (1L 0.20–0.58) and TB-1La-3L5267 (1L 0.20–0.72) have a more pronounced negative influence. The level of this protein is not altered by the dosage of 3L. These observations suggest that compensation is brought about by the cancellation of a positive structural gene dosage effect by the negative inverse effect. Other regions of the genome that contribute to the control of PRO levels are 4S and 5L. Total protein profiles were also compared in haploid, diploid and tetraploid maize as a comparison to the aneuploid series. Most proteins exhibit structural-gene-dosage effects through the ploidy series, but others show a positive effect greater than expected from varying the structural genes. Still others are negatively affected by ploidy changes. In general, the ploidy alterations are not as great as predicted from the cumulative action of the aneuploid effects. The bearing of these observations on the

  18. Elevation of serum heat-shock protein levels in amyotrophic lateral sclerosis.

    PubMed

    Miyazaki, Daigo; Nakamura, Akinori; Hineno, Akiyo; Kobayashi, Chinatsu; Kinoshita, Tomomi; Yoshida, Kunihiro; Ikeda, Shu-Ichi

    2016-08-01

    Heat-shock proteins (HSPs) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). In this study, we aimed to examine whether the serum levels of HSPs (HSP27, HSP70, and HSP90) are altered in patients with ALS. We included 58 patients diagnosed with ALS and 85 control individuals. Serum HSP levels of patients and controls were determined using enzyme-linked immunosorbent assay. The serum levels of HSP70 and HSP90 were significantly higher in patients than in controls. In contrast, serum levels of HSP27 did not differ significantly between the patient and control groups. Moreover, serum levels of HSP70 and HSP90 in patients remained high throughout the duration of the disease. Taken together, our findings suggest that HSPs might have a role in ALS progression throughout the course of the disease. Further studies are needed to clarify the role of HSPs in the pathogenesis of ALS. PMID:27112486

  19. Protein levels in beef cattle finishing diets: industry application, university research, and systems results.

    PubMed

    Galyean, M L

    1996-11-01

    Consulting nutritionists were surveyed to determine current formulation and management practices for finishing beef cattle. Among the six consultants surveyed, percentage of CP in finishing diets ranged from 12.5 to 14.4%, with urea levels ranging from .5 to 1.5% of DM. Finishing diets were based primarily on highly processed, rapidly fermented grains (steam-flaked and high-moisture grain), with roughage levels ranging from 3 to 11% of DM. All six consultants considered feed bunk management to be a critical factor affecting feed intake and performance; five of the six consultants used aggressive implant programs based on estrogen + trenbolone acetate. Recent university research was reviewed with respect to CP level and source in finishing diets. Finishing cattle managed on aggressive implant programs seem to respond to higher levels of CP better than would be expected from the factorial calculation approach. Moreover, improvements in performance noted in recent research seemed to be more consistent when supplemental CP was derived from ruminally degraded vs undegraded sources. Calculation of protein requirements with a metabolizable protein (MP) system yielded estimates of protein needs by finishing cattle that agreed more closely with current industry practices than did calculation based on the factorial method. The difference between the MP system and the factorial method was primarily a result of accounting for microbial N needs in the MP system. Reasons for production responses to CP levels that are greater than those calculated by the factorial method include increased accretion of protein by rapidly growing, implanted cattle, particularly during the initial phase of the finishing period, alleviation of a microbial N deficiency, and ruminal and systemic effects of ruminally degraded N on acid-base balance of beef cattle fed rapidly fermented, high-grain diets. Reasons for production responses to supplemental CP need further research. PMID:8923202

  20. Study of Serum Levels of Leptin, C-Reactive Protein and Nutritional Status in Hemodialysis Patients

    PubMed Central

    Montazerifar, Farzaneh; Karajibani, Mansour; Hassanpour, Zahra; Pourmofatteh, Mahla

    2015-01-01

    Background: Leptin is secreted by adipose tissue and decreases appetite. However, the role of leptin in the pathogenesis of hemodialysis (HD)-related malnutrition has not been fully evaluated. Objectives: The aim of study was to investigate the association between the serum leptin levels, serum C-reactive protein (CRP) levels, and nutritional status in hemodialysis patients. Patients and Methods: This analytical descriptive study included 45 hemodialysis patients and 40 healthy subjects. Biochemical parameters and serum leptin levels were measured. The nutritional status was evaluated using a food frequency questionnaire (FFQ) and the calculation of the body mass index (BMI). Results: Serum leptin (P < 0.05) and albumin (P < 0.0001) levels and BMI (P < 0.001) of HD patients were significantly lower, while CRP levels were significantly higher than those of controls (P < 0.0001). HD patients consumed the lower daily servings of the food groups compared to the control subjects (P < 0.0001). A significant positive correlation between serum levels of leptin and albumin and BMI was demonstrated. No significant correlations were identified between leptin level, CRP level, and other variables. Conclusions: The findings suggest that low levels of leptin may be a contributory factor for malnutrition in HD patients. Further studies are required to ascertain the significance of leptin levels in relation to nutritional factors in hemodialysis patients. PMID:26430525

  1. Ultrasensitive detection of proteins and sugars at single-cell level

    PubMed Central

    Watabe, Satoshi; Morikawa, Mika; Kaneda, Mugiho; Nakaishi, Kazunari; Nakatsuma, Akira; Ninomiya, Masaki; Yoshimura, Teruki; Miura, Toshiaki; Ito, Etsuro

    2016-01-01

    ABSTRACT Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine. PMID:27064305

  2. Amyloid beta protein levels in cerebrospinal fluid are elevated in early-onset Alzheimer's disease.

    PubMed

    Nakamura, T; Shoji, M; Harigaya, Y; Watanabe, M; Hosoda, K; Cheung, T T; Shaffer, L M; Golde, T E; Younkin, L H; Younkin, S G

    1994-12-01

    The 4-kd amyloid beta protein (A beta) deposited as amyloid in Alzheimer's disease (AD) is produced and released by normal proteolytic processing of the amyloid beta protein precursor (beta APP) and is readily detected in cerebrospinal fluid (CSF). Here, we present the levels of A beta in CSF from a total of 95 subjects, including 38 patients with AD, 14 with early-onset AD and 24 with late-onset AD, 25 normal control subjects, and 32 patients with other neurological diseases. The level of A beta decreased with normal aging, and there was a significant elevation in the level of A beta in the CSF of early-onset AD patients (4.14 +/- 1.37 pmol/ml, p < 0.01). Neither Mini-Mental State nor Functional Assessment Staging were correlated with the amount of A beta in the CSF. The A beta/secreted form of beta APP ratio was elevated, but the level of alpha 1-antichymotrypsin in the CSF did not correlate with the level of CSF A beta in early-onset AD patients. Thus, the level of A beta in the CSF is elevated in early-onset AD patients and is suggested to be correlated with the pathology in the brain that characterizes AD. PMID:7998778

  3. Correlating Flavivirus virulence and levels of intrinsic disorder in shell proteins: protective roles vs. immune evasion.

    PubMed

    Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

    2016-05-24

    Computational analyses revealed correlations between the intrinsic disorder propensity of shell proteins and case fatality rates (CFRs) among Flaviviruses and within at least two Flavivirus species, such as tick-borne encephalitis virus (TBEV) and dengue virus (DENV). The shell proteins analyzed in this study are capsid (C) and membrane (PrM, Pr, and M) proteins. The highest correlations can be found when regression analyses were conducted using Pr (Flavivirus: r(2) = 0.78, p < 0.01) or M (Flavivirus: r(2) = 0.91, p < 0.01) as an independent variable with C and CFR as co-explanatory and dependent variables, respectively. Interestingly, while predicted intrinsic disorder levels (PIDs) of both C and M are positively correlated with the virulence, the PIDs of Pr and CFR are negatively correlated. This is likely due to the fact that the Pr portion of PrM plays various roles in protecting the virion from damage, whereas M and C are assisted by greater potential in binding promiscuity as a result of greater disorder. The C protein of yellow fever virus (YFV), which is the most virulent virus in the sample, has the highest PID levels, whereas the second most virulent TBEV FE subtype has the second highest PID score due to its C protein, and the least virulent West Nile virus (WNV) has the least disordered C protein. This knowledge can be used while working on the development and identification of attenuated strains for vaccine. Curiously, unlike Flaviviruses, a disordered outer shell was described for hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human simplex virus 2 (HSV-2), which currently have no effective vaccine. PMID:27102744

  4. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    USGS Publications Warehouse

    Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

    2009-01-01

    Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

  5. Change in the protein level of mevalonate pyrophosphate decarboxylase in tissues of mouse by pravastatin.

    PubMed

    Michihara, Akihiro; Akasaki, Kenji; Yamori, Yukio; Tsuji, Hiroshi

    2003-08-01

    We previously reported that treatment of rats with a diet containing 0.1% pravastatin and 5% cholestyramine markedly increased mevalonate pyrophosphate decarboxylase (MPD) activity in liver crude extracts compared with nontreated rats. In this study, we examined the change in the protein level of MPD in the tissues of mice administered pravastatin. When MPD content in the tissues of nontreated mice was analyzed by quantitative immunoblotting, a single protein band with an apparent molecular weight of 46 kDa was detected in all tissues and the specific protein content of MPD in liver and kidney was markedly higher than that in other tissues. When MPD content in the tissues of pravastatin-treated mice was analyzed by immunoblotting, MPD was markedly increased (9-fold) only in the liver compared with nontreated mice. Next, when MPD activity was measured in the liver between nontreated and pravastatin-treated mice, MPD activity as well as protein levels were markedly increased (11-fold) in the liver of pravastatin-treated mice compared with nontreated mice. These data suggest that a marked induction of MPD in the liver by pravastatin is responsible for the tissue-specific effect of pravastatin. PMID:12913254

  6. Visualizing and quantifying protein polySUMOylation at the single-molecule level.

    PubMed

    Yang, Yong; Zhang, Chun-yang

    2014-01-21

    Protein polySUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is associated with a variety of physiological processes. However, the analysis of protein polySUMOylation is often complicated by the heterogeneity of SUMO-target conjugates. Here, we develop a new strategy to visualize and quantify polySUMOylation at the single-molecule level by integrating the tetracysteine (TC) tag labeling technology and total internal reflection fluorescence (TIRF)-based single-molecule imaging. As a proof-of-concept, we employ the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can specifically translate the SUMO-mediated modification into visible fluorescence signal without disturbing the function of SUMO-2. The SUMO monomers display homogeneous fluorescence spots at the single-molecule level, whereas the mixed SUMO chains exhibit nonuniform fluorescence spots with a wide range of intensities. Analysis of the number and the brightness of fluorescence spots enable quantitative measurement of the polySUMOylation degree inside the cells under different physiological conditions. Due to the frequent occurrence of posttranslational modification by polymeric chains in cells, this single-molecule strategy has the potential to be broadly applied for studying protein posttranslational modification in normal cellular physiology and disease etiology. PMID:24383460

  7. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  8. The Genomic Landscape of Position Effects on Protein Expression Level and Noise in Yeast.

    PubMed

    Chen, Xiaoshu; Zhang, Jianzhi

    2016-05-25

    Position effect, the influence of the chromosomal location of a gene on its activity, is a fundamental property of the genome. By placing a GFP gene cassette at 482 different locations across all chromosomes in budding yeast, we quantified the position effects on protein expression level and noise at the genomic scale. The position effects are significant, altering the mean protein expression level by up to 15 times and expression noise by up to 20 times. DNA replication timing, 3D chromosomal conformation, and several histone modifications are major covariates of position effects. Essential genes are enriched in genomic regions with inherently low expression noise, supporting the hypothesis that chromosomal clustering of essential genes results from selection against their expressional stochasticity. Position effects exhibit significant interactions with promoters. Together, our results suggest that position effects have shaped the evolution of chromosome organization and should inform future genome engineering efforts. PMID:27185547

  9. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  10. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels.

    PubMed

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m(2); all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  11. The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

    PubMed Central

    Jones, A L; Roberts, R C; Coughtrie, M W

    1993-01-01

    We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed. Images Figure 1 Figure 2 Figure 3 PMID:8257413

  12. Ileal apical sodium-dependent bile acid transporter protein levels are down-regulated through ubiquitin-dependent protein degradation induced by bile acids.

    PubMed

    Miyata, Masaaki; Yamakawa, Hiroki; Hayashi, Kenjiro; Kuribayashi, Hideaki; Yamazoe, Yasushi; Yoshinari, Kouichi

    2013-08-15

    The ileal apical sodium-dependent bile acid transporter (ASBT or SLC10A2) has a crucial role in intestinal bile acid absorption. We previously reported that enterobacteria-mediated bile acid conversion was involved in the alteration of ileal ASBT expression levels. In the present study, to investigate the hypothesis that ileal ASBT protein levels are post-translationally regulated by enterobacteria-associated bile acids, alteration of ileal ASBT protein levels was analysed in mice 12 h and 24 h after anti-bacterial drug ampicillin (ABPC) treatment (100 mg/kg, single shot) that altered bile acid composition in the intestinal lumen. In ABPC-treated mice, enterobacteria-biotransformed bile acid, taurodeoxycholic acid (TDCA) and cholic acid (CA) levels were decreased, whereas taurocholic acid (TCA) and tauro-β-muricholic acid levels were increased in the intestinal lumen. Ileal ASBT protein levels in brush-border membrane vesicles (BBMVs), but not ileal Asbt mRNA levels, were significantly increased in the ABPC-treated mice, and the extent of ubiquitination of the ileal ASBT protein was reduced in the ABPC-treated mice. Treatment of ABPC-pretreated mice with CA or TDCA, but not TCA, significantly decreased ileal ASBT protein levels and increased the extent of ubiquitination of ileal ASBT protein. Treatment of mice with the lysosome inhibitor, chloroquine, or the proteasome inhibitor, MG132, increased ileal ASBT protein levels in BBMVs. CA-mediated reduction of ASBT protein levels in the ABPC-pretreated mice was attenuated by co-treatment with chloroquine or MG132. These results suggest that ileal ASBT protein is degraded by a ubiquitin-dependent pathway in response to enterobacteria-associated bile acids. PMID:23872411

  13. Melamine Impairs Female Fertility via Suppressing Protein Level of Juno in Mouse Eggs

    PubMed Central

    Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Miao, Yilong; Zhou, Changyin; Sun, Shaochen; Xiong, Bo

    2015-01-01

    Melamine is an organic nitrogenous compound widely used as an industrial chemical, and it has been recently reported by us that melamine has a toxic effect on the female reproductive system in mice, and renders females subfertile; the molecular basis, however, has not been adequately assessed. In the present study, we explore the underlying mechanism regarding how melamine compromises fertility in the mouse. The data showed that melamine exposure significantly impaired the fertilization capability of the egg during in vitro fertilization. To further figure out the cause, we analyzed ovastacin localization and protein level, the sperm binding ability of zona pellucida, and ZP2 cleavage status in unfertilized eggs from melamine fed mice, and no obvious differences were found between control and treatment groups. However, the protein level of Juno on the egg plasma membrane in the high-dose feeding group indeed significantly decreased compared to the control group. Thus, these data suggest that melamine compromises female fertility via suppressing Juno protein level on the egg membrane. PMID:26633308

  14. Melamine Impairs Female Fertility via Suppressing Protein Level of Juno in Mouse Eggs.

    PubMed

    Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Miao, Yilong; Zhou, Changyin; Sun, Shaochen; Xiong, Bo

    2015-01-01

    Melamine is an organic nitrogenous compound widely used as an industrial chemical, and it has been recently reported by us that melamine has a toxic effect on the female reproductive system in mice, and renders females subfertile; the molecular basis, however, has not been adequately assessed. In the present study, we explore the underlying mechanism regarding how melamine compromises fertility in the mouse. The data showed that melamine exposure significantly impaired the fertilization capability of the egg during in vitro fertilization. To further figure out the cause, we analyzed ovastacin localization and protein level, the sperm binding ability of zona pellucida, and ZP2 cleavage status in unfertilized eggs from melamine fed mice, and no obvious differences were found between control and treatment groups. However, the protein level of Juno on the egg plasma membrane in the high-dose feeding group indeed significantly decreased compared to the control group. Thus, these data suggest that melamine compromises female fertility via suppressing Juno protein level on the egg membrane. PMID:26633308

  15. A Compendium of Caenorhabditis elegans RNA Binding Proteins Predicts Extensive Regulation at Multiple Levels

    PubMed Central

    Tamburino, Alex M.; Ryder, Sean P.; Walhout, Albertha J. M.

    2013-01-01

    Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs. PMID:23390605

  16. Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice.

    PubMed

    Kanda, Atsushi; Morifuji, Masashi; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2012-11-14

    Previously, we have shown that consuming carbohydrate plus whey protein hydrolysates (WPHs) replenished muscle glycogen after exercise more effectively than consuming intact whey protein or branched-chain amino acids (BCAAs). The mechanism leading to superior glycogen replenishment after consuming WPH is unclear. In this 5 week intervention, ddY mice were fed experimental diets containing WPH, a mixture of whey amino acids (WAAs), or casein (control). After the intervention, gastrocnemius muscle glycogen levels were significantly higher in the WPH group (4.35 mg/g) than in the WAA (3.15 mg/g) or control (2.51 mg/g) groups. In addition, total glycogen synthase (GS) protein levels were significantly higher in the WPH group (153%) than in the WAA (89.2%) or control groups, and phosphorylated GS levels were significantly decreased in the WPH group (51.4%). These results indicate that dietary WPH may increase the muscle glycogen content through increased GS activity. PMID:23113736

  17. Inclusion of Yucca schidigera extract in diets with different protein levels for dogs.

    PubMed

    Dos Reis, Jéssica S; Zangerônimo, Márcio G; Ogoshi, Rosana C S; França, Janine; Costa, Adriano C; Almeida, Thomás N; Dos Santos, João P F; Pires, Carolina P; Chizzotti, Ana F; Leite, Carlos A L; Saad, Flávia M O B

    2016-08-01

    This study evaluated the effects of inclusion of Yucca schidigera extract (YSE) in two diets with different levels of crude protein (CP) for dogs on facal odour, nutrient digestibility, ammonia concentration in feces and hematological and serum biochemical profiles. Twenty adults Beagles were used, distributed in a randomized block design in a 2 × 4 factorial design (two diets, 25% and 34% CP, and four YSE levels: 0, 250, 500 and 750 mg/kg) with five replicates, obtained during two experimental periods. The fecal odour reduced (P < 0.05) when 500 mg/kg of YSE was used in diets with higher CP. The inclusion of YSE reduced (P < 0.05) fecal ammonia, and the inclusion of 250 and 500 mg/kg YSE reduced intestinal gas. The inclusion of 750 mg/kg YSE increased the mean corpuscular hemoglobin (MCH), alanine aminotransferase (ALT) activity and tended to increase the serum cholesterol concentration, regardless of the protein level of the diets. There was no effect on the digestibility of nutrients, fecal consistency, nitrogen balance and thickness of the intestinal wall. The inclusion of 500 mg/kg YSE is effective in reducing fecal odour in dogs receiving diets with 34% of CP. Regardless of the protein content, YSE reduces fecal ammonia, but may cause adverse effects if included at higher doses. PMID:26800023

  18. Oxidative stress and hippocampal synaptic protein levels in elderly cognitively intact individuals with Alzheimer's disease pathology.

    PubMed

    Scheff, Stephen W; Ansari, Mubeen A; Mufson, Elliott J

    2016-06-01

    Neuritic amyloid plaques and neurofibrillary tangles are hallmarks of Alzheimer's disease (AD) and are major components used for the clinical diagnosis of this disorder. However, many individuals with no cognitive impairment (NCI) also present at autopsy with high levels of these AD pathologic hallmarks. In this study, we evaluated 15 autopsy cases from NCI individuals with high levels of AD-like pathology (high pathology no cognitive impairment) and compared them to age- and postmortem-matched cohorts of individuals with amnestic mild cognitive impairment and NCI cases with low AD-like pathology (low pathology no cognitive impairment [LPNCI]). Individuals classified as high pathology no cognitive impairment or amnestic mild cognitive impairment had a significant loss of both presynaptic and postsynaptic proteins in the hippocampus compared with those in the LPNCI cohort. In addition, these 2 groups had a significant increase in 3 different markers of oxidative stress compared with that in the LPNCI group. The changes in levels of synaptic proteins are strongly associated with levels of oxidative stress. These data suggest that cognitively older subjects without dementia but with increased levels of AD-like pathology may represent a very early preclinical stage of AD. PMID:27143416

  19. Studies of composition and major protein level in milk and colostrum of mares.

    PubMed

    Pecka, Ewa; Dobrzański, Zbigniew; Zachwieja, Andrzej; Szulc, Tadeusz; Czyż, Katarzyna

    2012-02-01

    The aim of the study was to determine the changes in composition and physicochemical features (pH, density, thermostability and acidity) of mare colostrum and milk, and of protein fraction contribution (serum albumin, β-casein, γ-casein, α-lactalbumin, G class immunoglobulins) depending on lactation stage. The research material was colostrum and milk samples from 12 Arabian mares. Colostrum samples were collected within 2 h after parturition and milk samples were collected twice, in the 3rd and 6th weeks of lactation. The level of basic milk components decreased significantly (only lactose content increased) as compared to colostrum. Total bacteria count and somatic cell count decreased significantly with an increase in resistance and urea level. The changes observed were connected to differentiated contribution of particular protein fractions and their relative proportions. Lower levels of γ-casein (P ≤ 0.05), β-casein, serum albumin as well as α-lactalbumin were observed in colostrum as compared to those in milk. Any relationship between lactation stage and β-casein content was observed. Serum albumin and α-lactalbumin content increased in subsequent milkings. The level of G class immunoglobulins decreased significantly and its highest level was noted in colostrum. Any significant differences between the 3rd and 6th lactation weeks were obtained. PMID:22339698

  20. Birdsong decreases protein levels of FoxP2, a molecule required for human speech.

    PubMed

    Miller, Julie E; Spiteri, Elizabeth; Condro, Michael C; Dosumu-Johnson, Ryan T; Geschwind, Daniel H; White, Stephanie A

    2008-10-01

    Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability. PMID:18701760

  1. Birdsong Decreases Protein Levels of FoxP2, a Molecule Required for Human Speech

    PubMed Central

    Miller, Julie E.; Spiteri, Elizabeth; Condro, Michael C.; Dosumu-Johnson, Ryan T.; Geschwind, Daniel H.; White, Stephanie A.

    2008-01-01

    Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability. PMID:18701760

  2. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar

    PubMed Central

    Sousa, Daniele O. B.; Carvalho, Ana F. U.; Oliveira, José Tadeu A.; Farias, Davi F.; Castelar, Ivan; Oliveira, Henrique P.; Vasconcelos, Ilka M.

    2015-01-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  3. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

    PubMed

    Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

    2015-07-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  4. Level of dietary protein does not impact whole body protein turnover during an exercise induced energy deficit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: This study examined the effect of a high protein diet on whole body protein turnover during an exercise-induced energy deficit. A sustained energy deficit induced by energy intake restriction increases protein catabolism which can cause lean-body mass loss. A high-protein diet has be...

  5. Optimal dietary protein level improved growth, disease resistance, intestinal immune and physical barrier function of young grass carp (Ctenopharyngodon idella).

    PubMed

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

    2016-08-01

    This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis

  6. Protein level identification of the Listeria monocytogenes Sigma H, Sigma L, and Sigma C regulons

    PubMed Central

    2013-01-01

    Background Transcriptional regulation by alternative sigma (σ) factors represents an important mechanism that allows bacteria to rapidly regulate transcript and protein levels in response to changing environmental conditions. While the role of the alternative σ factor σB has been comparatively well characterized in L. monocytogenes, our understanding of the roles of the three other L. monocytogenes alternative σ factors is still limited. In this study, we employed a quantitative proteomics approach using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to characterize the L. monocytogenes σL, σH, and σC protein regulons. Proteomic comparisons used a quadruple alternative σ factor mutant strain (ΔBCHL) and strains expressing a single alternative σ factor (i.e., σL, σH, and σC; strains ΔBCH, ΔBCL, and ΔBHL) to eliminate potential redundancies between σ factors. Results Among the three alternative σ factors studied here, σH provides positive regulation for the largest number of proteins, consistent with previous transcriptomic studies, while σL appears to contribute to negative regulation of a number of proteins. σC was found to regulate a small number of proteins in L. monocytogenes grown to stationary phase at 37°C. Proteins identified as being regulated by multiple alternative σ factors include MptA, which is a component of a PTS system with a potential role in regulation of PrfA activity. Conclusions This study provides initial insights into global regulation of protein production by the L. monocytogenes alternative σ factors σL, σH, and σC. While, among these σ factors, σH appears to positively regulate the largest number of proteins, we also identified PTS systems that appear to be co-regulated by multiple alternative σ factors. Future studies should not only explore potential roles of alternative σ factors in activating a “cascade” of PTS systems that potentially regulate PrfA, but also may want to explore the

  7. Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2005-04-01

    The effects of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCl4)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats maintained on a low-protein diet for 5 days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCl4 intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low-protein diet for the duration of the investigation. Groups of rats from the different subgroups were killed at 24, 48 and 72 h after their respective treatments. After 5 days on the low-protein diet the activity levels of all four enzymes were significantly higher than their counterparts on a normal balanced diet. CCl4 intoxication resulted in significant increases in the activity levels of all four enzymes investigated. The administration of pumpkin seed protein isolate after CCl4 intoxication resulted in significantly reduced activity levels of all four enzymes. It is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition. PMID:16041732

  8. Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner

    PubMed Central

    Christensen, Steen; Pérez Dulzaides, Ricardo; Hedrick, Victoria E.; Momtaz, A. J. M. Zehadee; Nakayasu, Ernesto S.; Paul, Lake N.

    2016-01-01

    ABSTRACT Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia. Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified

  9. Rapid Dietary Protein Changes and Relationships among Enzyme Activities, Their RNA's and Plasma Hormone Levels in the Broiler Chicken

    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%) and sampled three days following the switch. The purpose of these treat...

  10. [Relation between serum eosinophil cationic protein (ECP) level and asthma attack in children].

    PubMed

    Ishigaki, N; Masuhara, C; Sakamaki, K; Ishikawa, Y; Ohta, K; Koike, R; Mikuni, K; Haruna, H; Awa, S

    2000-11-01

    Serum eosinophil cationic protein (sECP) levels were measured in 339 patients with childhood asthma, and the clinical courses of these patients were followed for 57 weeks. While considering the history and characteristics of each patient, we examined the correlation between asthma attack frequency and sECP, blood eosinophil count, and serum total IgE (tIgE) to determine their usefulness in predicting asthma attacks. Among patients with no other allergic diseases, sECP levels in patients who had no asthma attacks two weeks before or after the measurement were significantly lower than those of patients who had attacks during the same four-week period. Among patients who had attacks, those patients with no attack for a year after the measurement were also found to have low sECP levels. Similarly, even among patients with asthma attacks and high sECP levels, there were cases where attacks were well controlled using nebulizer treatments with DSCG or BDP. The incident rate of attacks for patients with other allergic diseases and a low sECP was low. Yet, there was no common trend in patients with high sECP levels. Moreover, this study detected a significant correlation between sECP level and blood eosinophil count as well as between sECP level and serum tIgE. The most significant correlation with asthma attack frequency was sECP level. Thus, sECP level seems to reflect the allergy activity level, especially two weeks prior to and after the measurement. For patients without other allergic diseases, asthma attack prediction during the two weeks period after the measurement of sECP also seems possible. Therefore, periodic measurement of sECP level is useful in objectively monitoring the improvement of symptoms and establishing the treatment plan, including treatment with DSCG or BDP. PMID:11193461

  11. Serum levels of hypersensitive-C-reactive protein in moderate and severe acne

    PubMed Central

    Namazi, M. R.; Parhizkar, A. R.; Jowkar, F.

    2015-01-01

    Background: Elevation of C-reactive protein (CRP) has been reported to occur in psoriasis, urticaria, acne, rosacea and many other dermatological and nondermatological conditions. Chronic systemic inflammation has been implicated in the development of neuropsychiatric/degenerative disorders, atherosclerosis, coronary artery disease, diabetes mellitus and even carcinogenesis. The present study is designed to determine whether the level of inflammation created by acne vulgaris could be high enough to raise the serum levels of high-sensitive CRP. Materials and Methods: Forty-two patients with moderate and severe acne vulgaris were enrolled, along with 44 age and sex matched healthy blood donors as controls. Hypersensitive-CRP (Hs-CRP) was measured in both groups. Results: Hypersensitive-C-reactive protein levels in the case group varied between 0 and 28.1 μg/ml with an average of 2.24 ± 4.87 μg/ml (mean ± standard deviation) and a median of 0.6 μg/ml (interquartile range [IQR] =0.3, 1.4 μg/ml). Hs-CRP levels of the control group varied between 0 and 14 μg/ml with an average of 3.12 ± 3.67 μg/ml and a median of 1.5 μg/ml (IQR = 0.55, 5.0 μg/ml). No significant difference of Hs-CRP level between the two groups was seen (t = –0.961, 95% confidence interval: Lower = –2.6942, upper = 0.9377; P = 0.339). Additionally, no significant difference in the level of Hs-CRP was noted between the moderate and severe acne groups (95% confidence interval: Lower = –5.2495, upper = 1.6711; P = 0.165). Conclusion: Acne vulgaris, even in its severe grades (excluding acne fulminans and acne conglobata), does not induce significant inflammation at the systemic level. PMID:26225329

  12. Circulating growth arrest-specific protein 6 levels are associated with erythropoietin resistance in hemodialysis patients.

    PubMed

    Chen, Miao-Pei; Chen, Chien-Wen; Chen, Jin-Shuen; Mao, Hung-Chung; Chou, Chu-Lin

    2016-01-01

    Growth arrest-specific protein 6 (Gas6) works synergistically with erythropoietin (EPO) to increase the proliferation and maturation of erythroblasts. However, the role of Gas 6 levels on EPO resistance in hemodialysis (HD) patients remains unclear. Therefore, the objective of this study was the first to examine the correlation between plasma Gas6 levels and EPO resistance in HD patients. We enrolled 134 HD patients and 85 healthy individuals. The HD patients were divided into 2 groups: 98 non-EPO-resistant patients and 36 EPO-resistant patients. Plasma levels of Gas6, interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), and albumin were quantified. Compared with non-EPO-resistant patients, EPO-resistant patients had elevated plasma concentrations of Gas6 (15.4 ± 3.3 vs. 13.7 ± 3.2 ng/mL, P = 0.006), IL-6 (3.1 ± 3.1 vs. 2.1 ± 1.5 pg/mL, P = 0.009), and hs-CRP (12.7 ± 25.2 vs. 4.5 ± 5.5 mg/L, P = 0.002). In EPO-resistant HD patients, plasma Gas6 levels were negatively correlated with albumin levels (r = -0.388, P < 0.021). Elevated Gas6 levels are associated with EPO resistance in HD patients. Also, EPO resistance is related to inflammation and malnutrition. Thus, circulating Gas6 levels could be used as the potential marker in HD patients with EPO resistance. PMID:26788441

  13. Correlation between radiation dose and p53 protein expression levels in human lymphocytes.

    PubMed

    Cavalcanti, Mariana B; Fernandes, Thiago S; Silva, Edvane B; Amaral, Ademir

    2015-09-01

    The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed. PMID:26312422

  14. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure.

    PubMed

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-09-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (p<0.05) in CP 15% group among three CP levels. Relative abundance of Bacteroidetes phylum and bacterial genera including Leuconostoc, Bacillus, Atopostipes, Peptonphilus, Ruminococcaceae_uc, Bacteroides, and Pseudomonas was lower (p<0.05) in CP 15% than in CP 20% group. There was a positive correlation (p<0.05) between odorous compounds and bacterial genera: phenol, indole, iso-butyric acid, and iso-valeric acid with Atopostipes, p-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  15. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure

    PubMed Central

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-01-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (p<0.05) in CP 15% group among three CP levels. Relative abundance of Bacteroidetes phylum and bacterial genera including Leuconostoc, Bacillus, Atopostipes, Peptonphilus, Ruminococcaceae_uc, Bacteroides, and Pseudomonas was lower (p<0.05) in CP 15% than in CP 20% group. There was a positive correlation (p<0.05) between odorous compounds and bacterial genera: phenol, indole, iso-butyric acid, and iso-valeric acid with Atopostipes, p-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  16. Development of Multigene Expression Signature Maps at the Protein Level from Digitized Immunohistochemistry Slides

    PubMed Central

    Metzger, Gregory J.; Dankbar, Stephen C.; Henriksen, Jonathan; Rizzardi, Anthony E.; Rosener, Nikolaus K.; Schmechel, Stephen C.

    2012-01-01

    Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions. PMID:22438942

  17. Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells.

    PubMed

    Nishimura, Kazuhiro; Okudaira, Hiroyuki; Ochiai, Eriko; Higashi, Kyohei; Kaneko, Mayumi; Ishii, Itsuko; Nishimura, Tomoe; Dohmae, Naoshi; Kashiwagi, Keiko; Igarashi, Kazuei

    2009-11-01

    In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [(35)S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, beta subunit (Cct2); heterogeneous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5'-UTR of Cct2 mRNA. PMID:19427401

  18. Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

    PubMed Central

    Nicolau Jr., Dan V.; Paszek, Ewa; Fulga, Florin; Nicolau, Dan V.

    2014-01-01

    A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced “leopard skin”-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions

  19. Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation.

    PubMed

    Shiba, T; Inoguchi, T; Sportsman, J R; Heath, W F; Bursell, S; King, G L

    1993-11-01

    The increases in diacylglycerol (DAG) level and protein kinase C (PKC) activity have been characterized biochemically and functionally in the retina and the brain of diabetic rats as well as in cultured vascular cells. PKC specific activities were increased in the membraneous fraction of retina from streptozotocin (STZ)-induced diabetic rats and the genetically determined diabetic BB rats, respectively, after 1 or 2 wk of diabetes, compared with control. The ratio of total PKC activities from membraneous and cytosol fractions was also increased in the retina of diabetic rats. With diabetes, all the isoenzymes and the total DAG level were increased in the rat retina, whereas no changes were found in the rat brain. Insulin treatment normalized plasma glucose levels and partially prevented the increases in the membraneous PKC activity and all the isoenzymes in the retina. In the retinal endothelial cells, the total DAG level and PKC specific activities are increased by 36 and 22%, respectively, in the membraneous pool when the glucose levels are changed from 5.5 to 22 mM. Activation of PKC activity and isoform beta II by the vitreal injection of phorbol dibutyrate mimicked the abnormal retinal blood circulation observed in diabetic rats (2.22 +/- 0.24 vs. 1.83 +/- 0.40 s). Thus diabetes and elevated glucose levels will increase DAG level and PKC activities and its isoenzyme specifically in vascular cells and may affect retinal hemodynamics. PMID:8238505

  20. Intermediate divergence levels maximize the strength of structure-sequence correlations in enzymes and viral proteins.

    PubMed

    Jackson, Eleisha L; Shahmoradi, Amir; Spielman, Stephanie J; Jack, Benjamin R; Wilke, Claus O

    2016-07-01

    Structural properties such as solvent accessibility and contact number predict site-specific sequence variability in many proteins. However, the strength and significance of these structure-sequence relationships vary widely among different proteins, with absolute correlation strengths ranging from 0 to 0.8. In particular, two recent works have made contradictory observations. Yeh et al. (Mol. Biol. Evol. 31:135-139, 2014) found that both relative solvent accessibility (RSA) and weighted contact number (WCN) are good predictors of sitewise evolutionary rate in enzymes, with WCN clearly out-performing RSA. Shahmoradi et al. (J. Mol. Evol. 79:130-142, 2014) considered these same predictors (as well as others) in viral proteins and found much weaker correlations and no clear advantage of WCN over RSA. Because these two studies had substantial methodological differences, however, a direct comparison of their results is not possible. Here, we reanalyze the datasets of the two studies with one uniform analysis pipeline, and we find that many apparent discrepancies between the two analyses can be attributed to the extent of sequence divergence in individual alignments. Specifically, the alignments of the enzyme dataset are much more diverged than those of the virus dataset, and proteins with higher divergence exhibit, on average, stronger structure-sequence correlations. However, the highest structure-sequence correlations are observed at intermediate divergence levels, where both highly conserved and highly variable sites are present in the same alignment. PMID:26971720

  1. Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells.

    PubMed

    Paulo, Joao A; Gaun, Aleksandr; Gygi, Steven P

    2015-10-01

    Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  2. The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart.

    PubMed

    Gerber, W V; Yatskievych, T A; Antin, P B; Correia, K M; Conlon, R A; Krieg, P A

    1999-01-01

    In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development. PMID:10096065

  3. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells

    PubMed Central

    Paulo, Joao A.; Gaun, Aleksandr; Gygi, Steven P.

    2016-01-01

    Smoking is a risk factor in pancreatic disease, however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 proteins were quantified across all nine samples of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease and lysosomal proteins. In addition, we measured differences for ∼16,000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  4. Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D.

    PubMed

    Fukuzawa, Taku; Ishida, Junji; Kato, Akira; Ichinose, Taro; Ariestanti, Donna Maretta; Takahashi, Tomoya; Ito, Kunitoshi; Abe, Jumpei; Suzuki, Tomohiro; Wakana, Shigeharu; Fukamizu, Akiyoshi; Nakamura, Nobuhiro; Hirose, Shigehisa

    2013-01-01

    Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta(+/+) and Ig-Hepta(-/-) mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space. PMID:23922714

  5. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  6. 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

  7. 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

  8. No association between lck gene polymorphisms and protein level in type 1 diabetes.

    PubMed

    Nervi, Solange; Nicodeme, Sandra; Gartioux, Corinne; Atlan, Catherine; Lathrop, Marc; Reviron, Denis; Naquet, Philippe; Matsuda, Fumihiko; Imbert, Jean; Vialettes, Bernard

    2002-11-01

    We previously described a reduced expression of the protein tyrosine kinase Lck in T-cells from type 1 diabetic patients, the origin of which is still unknown. The human lck gene, located on chromosome 1p35-34.3, was evaluated as a candidate susceptibility gene for type 1 diabetes. A molecular scan of the sequence variations in the coding, the relevant promoter, and most of the intronic sequences of the lck gene (representing a total of 10.5 kb fragment) was performed in 187 Caucasian subjects including 91 type 1 diabetic patients and 96 normoglycemic control subjects. We identified 35 sequence variations, including one deletion and 34 single nucleotide polymorphisms (SNPs), 33 of them being new. Four variants were frequent but not significantly associated with diabetes or Lck protein level. Of the SNP variants, 11 were only found within the diabetic population and some were associated with low Lck protein levels. The low frequency of these polymorphisms did not permit any statistically significant correlations with the disease status, suggesting that the lck gene probably does not contribute to genetic susceptibility to type 1 diabetes. PMID:12401726

  9. 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

  10. Telomere protein RAP1 levels are affected by cellular aging and oxidative stress

    PubMed Central

    Swanson, Mark J.; Baribault, Michelle E.; Israel, Joanna N.; Bae, Nancy S.

    2016-01-01

    Telomeres are important for maintaining the integrity of the genome through the action of the shelterin complex. Previous studies indicted that the length of the telomere did not have an effect on the amount of the shelterin subunits; however, those experiments were performed using immortalized cells with stable telomere lengths. The interest of the present study was to observe how decreasing telomere lengths over successive generations would affect the shelterin subunits. As neonatal human dermal fibroblasts aged and their telomeres became shorter, the levels of the telomere-binding protein telomeric repeat factor 2 (TRF2) decreased significantly. By contrast, the levels of one of its binding partners, repressor/activator protein 1 (RAP1), decreased to a lesser extent than would be expected from the decrease in TRF2. Other subunits, TERF1-interacting nuclear factor 2 and protection of telomeres protein 1, remained stable. The decrease in RAP1 in the older cells occurred in the nuclear and cytoplasmic fractions. Hydrogen peroxide (H2O2) stress was used as an artificial means of aging in the cells, and this resulted in RAP1 levels decreasing, but the effect was only observed in the nuclear portion. Similar results were obtained using U251 glioblastoma cells treated with H2O2 or grown in serum-depleted medium. The present findings indicate that TRF2 and RAP1 levels decrease as fibroblasts naturally age. RAP1 remains more stable compared to TRF2. RAP1 also responds to oxidative stress, but the response is different to that observed in aging. PMID:27446538

  11. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    PubMed

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions. PMID:26478262

  12. Serum Levels of Surfactant Proteins in Patients with Combined Pulmonary Fibrosis and Emphysema (CPFE)

    PubMed Central

    Papaioannou, Andriana I.; Kostikas, Konstantinos; Manali, Effrosyni D.; Papadaki, Georgia; Roussou, Aneza; Spathis, Aris; Mazioti, Argyro; Tomos, Ioannis; Papanikolaou, Ilias; Loukides, Stelios; Chainis, Kyriakos; Karakitsos, Petros; Griese, Matthias; Papiris, Spyros

    2016-01-01

    Introduction Emphysema and idiopathic pulmonary fibrosis (IPF) present either per se or coexist in combined pulmonary fibrosis and emphysema (CPFE). Serum surfactant proteins (SPs) A, B, C and D levels may reflect lung damage. We evaluated serum SP levels in healthy controls, emphysema, IPF, and CPFE patients and their associations to disease severity and survival. Methods 122 consecutive patients (31 emphysema, 62 IPF, and 29 CPFE) and 25 healthy controls underwent PFTs, ABG-measurements, 6MWT and chest HRCT. Serum levels of SPs were measured. Patients were followed-up for 1-year. Results SP-A and SP-D levels differed between groups (p = 0.006 and p<0.001 respectively). In post-hoc analysis, SP-A levels differed only between controls and CPFE (p<0.05) and CPFE and emphysema (p<0.05). SP-D differed between controls and IPF or CPFE (p<0.001 for both comparisons). In IPF SP-B correlated to pulmonary function while SP-A, correlated to the Composite Physiological Index (CPI). Controls current smokers had higher SP-A and SP-D levels compared to non-smokers (p = 0.026 and p = 0.023 respectively). SP-D levels were higher in CPFE patients with extended emphysema (p = 0.042). In patients with IPF, SP-B levels at the upper quartile of its range (≥26 ng/mL) presented a weak association with reduced survival (p = 0.05). Conclusion In conclusion, serum SP-A and SP-D levels were higher where fibrosis exists or coexists and related to disease severity, suggesting that serum SPs relate to alveolar damage in fibrotic lungs and may reflect either local overproduction or overleakage. The weak association between high levels of SP-B and survival needs further validation in clinical trials. PMID:27337142

  13. EFFECT OF DIETARY PROTEIN AND CARBOHYDRATE LEVELS ON WEIGHT GAIN AND GONAD PRODUCTION IN THE SEA URCHIN LYTECHINUS VARIEGATUS

    PubMed Central

    Heflin, Laura E.; Gibbs, Victoria K.; Powell, Mickie L; Makowsky, Robert; Lawrence, John M.; Lawrence, Addison L.; Watts, Stephen A.

    2014-01-01

    Adult Lytechinus variegatus were fed eight formulated diets with different protein (ranging from 12 to 36%) and carbohydrate (ranging from 21 to 39 %) levels. Each sea urchin (n = 8 per treatment) was fed a daily sub-satiation ration of 1.5% of average body weight for 9 weeks. Akaike information criterion analysis was used to compare six different hypothesized dietary composition models across eight growth measurements. Dietary protein level and protein: energy ratio were the best models for prediction of total weight gain. Diets with the highest (> 68.6 mg P kcal−-1) protein: energy ratios produced the most wet weight gain after 9 weeks. Dietary carbohydrate level was a poor predictor for most growth parameters examined in this study. However, the model containing a protein × carbohydrate interaction effect was the best model for protein efficiency ratio (PER). PER decreased with increasing dietary protein level, more so at higher carbohydrate levels. Food conversion ratio (FCR) was best modeled by total dietary energy levels: Higher energy diets produced lower FCRs. Dietary protein level was the best model of gonad wet weight gain. These data suggest that variations in dietary nutrients and energy differentially affect organismal growth and growth of body components. PMID:24994942

  14. Analysis of the Sperm Head Protein Profiles in Fertile Men: Consistency across Time in the Levels of Expression of Heat Shock Proteins and Peroxiredoxins

    PubMed Central

    Kichine, Elsa; Di Falco, Marcos; Hales, Barbara F.; Robaire, Bernard; Chan, Peter

    2013-01-01

    We investigated the identity and quantitative variations of proteins extracted from human sperm heads using a label-free Gel-MS approach. Sperm samples were obtained from three men with high sperm counts at three different time points. This design allowed us to analyse intra-individual and inter-individual variations of the human sperm head proteome. Each time point was analyzed in triplicate to minimize any background artifactual effects of the methodology on the variation analyses. Intra-individual analysis using the spectral counting method revealed that the expression levels of 90% of the common proteins identified in three samples collected at various time-points, separated by several months, had a coefficient of variation of less than 0.5 for each man. Across individuals, the expression level of more than 80% of the proteins had a CV under 0.7. Interestingly, 83 common proteins were found within the core proteome as defined by the intra- and inter-variation analyses set criteria (CV<0.7). Some of these uniformly expressed proteins were chaperones, peroxiredoxins, isomerases, and cytoskeletal proteins. Although there is a significant level of inter-individual variation in the protein profiles of human sperm heads even in a well-defined group of men with high sperm counts, the consistent expression levels of a wide range of proteins points to their essential role during spermatogenesis. PMID:24204839

  15. Effect of altered eating pattern on serum fructosamine: total protein ratio and plasma glucose level.

    PubMed

    Ch'ng, S L; Cheah, S H; Husain, R; Duncan, M T

    1989-05-01

    The effect of alteration of eating pattern during Ramadan on body mass index (BMI), serum fructosamine: total protein ratio (F/TP), and glucose level in 18 healthy male Asiatic Moslems were studied. The results showed a significant decrease (p less than 0.025) in F/TP at the second week of Ramadan in 11 subjects who experienced continuous decrease in BMI throughout Ramadan. The remaining 7 subjects showed no significant changes in BMI and F/TP. No evidence of hypoglycaemia was observed in the subjects during the study. Serum fructosamine: total protein ratio in subjects with altered eating pattern preferably should be interpreted along with the change in body mass index. PMID:2774480

  16. A Theoretical Lower Bound for Selection on the Expression Levels of Proteins.

    PubMed

    Price, Morgan N; Arkin, Adam P

    2016-01-01

    We use simple models of the costs and benefits of microbial gene expression to show that changing a protein's expression away from its optimum by 2-fold should reduce fitness by at least [Formula: see text], where P is the fraction the cell's protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 10(6), this implies that 2-fold changes to gene expression levels are under strong selection, as [Formula: see text], where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral. PMID:27289091

  17. Utility of C-Reactive Protein Levels for Early Prediction of Dengue Severity in Adults

    PubMed Central

    Chen, Chien-Chih; Lee, Ing-Kit; Liu, Jien-Wei; Huang, Shi-Yu; Wang, Lin

    2015-01-01

    Dengue has broad clinical presentation with unpredictable clinical evolution and outcome. We aimed to evaluate the utility of C-reactive protein (CRP) levels for distinguishing between mild and severe cases in the early phase of the dengue illness. We retrospectively evaluated adults with dengue from 2006 to 2014, according to 1997 and 2009 World Health Organization (WHO) criteria for severity. Of 191 included patients, 32.9% had nonshock dengue hemorrhagic fever (DHF), 3.1% dengue shock syndrome (DSS), and 7.9% severe dengue. The risk of DHF/DSS and severe dengue is significantly related to the increasing levels of CRP. Of 191 patients, 97 had CRP levels measured during the febrile (days 1–3); 85 during the critical (days 4–6); and 9 during the convalescent (days 7–10) illness phases. During the febrile phase, there was significant higher CRP level for DSS versus DF/nonshock DHF and severe dengue versus nonsevere dengue, with CRP cutoff level 30.1 mg/L (area under the receiver operating characteristic curve (AUC), 0.938; 100% sensitivity, 76.3% specificity) and 24.2 mg/L (AUC, 0.717; 70% sensitivity, 71.3% specificity), respectively. Our study highlights the utility of the CRP levels in early prediction of DSS and severe dengue in adult patients. PMID:26247033

  18. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level.

    PubMed

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  19. An alternative method of enhancing the expression level of heterologous protein in Escherichia coli.

    PubMed

    Yin, Jun; Tian, Hong; Bao, Lichen; Dai, Xin; Gao, Xiangdong; Yao, Wenbing

    2014-12-12

    Though numerous strategy options are available for achieving high expression levels of genes in Escherichia coli, not every gene can be efficiently expressed in this organism. By investigating the relationship between the mRNA secondary structure of translational initiation region (TIR) and gene expression in E.coli, we establish a simple method to design sequences of appropriate TIR (from -35 to +36) that meet a specific expression level as we need. Using this method, overexpression of native human humor necrosis factor α and extracellular domain of Her2/neu protein (aa 23-146) in E. coli were achieved. Differences in expression appeared was mainly related to the efficiency of translation initiation and the stability of mRNA secondary structure, because the intracellular mRNA levels analyzed by real-time RT-PCR were quite similar. Our approach can overcome the steric hindrance of translation startup, and therefore promote translation smoothly to acquire high expression of exogenous protein. PMID:25449272

  20. Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels.

    PubMed

    Spellman, David; O'Cuinn, Gerard; FitzGerald, Richard J

    2005-05-01

    Whey protein hydrolysates were generated at different total solids (TS) levels (50-300 g/l) using the commercially available proteolytic preparation Debitrase HYW20, while enzyme to substrate ratio, pH and temperature were maintained constant. Hydrolysis proceeded at a faster rate at lower TS reaching a degree of hydrolysis (DH) of 16.6% at 300 g TS/l, compared with a DH of 22.7% at 50 g TS/l after 6 h hydrolysis. The slower breakdown of intact whey proteins at high TS was quantified by gel-permeation HPLC. Reversed-phase (RP) HPLC of hydrolysate samples of equivalent DH (approximately 15%) generated at different TS levels indicated that certain hydrophobic peptide peaks were present at higher levels in hydrolysates generated at low TS. Sensory evaluation showed that hydrolysates with equivalent DH values were significantly (P < 0.0005) less bitter when generated at 300 g TS/l (mean bitterness score = 25.4%) than hydrolysates generated at 50 g TS/l (mean bitterness score = 39.9%). A specific hydrophobic peptide peak present at higher concentrations in hydrolysates generated at low TS was isolated and identified as beta-lactoglobulin f(43-57), a fragment having the physical and chemical characteristics of a bitter peptide. PMID:15909678

  1. Association Between Serum Levels of Adipocyte Fatty Acid-binding Protein and Free Thyroxine

    PubMed Central

    Tseng, Fen-Yu; Chen, Pei-Lung; Chen, Yen-Ting; Chi, Yu-Chao; Shih, Shyang-Ron; Wang, Chih-Yuan; Chen, Chi-Ling; Yang, Wei-Shiung

    2015-01-01

    Abstract Adipocyte fatty acid-binding protein (AFABP) has been shown to be a biomarker of body weight change and atherosclerosis. Changes in thyroid function are associated with changes in body weight and risks of cardiovascular diseases. The association between AFABP and thyroid function status has been seldom evaluated. The aim of this study was to compare the serum AFABP concentrations in hyperthyroid patients and those in euthyroid individuals, and to evaluate the associations between serum AFABP and free thyroxine (fT4) levels. For this study, 30 hyperthyroid patients and 30 euthyroid individuals at a referral medical center were recruited. The patients with hyperthyroidism were treated with antithyroid regimens as clinically indicated. No medication was given to the euthyroid individuals. The body weight, body mass index, thyroid function, serum levels of AFABP, and biochemical data of both groups at baseline and at the 6th month were compared. Associations between AFABP and fT4 levels were also analyzed. At the baseline, the hyperthyroid patients had significantly higher serum AFABP levels than the euthyroid individuals (median [Q1, Q3]: 22.8 [19.4, 30.6] ng/mL vs 18.6 [15.3, 23.2] ng/mL; P = 0.038). With the antithyroid regimens, the AFABP serum levels of the hyperthyroid patients decreased to 16.6 (15.0, 23.9) ng/mL at the 6th month. No difference in the AFABP level was found between the hyperthyroid and the euthyroid groups at the 6th month. At baseline, sex (female vs male, ß = 7.65, P = 0.022) and fT4 level (ß = 2.51, P = 0.018) were significantly associated with AFABP levels in the univariate regression analysis. At the 6th month, sex and fT4 level (ß = 8.09, P < 0.001 and ß = 3.61, P = 0.005, respectively) were also significantly associated with AFABP levels. The associations between sex and fT4 level with AFABP levels remained significant in the stepwise multivariate regression analysis, both at baseline and at

  2. In vitro effect of dietary protein level and nondigestible oligosaccharides on feline fecal microbiota.

    PubMed

    Pinna, C; Stefanelli, C; Biagi, G

    2014-12-01

    The aim of the present study was to evaluate in vitro the effect of some prebiotic substances and 2 dietary protein levels on the composition and activity of feline fecal microbiota. Two in vitro studies were conducted. First, 6 nondigestible oligosaccharides were studied; treatments were control diet (CTRL), gluconic acid (GA), carrot fiber (CF), fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactitol (LAC), and pectins from citrus fruit (PEC). Substrates were added to feline fecal cultures at 2 g/L for 24 h incubation. Compared with the CTRL, ammonia had been reduced (P<0.05) by GOS (-9%) after 6 h and by GA (-14%), LAC (-12%), and PEC (-10%) after 24 h. After 24 h, all treatments had resulted in a lower pH versus the CTRL. Putrescine concentrations at 24 h were greater (P<0.05) in cultures treated with FOS (+90%), GOS (+96%), and LAC (+87%). Compared with the CTRL, total VFA were higher (P<0.05) in bottles containing CF (+41%), whereas the acetic to propionic acid ratio was reduced by LAC (-51%; P<0.05). After 24 h, Enterobacteriaceae had been reduced (P<0.05) by LAC and PEC. In a second study, LAC and FOS were selected to be tested in the presence of 2 diets differing in their protein content. There were 6 treatments: low-protein (LP) CTRL with no addition of prebiotics (CTRL-LP), high-protein (HP) CTRL with no addition of prebiotics (CTRL-HP), LP diet plus FOS, CTRL-HP plus FOS, LP diet plus LAC, and CTRL-HP plus LAC. Both FOS and LAC were added to feline fecal cultures at 2 g/L for 24 h incubation. Ammonia at 24 h was affected (P<0.05) by the protein level (36.2 vs. 50.2 mmol/L for LP and HP, respectively). The CTRL-HPs resulted in a higher pH and increased concentrations of biogenic amines were found after 6 and 24 h of incubation (P<0.05); putrescine at 24 h showed an increase (P<0.05) in cultures treated with FOS. Total VFA were influenced (P<0.05) by the protein level (40.9 vs. 32.6 mmol/L for LP and HP, respectively). At 24 h, the CTRL

  3. The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

    PubMed

    Ishikura, Shuhei; Iwaihara, Yuri; Tanaka, Yoko; Luo, Hao; Nishi, Kensuke; Doi, Keiko; Koyanagi, Midori; Okamura, Tadashi; Tsunoda, Toshiyuki; Shirasawa, Senji

    2016-07-15

    Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway. PMID:27226588

  4. The Prognostic Value of C-Reactive Protein Serum Levels in Patients with Uterine Leiomyosarcoma

    PubMed Central

    Schwameis, Richard; Grimm, Christoph; Petru, Edgar; Natter, Camilla; Staudigl, Christine; Lamm, Wolfgang; Koelbl, Heinz; Krainer, Michael; Brodowicz, Thomas; Reinthaller, Alexander; Polterauer, Stephan

    2015-01-01

    Objective C-reactive protein (CRP) has previously been shown to serve as a prognostic parameter in women with gynecologic malignancies. Due to the lack of valid prognostic markers for uterine leiomyosarcoma (ULMS) this study set out to investigate the value of pre-treatment CRP serum levels as prognostic parameter. Methods Data of women with ULMS were extracted from databases of three Austrian centres for gynaecologic oncology. Pre-treatment CRP serum levels were measured and correlated with clinico-pathological parameters. Univariate and multivariable survival analyses were performed. Results In total, 53 patients with ULMS were included into the analysis. Mean (SD) CRP serum level was 3.46 mg/dL (3.96). Solely, an association between pre-treatment CRP serum levels and tumor size (p = 0.04) but no other clinic-pathologic parameter such as tumor stage (p = 0.16), or histological grade (p = 0.07), was observed. Univariate and multivariable survival analyses revealed that CRP serum levels (HR 2.7 [1.1–7.2], p = 0.037) and tumor stage (HR 6.1 [1.9–19.5], p = 0.002) were the only independent prognostic factors for overall survival (OS) in patients with ULMS. Patients with high pre-treatment CRP serum levels showed impaired OS compared to women with low levels (5-year-OS rates: 22.6% and 52.3%, p = 0.007). Conclusion High pre-treatment CRP serum levels were independently associated with impaired prognosis in women with ULMS and might serve as a prognostic parameter in these patients. PMID:26248232

  5. Generalized Anxiety and C-Reactive Protein Levels: A Prospective, Longitudinal Analysis

    PubMed Central

    Copeland, William E.; Shanahan, Lilly; Worthman, Carol; Angold, Adrian; Costello, E. Jane

    2012-01-01

    Background Generalized Anxiety Disorder (GAD) is highly comorbid with depression. Depression is associated with elevated levels of the inflammation marker C-reactive protein (CRP), cross-sectionally and over time. To date, no studies have looked at the association of CRP with GAD. Methods Ten waves of data from the prospective population-based Great Smoky Mountains Study (N = 1,420) were used, covering children in the community aged 9-16, 19, and 21 years old. Structured interviews were used at each assessment to assess GAD symptoms, diagnosis, and cumulative episodes. Bloodspots were collected and assayed for CRP levels. Results GAD was associated with increased levels of CRP in bivariate cross-sectional analyses. These bivariate associations, however, were attenuated after accounting for demographic, substance use, and health-related covariates. In longitudinal models, there was little evidence that CRP predicted later GAD. Associations from GAD to later CRP were attenuated in models adjusted for health-related coavariates and there was evidence that the GAD-CRP association was mediated by BMI and medication use. Conclusions Similar to depression, GAD was associated with elevated levels of CRP, but the effect of GAD on CRP levels was explained by the effect of GAD on health-related behaviors such as BMI and medication use. This study suggests differences in the association between inflammation and depression and GAD. PMID:22716910

  6. The impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in eventing horses.

    PubMed

    Oliveira, C A A; Azevedo, J F; Martins, J A; Barreto, M P; Silva, V P; Julliand, V; Almeida, F Q

    2015-01-01

    This study was performed to evaluate the impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in conditioning eventing horses. Twenty-four Brazilian Sport Horses, male and female (8.0 to 15.0 yr; 488 ± 32 kg BW), were used in a randomized design with 4 levels of CP diets: 7.5%, 9.0%, 11.0%, and 13.0%. A digestion assay was performed with partial feces collection over 4 d, followed by 1 d of total urine collection. Data were submitted to regression analysis and adjusted to linear and quadratic models (P < 0.05). No differences were observed in the intake of DM, OM, EE, ADF, and NDF as a function of dietary protein levels. Dry matter intake average was 1.7% of BW. CP and N intake showed a linear increase as a function of increasing protein level in diets. A quadratic response (P < 0.05) was observed on the CP and NDF digestibility coefficients, with the maximum estimated level of digestibility at 11.6% and 11.4% CP in the diet, respectively. There was a linear effect on ADF digestibility coefficients, digestible DM and protein intake, and CP/DE ratio according to dietary protein levels. There was no impact of dietary protein levels on daily water intake, total water intake, or fecal water excretion. Urinary excretion values showed a linear increase in response to increased dietary protein levels, but no impact was observed on water balance, with an average of 8.4 L/d. Nitrogen intake (NI), N absorption (NA), and urinary N increased linearly as a function of increasing dietary protein levels. There was no impact of dietary protein levels on N retention (NR), with an average of 7.5 g N/d. Nitrogen retention as a percentage of NI or NA showed no significant changes in the function of dietary protein levels. There was an impact of dietary protein levels on the digestibility coefficient of CP, NDF, ADF, and digestible protein intake on conditioning eventing horses. The 11.6% CP level in the diet provided an intake of 2.25 g CP/kg BW

  7. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    PubMed

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  8. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate

    PubMed Central

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  9. BAG2 promotes tumorigenesis through enhancing mutant p53 protein levels and function

    PubMed Central

    Yue, Xuetian; Zhao, Yuhan; Liu, Juan; Zhang, Cen; Yu, Haiyang; Wang, Jiabei; Zheng, Tongsen; Liu, Lianxin; Li, Jun; Feng, Zhaohui; Hu, Wenwei

    2015-01-01

    Tumor suppressor p53 is the most frequently mutated gene in tumors. Many mutant p53 (mutp53) proteins promote tumorigenesis through the gain-of-function (GOF) mechanism. Mutp53 proteins often accumulate to high levels in tumors, which is critical for mutp53 GOF. Its underlying mechanism is poorly understood. Here, we found that BAG2, a protein of Bcl-2 associated athanogene (BAG) family, promotes mutp53 accumulation and GOF in tumors. Mechanistically, BAG2 binds to mutp53 and translocates to the nucleus to inhibit the MDM2-mutp53 interaction, and MDM2-mediated ubiquitination and degradation of mutp53. Thus, BAG2 promotes mutp53 accumulation and GOF in tumor growth, metastasis and chemoresistance. BAG2 is frequently overexpressed in tumors. BAG2 overexpression is associated with poor prognosis in patients and mutp53 accumulation in tumors. These findings revealed a novel and important mechanism for mutp53 accumulation and GOF in tumors, and also uncovered an important role of BAG2 in tumorigenesis through promoting mutp53 accumulation and GOF. DOI: http://dx.doi.org/10.7554/eLife.08401.001 PMID:26271008

  10. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    SciTech Connect

    Zangar, Richard C. ); Kocarek, Thomas A.; Shen, Shang; Bollinger, Nikki ); Dahn, Michael S.; Lee, Donna W.

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3 A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  11. The level of major urinary proteins is socially regulated in wild Mus musculus musculus.

    PubMed

    Janotova, Katerina; Stopka, Pavel

    2011-06-01

    Major urinary proteins (MUPs) are highly polymorphic proteins that have been shown to perform several important functions in the chemical communication of the house mouse, Mus musculus. Production of these proteins in C57Bl/6 females is cyclic, reaching the maximum just before the beginning of estrus. Social environment is an important factor that increases MUP production in both sexes. We examined responsiveness of MUP production to social stimuli in wild mice, Mus musculus musculus. The direction of change of MUP production in males depended on the sex of the stimulus animal. Males up-regulated MUP production when caged with a female, but down-regulated MUP production when caged with a male. Down-regulation was more pronounced in males that were defeated in a male-male encounter. Females responded to a male's presence with a decrease in MUP production. We conclude that social modulation of MUP production is specific and, in coordination with other mechanisms, facilitates adjustment of the animal's odor profile to different social contexts. Our results also suggest that in males, MUPs may play an important role in advertizing the male's quality to females. Furthermore, we highlight the importance of analyzing data corrected with creatinine, which show MUP production on the (post)translational level as well as raw data (non-corrected with creatinine), which represent actual concentrations of MUPs in the urine. PMID:21594616

  12. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    PubMed Central

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, Chris S.; Giloteaux, Ludovic; Montgomery, Alison P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  13. Postnatal alterations in GABAB receptor tone produce sensorimotor gating deficits and protein level differences in adulthood.

    PubMed

    Bolton, Monica M; Heaney, Chelcie F; Murtishaw, Andrew S; Sabbagh, Jonathan J; Magcalas, Christy M; Kinney, Jefferson W

    2015-04-01

    The GABA transmitter system plays a vital role in modulating synaptic formation and activity during development. The GABAB receptor subtype in particular has been implicated in cell migration, promotion of neuronal differentiation, neurite outgrowth, and synapse formation but it's role in development is not well characterized. In order to investigate the effects of brief alterations in GABAB signaling in development, we administered to rats the GABAB agonist baclofen (2.0mg/kg) or antagonist phaclofen (0.3mg/kg) on postnatal days 7, 9, and 12, and evaluated sensorimotor gating in adulthood. We also examined tissue for changes in multiple proteins associated with GABAB receptor function and proteins associated with synapse formation. Our data indicate that early postnatal alterations to GABAB receptor-mediated signaling produced sex differences in sensorimotor gating in adulthood. Additionally, we found differences in GABAB receptor subunits and kalirin protein levels in the brain versus saline treated controls. Our data demonstrate that a subtle alteration in GABAB receptor function in early postnatal life induces changes that persist into adulthood. PMID:25314921

  14. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations.

  15. Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface.

    PubMed

    Wilkins, Michael J; Wrighton, Kelly C; Nicora, Carrie D; Williams, Kenneth H; McCue, Lee Ann; Handley, Kim M; Miller, Chris S; Giloteaux, Ludovic; Montgomery, Alison P; Lovley, Derek R; Banfield, Jillian F; Long, Philip E; Lipton, Mary S

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  16. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    SciTech Connect

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-03-05

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

  17. Racial Differences in C-Reactive Protein Levels During Normal Pregnancy

    PubMed Central

    Picklesimer, Amy H; Jared, Heather L.; Moss, Kevin; Offenbacher, Steven; Beck, James D; Boggess, Kim A

    2008-01-01

    Objective Characterization of serum C-Reactive Protein (CRP) levels in a diverse population of healthy pregnant women using a high sensitivity assay. Study Design Cross-sectional analysis of a cohort of 775 pregnant women. CRP measured on serum specimens drawn <26 weeks gestation using highly sensitive ELISA kits. Results Median CRP was 4.8 mg/L (inter-quartile range 0.63 – 15.7). Black women had higher median CRP values than whites (7.68 mg/L vs 2.59 mg/L, p<.001). Black women demonstrated higher levels of CRP even after controlling for known confounding factors such as smoking and maternal weight. Conclusion Pregnancy is an inflammatory stressor. The etiology of racial differences is unclear, but may be important for understanding racial disparities in the incidence inflammatory disorders such as preterm labor and pre-eclampsia. PMID:18539258

  18. Cypermethrin alters Glial Fibrillary Acidic Protein levels in the rat brain.

    PubMed

    Malkiewicz, Katarzyna; Koteras, Marcin; Folkesson, Ronnie; Brzezinski, Jacek; Winblad, Bengt; Szutowski, Miroslaw; Benedikz, Eirikur

    2006-01-01

    Pyrethroids, widely used insecticides, are biologically active in neurons. Whether they act on the non-neuronal brain cells remains an open question. Thus, the aim of this study was to examine whether Cypermethrin intoxication affects astroglial cells in the rat brain. The levels of Glial Fibrillary Acidic Protein (GFAP) in different brain regions were measured by ELISA following oral treatment with 5 or 10% of LD(50) of Cypermethrin per day for 6 days. A significant decrease of GFAP was observed in different brain regions of treated animals. The cerebral cortex showed the most pronounced effect with GFAP levels reduced to 81% of the controls 2 days after treatment and 77% 21 days after treatment. Although we did not find profound changes in the morphology of astrocytes in Cypermethrin treated animals, the decrease in GFAP suggests that astrocytes were affected by low doses of pyrethroids. The possible consequences were discussed. PMID:21783638

  19. 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

  20. 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

  1. Increased CSF Levels of Phosphorylated Neurofilament Heavy Protein following Bout in Amateur Boxers

    PubMed Central

    Neselius, Sanna; Zetterberg, Henrik; Blennow, Kaj; Marcusson, Jan; Brisby, Helena

    2013-01-01

    Introduction Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI. Materials and Methods Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed. Results CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001). No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers. Conclusions Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI. PMID:24260563

  2. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors

    PubMed Central

    González, Natalia; Moresco, James J.; Bustos, Francisco; Yates, John R.; Olguín, Hugo C.

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  3. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

    PubMed

    González, Natalia; Moresco, James J; Cabezas, Felipe; de la Vega, Eduardo; Bustos, Francisco; Yates, John R; Olguín, Hugo C

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  4. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels. PMID:26244518

  5. Infection and immunoglobulin levels in Sudanese children with severe protein-energy malnutrition*

    PubMed Central

    Suliman, Omer S. M.; Salih, Mustafa A. M.; Karrar, Zein A.; Mohammed, Abdelrahim O.; Helsing, Chrestover

    2011-01-01

    A hospital-based case control study was carried out to determine the pattern of infections and immunoblobulin levels in Sudanese children with severe protein energy malnutrition (PEM). The pre-dietary rehabilitation levels of the three major immunoglobulins (IgG, IgA and IgM) were compared with those of normal controls, and with the levels after dietary rehabilitation. Eighty one children were included in the study: 49 with severe PEM (23 with marasmus, 17 with marasmic – kwashiorkor and 9 with kwashiorkor), 13 with tuberculosis and 19 healthy children as controls. The study showed high incidence of infections, especially pneumonia and gastrointestinal infections in the malnourished children. Of special concern was the high incidence of urinary tract infection: 13 (26.5%) had significant pyuria and 9 of them had positive urine cultures, mainly Escherichia coli. Eight of the malnourished children also had pulmonary TB, and the ESR and Mantoux tests were not helpful in the diagnosis. The Mantoux test was negative in 88.8% of the malnourished group compared to 62.5% in those malnourished with TB. The malnourished groups had significantly higher plasma levels of the 3 immunoglobulins. While the maramic group attained significantly higher levels of IgG and IgA compared to the marasmic –kwashiorkor and kwashiorkor groups, the 3 groups of PEM showed a uniformly higher level of the IgM. After 2 weeks of rehabilitation, the levels of the 3 immunoglobulins showed no significant changes, except for the IgA which significantly decreased in all malnourished and the oedematous groups, and the IgM which increased significantly in the oedematous group.

  6. Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae.

    PubMed Central

    Hood, M A; Guckert, J B; White, D C; Deck, F

    1986-01-01

    The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation. Images PMID:2430523

  7. Myb-domain protein Teb1 controls histone levels and centromere assembly in fission yeast

    PubMed Central

    Valente, Luis P; Dehé, Pierre-Marie; Klutstein, Michael; Aligianni, Sofia; Watt, Stephen; Bähler, Jürg; Promisel Cooper, Julia

    2013-01-01

    The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function—the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity. PMID:23314747

  8. Myb-domain protein Teb1 controls histone levels and centromere assembly in fission yeast.

    PubMed

    Valente, Luis P; Dehé, Pierre-Marie; Klutstein, Michael; Aligianni, Sofia; Watt, Stephen; Bähler, Jürg; Cooper, Julia Promisel

    2013-02-01

    The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function-the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity. PMID:23314747

  9. Maternal High Fat Diet Affects Offspring’s Vitamin K-Dependent Proteins Expression Levels

    PubMed Central

    Lanham, Stuart; Cagampang, Felino R.; Oreffo, Richard O. C.

    2015-01-01

    Studies suggest bone growth & development and susceptibility to vascular disease in later life are influenced by maternal nutrition, during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including Osteocalcin, Matrix-gla protein, Periostin, and Gas6, in bone and vascular development. This study extends the analysis of VKDPs previously conducted in 6 week old offspring, into offspring of 30 weeks of age, to assess the longer term effects of a maternal and postnatal high fat (HF) diet on VKDP expression. Overall a HF maternal diet and offspring diet exacerbated the bone changes observed. Sex specific and tissue specific differences were observed in VKDP expression for both aorta and femoral tissues. In addition, significant correlations were observed between femoral OCN, Periostin Gas6, and Vkor expression levels and measures of femoral bone structure. Furthermore, MGP, OCN, Ggcx and Vkor expression levels correlated to mass and fat volume, in both sexes. In summary the current study has highlighted the importance of the long-term effects of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure. PMID:26381752

  10. Heat shock proteins in relation to heat stress tolerance of creeping bentgrass at different N levels.

    PubMed

    Wang, Kehua; Zhang, Xunzhong; Goatley, Mike; Ervin, Erik

    2014-01-01

    Heat stress is a primary factor causing summer bentgrass decline. Changes in gene expression at the transcriptional and/or translational level are thought to be a fundamental mechanism in plant response to environmental stresses. Heat stress redirects protein synthesis in higher plants and results in stress protein synthesis, particularly heat shock proteins (HSPs). The goal of this work was to analyze the expression pattern of major HSPs in creeping bentgrass (Agrostis stolonifera L.) during different heat stress periods and to study the influence of nitrogen (N) on the HSP expression patterns. A growth chamber study on 'Penn-A4' creeping bentgrass subjected to 38/28°C day/night for 50 days, was conducted with four nitrate rates (no N-0, low N-2.5, medium N-7.5, and high N-12.5 kg N ha-1) applied biweekly. Visual turfgrass quality (TQ), normalized difference vegetation index (NDVI), photochemical efficiency of photosystem II (Fv/Fm), shoot electrolyte leakage (ShEL), and root viability (RV) were monitored, along with the expression pattern of HSPs. There was no difference in measured parameters between treatments until week seven, except TQ at week five. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower ShEL and higher RV, suggesting a major role in improved heat tolerance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and their roles in bentgrass thermotolerance. In addition, their production seems to be resource dependent. This study could further improve our understanding about how different N levels affect bentgrass thermotolerance. PMID:25050702

  11. Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.

    PubMed

    Martínez-de la Cruz, Enrique; García-Ramírez, Elpidio; Vázquez-Ramos, Jorge M; Reyes de la Cruz, Homero; López-Bucio, José

    2015-03-15

    Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins. PMID:25615607

  12. Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

    PubMed

    Kawabata, Fuminori; Mizushige, Takafumi; Uozumi, Keisuke; Hayamizu, Kohsuke; Han, Li; Tsuji, Tomoko; Kishida, Taro

    2015-01-01

    In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type. PMID:25198797

  13. Effects of Different Exercise Intensities with Isoenergetic Expenditures on C-Reactive Protein and Blood Lipid Levels

    ERIC Educational Resources Information Center

    Tsao, Te Hung; Yang, Chang Bin; Hsu, Chin Hsing

    2012-01-01

    We investigated the effects of different exercise intensities on C-reactive protein (CRP), and whether changes in CRP levels correlated with blood lipid levels. Ten men exercised at 25%, 65%, and 85% of their maximum oxygen consumption rates. Participants' blood was analyzed for CRP and blood lipid levels before and after the exercise sessions.…

  14. Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

    2016-03-01

    Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

  15. Increased Circulating Levels of Vitamin D Binding Protein in MS Patients

    PubMed Central

    Rinaldi, Arturo Ottavio; Sanseverino, Isabella; Purificato, Cristina; Cortese, Antonio; Mechelli, Rosella; Francisci, Silvia; Salvetti, Marco; Millefiorini, Enrico; Gessani, Sandra; Gauzzi, Maria Cristina

    2015-01-01

    Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS. PMID:25590278

  16. Serum C-reactive protein level as a biomarker for differentiation of ischemic from hemorrhagic stroke.

    PubMed

    Roudbary, Seyed Ali; Saadat, Farshid; Forghanparast, Kambiz; Sohrabnejad, Reza

    2011-01-01

    Cerebrovascular accidents rank first in the frequency and importance among all neurological disease. Although a number of studies had shown increased level of the high sensitive C-reactive protein (hs-CRP) in patients with ischemic stroke, the association of increased hs-CRP with various type of stroke especially the assessment hs-CRP level in ischemic and hemorrhagic stroke have not been investigated. In the present study, we assessed the concentration of hs-CRP in patients with documented ischemic and hemorrhagic stroke in the first 24 hours of the onset of symptoms. Thirty-two patients with Ischemic and hemorrhagic stroke were evaluated at neurology department of Poursina Hospital. The presence of baseline vascular risk factors, including hypertension, diabetes mellitus, hypercholesterolemia, obesity, and smoking, was determined. The blood samples were then collected and routine hematology and biochemistry tests were done. hs-CRP levels were determined using a highly sensitive immunonephelometric method. In this cross sectional study, the age of patient varied from 45-85 years (Mean 70.9 ± 9.4). Serum level of hs-CRP in Ischemic patients were 18.92 ± 11.28 and in hemorrhagic group was 2.65 ± 1.7. This relationship was statistically significant (P<0.0001). It might be concluded that hs-CRP might be considered as a usefully adjunct method for the initial diagnosis of the type of stroke. PMID:21681701

  17. ELEVATED LEVELS OF SOLUBLE ST2 PROTEIN IN DENGUE VIRUS INFECTED PATIENTS

    PubMed Central

    Becerra, Aniuska; Warke, Rajas V.; de Bosch, Norma; Rothman, Alan L.; Bosch, Irene

    2008-01-01

    Levels of the soluble form of the interleukin-1 receptor like 1 protein (IL-1RL-1 / ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty four patients with confirmed dengue infection, classified as dengue fever, and eleven patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004 respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections (p=0.047 at last day of fever and p=0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection. PMID:18226917

  18. Association of exome sequences with plasma C-reactive protein levels in >9000 participants

    PubMed Central

    Schick, Ursula M.; Auer, Paul L.; Bis, Joshua C.; Lin, Honghuang; Wei, Peng; Pankratz, Nathan; Lange, Leslie A.; Brody, Jennifer; Stitziel, Nathan O.; Kim, Daniel S.; Carlson, Christopher S.; Fornage, Myriam; Haessler, Jeffery; Hsu, Li; Jackson, Rebecca D.; Kooperberg, Charles; Leal, Suzanne M.; Psaty, Bruce M.; Boerwinkle, Eric; Tracy, Russell; Ardissino, Diego; Shah, Svati; Willer, Cristen; Loos, Ruth; Melander, Olle; Mcpherson, Ruth; Hovingh, Kees; Reilly, Muredach; Watkins, Hugh; Girelli, Domenico; Fontanillas, Pierre; Chasman, Daniel I.; Gabriel, Stacey B.; Gibbs, Richard; Nickerson, Deborah A.; Kathiresan, Sekar; Peters, Ulrike; Dupuis, Josée; Wilson, James G.; Rich, Stephen S.; Morrison, Alanna C.; Benjamin, Emelia J.; Gross, Myron D.; Reiner, Alex P.

    2015-01-01

    C-reactive protein (CRP) concentration is a heritable systemic marker of inflammation that is associated with cardiovascular disease risk. Genome-wide association studies have identified CRP-associated common variants associated in ∼25 genes. Our aims were to apply exome sequencing to (1) assess whether the candidate loci contain rare coding variants associated with CRP levels and (2) perform an exome-wide search for rare variants in novel genes associated with CRP levels. We exome-sequenced 6050 European-Americans (EAs) and 3109 African-Americans (AAs) from the NHLBI-ESP and the CHARGE consortia, and performed association tests of sequence data with measured CRP levels. In single-variant tests across candidate loci, a novel rare (minor allele frequency = 0.16%) CRP-coding variant (rs77832441-A; p.Thr59Met) was associated with 53% lower mean CRP levels (P = 2.9 × 10−6). We replicated the association of rs77832441 in an exome array analysis of 11 414 EAs (P = 3.0 × 10−15). Despite a strong effect on CRP levels, rs77832441 was not associated with inflammation-related phenotypes including coronary heart disease. We also found evidence for an AA-specific association of APOE-ε2 rs7214 with higher CRP levels. At the exome-wide significance level (P < 5.0 × 10−8), we confirmed associations for reported common variants of HNF1A, CRP, IL6R and TOMM40-APOE. In gene-based tests, a burden of rare/lower frequency variation in CRP in EAs (P ≤ 6.8 × 10−4) and in retinoic acid receptor-related orphan receptor α (RORA) in AAs (P = 1.7 × 10−3) were associated with CRP levels at the candidate gene level (P < 2.0 × 10−3). This inquiry did not elucidate novel genes, but instead demonstrated that variants distributed across the allele frequency spectrum within candidate genes contribute to CRP levels. PMID:25187575

  19. Association of exome sequences with plasma C-reactive protein levels in >9000 participants.

    PubMed

    Schick, Ursula M; Auer, Paul L; Bis, Joshua C; Lin, Honghuang; Wei, Peng; Pankratz, Nathan; Lange, Leslie A; Brody, Jennifer; Stitziel, Nathan O; Kim, Daniel S; Carlson, Christopher S; Fornage, Myriam; Haessler, Jeffery; Hsu, Li; Jackson, Rebecca D; Kooperberg, Charles; Leal, Suzanne M; Psaty, Bruce M; Boerwinkle, Eric; Tracy, Russell; Ardissino, Diego; Shah, Svati; Willer, Cristen; Loos, Ruth; Melander, Olle; Mcpherson, Ruth; Hovingh, Kees; Reilly, Muredach; Watkins, Hugh; Girelli, Domenico; Fontanillas, Pierre; Chasman, Daniel I; Gabriel, Stacey B; Gibbs, Richard; Nickerson, Deborah A; Kathiresan, Sekar; Peters, Ulrike; Dupuis, Josée; Wilson, James G; Rich, Stephen S; Morrison, Alanna C; Benjamin, Emelia J; Gross, Myron D; Reiner, Alex P

    2015-01-15

    C-reactive protein (CRP) concentration is a heritable systemic marker of inflammation that is associated with cardiovascular disease risk. Genome-wide association studies have identified CRP-associated common variants associated in ∼25 genes. Our aims were to apply exome sequencing to (1) assess whether the candidate loci contain rare coding variants associated with CRP levels and (2) perform an exome-wide search for rare variants in novel genes associated with CRP levels. We exome-sequenced 6050 European-Americans (EAs) and 3109 African-Americans (AAs) from the NHLBI-ESP and the CHARGE consortia, and performed association tests of sequence data with measured CRP levels. In single-variant tests across candidate loci, a novel rare (minor allele frequency = 0.16%) CRP-coding variant (rs77832441-A; p.Thr59Met) was associated with 53% lower mean CRP levels (P = 2.9 × 10(-6)). We replicated the association of rs77832441 in an exome array analysis of 11 414 EAs (P = 3.0 × 10(-15)). Despite a strong effect on CRP levels, rs77832441 was not associated with inflammation-related phenotypes including coronary heart disease. We also found evidence for an AA-specific association of APOE-ε2 rs7214 with higher CRP levels. At the exome-wide significance level (P < 5.0 × 10(-8)), we confirmed associations for reported common variants of HNF1A, CRP, IL6R and TOMM40-APOE. In gene-based tests, a burden of rare/lower frequency variation in CRP in EAs (P ≤ 6.8 × 10(-4)) and in retinoic acid receptor-related orphan receptor α (RORA) in AAs (P = 1.7 × 10(-3)) were associated with CRP levels at the candidate gene level (P < 2.0 × 10(-3)). This inquiry did not elucidate novel genes, but instead demonstrated that variants distributed across the allele frequency spectrum within candidate genes contribute to CRP levels. PMID:25187575

  20. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    PubMed

    Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

    2015-01-01

    Dietary protein levels and cysteamine (CS) supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg) were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP) levels (14% or 10%) and CS supplemental levels (0 or 700 mg/kg). The low-protein (LP) diets (10% CP) were supplemented with enough essential amino acids (EAA) to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS) (P<0.01) and plasma urea nitrogen (PUN) (P<0.001), while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001) and lean percentage (P<0.05), and decreased the feed conversion ratio (P<0.05) and back fat (P<0.05). CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1) (P<0.001), and reduced the concentrations of leptin, SS, and PUN (P<0.001). Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001) and decreased mRNA abundance of Forkhead Box O (FOXO) 4 (P<0.01) and muscle atrophy F-box (P<0.001) were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target

  1. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs

    PubMed Central

    Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

    2015-01-01

    Dietary protein levels and cysteamine (CS) supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg) were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP) levels (14% or 10%) and CS supplemental levels (0 or 700 mg/kg). The low-protein (LP) diets (10% CP) were supplemented with enough essential amino acids (EAA) to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS) (P<0.01) and plasma urea nitrogen (PUN) (P<0.001), while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001) and lean percentage (P<0.05), and decreased the feed conversion ratio (P<0.05) and back fat (P<0.05). CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1) (P<0.001), and reduced the concentrations of leptin, SS, and PUN (P<0.001). Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001) and decreased mRNA abundance of Forkhead Box O (FOXO) 4 (P<0.01) and muscle atrophy F-box (P<0.001) were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target

  2. mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells

    PubMed Central

    2014-01-01

    Background Glioblastoma multiforme (GBM), the most common and most aggressive type of primary adult brain tumour, responds poorly to conventional treatment. Temozolomide (TMZ) chemotherapy remains the most commonly used treatment, despite a large proportion of tumours displaying TMZ resistance. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Tumours also often exhibit hyperactive PI3-kinase/mTOR signalling, which enables them to resynthesise proteins quickly. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity. Methods MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro. Results We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis. Western blots show that despite this, relative to loading control proteins, steady state levels of MGMT protein increased and MGMT mRNA was retained in heavy polysomes. Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels. Conclusions These in vitro data suggest that, counterintuitively, mTOR inhibition may not be a useful adjunct to TMZ therapy and that more investigation is needed before applying mTOR inhibitors in a clinical setting. PMID:24909675

  3. Cumulative Depression Episodes Predicts Later C-Reactive Protein Levels: A Prospective Analysis

    PubMed Central

    Copeland, William E.; Shanahan, Lilly; Worthman, Carol; Angold, Adrian; Costello, E. Jane

    2013-01-01

    Background Depression is associated with elevated levels of the inflammation marker C -reactive protein (CRP), yet the direction of this association remains unclear. This study tested bi-directional longitudinal associations between CRP and depression in a sample of adolescent and young adults. The study compared the effects of current depression to the cumulative episodes of depression over time. Methods Nine waves of data from the prospective population-based Great Smoky Mountains Study (N = 1,420) were used, covering children in the community aged 9–16, 19, and 21 years old. Structured interviews were used to assess depressive symptoms, depression diagnosis, and cumulative depressive episodes. Bloodspots were collected at each observation and assayed for CRP levels. Results CRP levels were not associated with later depression status. In contrast, all depression-related variables displayed evidence of association with later CRP levels. The associations with depressive symptoms and diagnostic status were attenuated after controlling for covariates particularly body mass index, smoking, and medication use. The effect of cumulative depressive episodes, however, continued to be significant after accounting for a range of covariates. Body mass index, smoking behavior and recent infections may mediate a portion of the effect of cumulative episodes on later CRP, but cumulative depressive episodes continued to predict CRP levels independently. Conclusions The occurrence of multiple depressive episodes exerted the greatest effect on later CRP levels. This suggests that risk for the diseases of middle age - cardiovascular and metabolic disease – may begin in childhood and depend, in part, upon long-term emotional functioning. PMID:22047718

  4. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    SciTech Connect

    Sampson, D.A.; Jansen, G.R.

    1985-04-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

  5. Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

    SciTech Connect

    Celis, J.E.; Madsen, P.; Nielsen, S.; Ratz, G.P.; Lauridsen, J.B.; Celis, A.

    1987-02-01

    A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of (/sup 35/S)methionine-labelled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.

  6. Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype.

    PubMed

    Taylor, Janice M W; Allen, Anne-Marie; Graham, Annette

    2014-11-01

    The aim of the present study was to establish mitochondrial cholesterol trafficking 18 kDa translocator protein (TSPO) as a potential therapeutic target, capable of increasing macrophage cholesterol efflux to (apo)lipoprotein acceptors. Expression and activity of TSPO in human (THP-1) macrophages were manipulated genetically and by the use of selective TSPO ligands. Cellular responses were analysed by quantitative PCR (Q-PCR), immunoblotting and radiolabelling, including [3H]cholesterol efflux to (apo)lipoprotein A-I (apoA-I), high-density lipoprotein (HDL) and human serum. Induction of macrophage cholesterol deposition by acetylated low-density lipoprotein (AcLDL) increased expression of TSPO mRNA and protein, reflecting findings in human carotid atherosclerosis. Transient overexpression of TSPO enhanced efflux (E%) of [3H]cholesterol to apoA-I, HDL and human serum compared with empty vector (EV) controls, whereas gene knockdown of TSPO achieved the converse. Ligation of TSPO (using PK11195, FGIN-1-27 and flunitrazepam) triggered increases in [3H]cholesterol efflux, an effect that was amplified in TSPO-overexpressing macrophages. Overexpression of TSPO induced the expression of genes [PPARA (peroxisome-proliferator-activated receptor α), NR1H3 (nuclear receptor 1H3/liver X receptor α), ABCA1 (ATP-binding cassette A1), ABCG4 (ATP-binding cassette G4) and APOE (apolipoprotein E)] and proteins (ABCA1 and PPARα) involved in cholesterol efflux, reduced macrophage neutral lipid mass and lipogenesis and limited cholesterol esterification following exposure to AcLDL. Thus, targeting TSPO reduces macrophage lipid content and prevents macrophage foam cell formation, via enhanced cholesterol efflux to (apo)lipoprotein acceptors. PMID:24814875

  7. Identification of a functional SNP in the 3'-UTR of caprine MTHFR gene that is associated with milk protein levels.

    PubMed

    An, Xiaopeng; Song, Yuxuan; Hou, Jinxing; Wang, Shan; Gao, Kexin; Cao, Binyun

    2016-08-01

    Xinong Saanen (n = 305) and Guanzhong (n = 317) dairy goats were used to detect SNPs in the caprine MTHFR 3'-UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494 G>A locus in both dairy goat breeds (P < 0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta-miR-370 with the MTHFR 3'-UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level. PMID:27062401

  8. Chronic Low Level Complement Activation within the Eye Is Controlled by Intraocular Complement Regulatory Proteins

    PubMed Central

    Sohn, Jeong-Hyeon; Kaplan, Henry J.; Suk, Hye-Jung; Bora, Puran S.; Bora, Nalini S.

    2007-01-01

    -Crry mAb. Intracameral injection of anti-rat CD59 (6D1), anti-rat MHC class I antigen (OX-18), anti-rat Ig (G-16-510E3), or MOPC-21 caused no inflammatory reaction. Conclusions The results suggest that the complement system is continuously active at a low level in the normal eye and is tightly regulated by intraocular complement regulatory proteins. PMID:11006244

  9. C-reactive protein levels in stable COPD patients: a case-control study

    PubMed Central

    Silva, Denise Rossato; Gazzana, Marcelo Basso; Knorst, Marli Maria

    2015-01-01

    Background Previous studies have documented that C-reactive protein (CRP) levels are increased in stable COPD patients. However, most studies have also shown that higher CRP levels are observed in patients with comorbidities like diabetes mellitus and cardiovascular disease. We aimed to investigate if CRP levels are increased in stable COPD patients, and if there is an association between CRP levels and pulmonary function tests and clinical characteristics. Methods We conducted a case-control study in a tertiary care, university-affiliated hospital. COPD patients and controls were matched for sex and age in a 2:1 matching ratio. We included only those patients who had quit smoking. CRP levels were determined and pulmonary function tests were performed in both the groups. Results A total of 60 COPD patients and 30 controls were included in the analysis. The study subjects had a mean age of 64.8±8.5 years in COPD group and 64.3±9.2 years in control group (P=0.214). The median of CRP levels was 3.17 mg/L (interquartile range [IQR]: 1.73–5.99 mg/L) in COPD group and 2.13 mg/L (IQR: 1.18–7.69 mg/L) in control group (P=0.370). There were 34 (56.7%) patients in COPD group and 14 (46.7%) patients in control group with CRP levels greater than 3 mg/dL (P=0.382). Using bivariate correlations, we found significant positive correlations in COPD patients between body mass index (BMI) and CRP (r=0.3, P=0.045), and between CRP and forced vital capacity (FVC, % of predicted) (r=−0.3; P=0.023). In a multivariate model, female sex and FVC (% of predicted) were associated with a CRP value greater than 3 mg/dL in the COPD group. Conclusion The levels of CRP in the stable COPD patients were not significantly different when compared to those in the control subjects. Female sex and FVC (% predicted) were associated with CRP levels greater than 3 mg/dL in the COPD group. PMID:26357470

  10. A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels.

    PubMed

    Juhas, Matthias; von Zadow, Andrea; Spexard, Meike; Schmidt, Matthias; Kottke, Tilman; Büchel, Claudia

    2014-05-01

    Diatoms possess several genes for proteins of the cryptochrome/photolyase family. A typical sequence for a plant cryptochrome was not found in our analysis of the Phaeodactylum tricornutum genome, but one protein grouped with higher plant and green algal cryptochromes. This protein, CryP, binds FAD and 5,10-methenyltetrahydrofolate, according to our spectroscopic studies on heterologously expressed protein. 5,10-Methenyltetrahydrofolate binding is a feature common to both cyclobutane pyrimidine dimer photolyases and DASH cryptochromes. In recombinant CryP, however, the FAD chromophore was present in its neutral radical state and had a red-shifted absorption maximum at 637 nm, which is more characteristic for a DASH cryptochrome than a cyclobutane pyrimidine dimer photolyase. Upon illumination with blue light, the fully reduced state of FAD was formed in the presence of reductant. Expression of CryP was silenced by antisense approaches, and the resulting cell lines showed increased levels of proteins of light-harvesting complexes, the Lhcf proteins, in vivo. In contrast, the levels of proteins active in light protection, the Lhcx proteins, were reduced. Thus, CryP cannot be directly grouped with known members of the cryptochrome/photolyase family. Of all P. tricornutum proteins, it is the most similar in sequence to a plant cryptochrome, and is involved in the regulation of light-harvesting protein expression, but shows spectroscopic features and a chromophore composition that are most typical of a DASH cryptochrome. PMID:24628952

  11. Prediction of the Grade of Acute Cholecystitis by Plasma Level of C-Reactive Protein

    PubMed Central

    Kabul Gurbulak, Esin; Gurbulak, Bunyamin; Akgun, Ismail Ethem; Duzkoylu, Yigit; Battal, Muharrem; Fevzi Celayir, Mustafa; Demir, Uygar

    2015-01-01

    Background: Acute cholecystitis is the most common complication of gallbladder stones. Today, Tokyo guidelines criteria are recommended for diagnosis, grading, and management of acute cholecystitis. Objectives: We aimed to evaluate the levels of C-reactive protein (CRP) at different cut-off values to predict the severity of the disease and its possible role in grading the disease with regard to the guideline. Patients and Methods: This is a retrospective study, analyzing 682 cases out of consecutive 892 patients with acute cholecystitis admitted to two different general surgery clinics in Istanbul, Turkey. Records of patients diagnosed with acute cholecystitis were screened retrospectively from the hospital computer database between January 2011 and July 2014. A total of 210 patients with concomitant diseases causing high CRP levels were excluded from the study. The criteria of Tokyo guidelines were used in grading the severity of acute cholecystitis, and patients were divided into 3 groups. CRP values at the time of admission were analyzed and compared among the groups. Results: Mean CRP levels of groups were found to be significantly different, 18.96 mg/L in Group I, 133.51 mg/L in Group II, and 237.23 mg/L in Group III (P < 0.001). Having examined CRP values among the groups, they were found to be highly and significantly correlated with the disease grade (P < 0.0001). After evaluating CRP levels according to the grade of the disease, group 2 was distinguished from group 1 with a cut-off CRP level of 70.65 mg/L, and from group 3 with a value of 198.95 mg/L. Those results were found to be statistically significant (P < 0.001). Conclusions: CRP, a well-known acute phase reactant that increases rapidly in various inflammatory processes, can be accepted as a strong predictor in classifying different grades of the disease, and treatment can be reliably planned according to this classification. PMID:26023353

  12. Retinol Binding Protein-4 and Adiponectin Levels in Thyroid Overt and Subclinical Dysfunction.

    PubMed

    Kokkinos, S; Papazoglou, D; Zisimopoulos, A; Papanas, N; Tiaka, E; Antonoglou, C; Maltezos, E

    2016-02-01

    Thyroid dysfunction is accompanied by numerous changes in intermediary metabolism. Retinol binding protein-4 (RBP-4) and adiponectin are 2 adipocytokines that have multiple metabolic functions. The aim of our study was to examine serum RBP4 and adiponectin levels in clinical (before and after therapy) and subclinical hyperthyroid and hypothyroid subjects as compared to controls.150 patients with thyroid dysfunction were recruited (65 hyperthyroid and 85 hypothyroid) while 28 euthyroid subjects served as a control group. We measured anthropometric, biochemical and hormonal (free T4, free T3, TSH, insulin) parameters in all participants. RBP-4 and adiponectin were measured using commercial ELISA kits.Mean baseline levels of RBP-4 were higher in patients with clinical hypothyroidism (29.0±10.2 ng/ml, 25.1±12.6 ng/ml, 38.8±16.5 ng/ml, 31.9±13.2 ng/ml, 20.4±8.2 ng/ml in patients with hyperthyroidism, subclinical hyperthryrodism, hypothyroidism, subclinical hypothyroidism and controls respectively, F=4.86, P<0.001) and decreased significantly in patients with clinical hyperthyroidism and hypothyroidism after normalization of thyroid hormones' levels (from 29.0±10.2 to 24.9±8.4 ng/ml, p=0.003 and from 38.8±16.5 to 29.0±10.8 ng/ml, p=0.001 respectively). We did not observe analogous changes in adiponectin levels in any of the studied groups.RBP-4 levels are higher in patients with clinical hypothyroidism and exhibit a marked decrease after normalization of thyroid function in both hyper and hypothyroid patients. We suggest that RBP-4 may play a role in the metabolic disturbances which accompany thyroid dysfunction. PMID:26575118

  13. A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics.

    PubMed

    Aydin, Suleyman

    2007-01-31

    During the past decade, many salivary parameters have been used to characterize disease states. Ghrelin (GAH) is recently-discovered peptide hormone secreted mainly from the stomach but also produced in a number of other tissues including salivary glands. The aim of this work was to examine the relationship between active (aGAH) and inactive (dGAH) ghrelin in the saliva and other salivary parameters in type II diabetic patients and healthy controls. Salivary parameters were assessed in a single measurement of unstimulated whole saliva from 20 obese and 20 non-obese type II diabetes patients, and in 22 healthy controls. Total protein and alpha-amylase were determined by colorimetric methods, and glucose by the glucose-oxidase method. Saliva aGAH and dGAH levels were measured using a commercial radioimmunoassay (RIA) kit. Salivary concentrations of aGAH and dGAH ghrelin were more markedly decreased in obese diabetic subjects than in the two other groups. Glucose and alpha-amylase levels were higher in diabetic subjects than in controls. Furthermore, there were correlations between GAH levels and BMI, and between GAH and blood pressure. However, there was no marked variability in saliva flow rates among the groups. These results indicate that measurement of salivary GAH and its relationship to other salivary parameters might help to provide insight into the role of ghrelin in diabetes. PMID:17244479

  14. Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila.

    PubMed

    Isabella, Adam J; Horne-Badovinac, Sally

    2015-10-15

    Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC's conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis. PMID:26348027

  15. Measurement of pulmonary status and surfactant protein levels during dexamethasone treatment of neonatal respiratory distress syndrome.

    PubMed Central

    Wang, J. Y.; Yeh, T. F.; Lin, Y. C.; Miyamura, K.; Holmskov, U.; Reid, K. B.

    1996-01-01

    BACKGROUND: Early postnatal use of dexamethasone in infants with respiratory distress syndrome (RDS) has been shown effectively to improve pulmonary status and to allow early weaning off mechanical ventilation. However, the mechanisms to explain the beneficial effects of dexamethasone in ventilatory dependent preterm infants remain unclear. METHODS: A double blind, placebo controlled study was performed to determine the change in pulmonary ventilation of premature infants with RDS as a result of dexamethasone treatment, and to evaluate the effect of dexamethasone on the levels of surfactant-associated proteins A (SP-A) and D (SP-D) in the tracheal fluid from 34 premature infants with RDS and 29 control subjects. RESULTS: Dexamethasone treatment decreased fractional inspired oxygen concentration (FIO2), arterial carbon dioxide tension (PCO2), mean airway pressure (MAP), and facilitated successful weaning from mechanical ventilation. SP-A concentrations in the tracheal aspirates were increased at days 7 and 14, and SP-D concentrations were increased during the period from days 3 to 14 in the dexamethasone treated group compared with the control group. However, albumin levels in the tracheal aspirate samples were decreased after dexamethasone treatment over the period from days 3 to 14. There was an inverse correlation between PCO2 values and SP-A concentrations. CONCLUSIONS: These results suggest that early use of dexamethasone can improve pulmonary status and also increase SP-A and SP-D levels in the tracheal fluid in premature infants with RDS. PMID:8984701

  16. Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

    PubMed Central

    Khermesh, Khen; D'Erchia, Anna Maria; Barak, Michal; Annese, Anita; Wachtel, Chaim; Levanon, Erez Y.; Picardi, Ernesto; Eisenberg, Eli

    2016-01-01

    Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing. PMID:26655226

  17. Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level.

    PubMed

    Bahrami, Soroush Alaghehband; Bakhtiari, Nuredin

    2016-08-01

    We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1β and α-Klotho. In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20mg/ml) and then administrated (200mg/Kg i.p injection) to mice, twice daily for 7days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy. The data illustrated that UA significantly increased SIRT1 (∼3.5±0.3 folds) and SIRT-6 (∼1.5±0.2 folds) proteins overexpression (P<0.001). In addition, our results showed that UA enhanced α-Klotho (∼3.3±0.3) and PGC-1β (∼2.6±0.2 folds) proteins levels (P<0. 01). In this study, data were analyzed using SPSS 16 (ANOVA test). To the best of our knowledge, it seems that UA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1β in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging. PMID:27470332

  18. Increased levels of myelin basic protein transcripts in virus-induced demyelination.

    PubMed

    Kristensson, K; Holmes, K V; Duchala, C S; Zeller, N K; Lazzarini, R A; Dubois-Dalcq, M

    In multiple sclerosis, a demyelinating disease of young adults, there is a paucity of myelin repair in the central nervous system (CNS) which is necessary for the restoration of fast saltatory conduction in axons. Consequently, this relapsing disease often causes marked disability. In similar diseases of small rodents, however, remyelination can be quite extensive, as in the demyelinating disease caused by the A59 strain of mouse hepatitis virus (MHV-A59), a coronavirus of mice. To investigate when and where oligodendrocytes are first triggered to repair CNS myelin in such disease, we have used a complementary DNA probe specific for one major myelin protein gene, myelin basic protein (MBP), which hybridizes with the four forms of MBP messenger RNA in rodents. Using Northern blot and in situ hybridization techniques, we previously found that MBP mRNA is first detected at about 5 days after birth, peaks at 18 days and progressively decreases to 25% of the peak levels in the adult. We now report that in spinal cord sections of adult animals with active demyelination and inflammatory cells, in situ hybridization reveals a dramatic increase in probe binding to MBP-specific mRNA at 2-3 weeks after virus inoculation and before remyelination can be detected by morphological methods. This increase of MBP-specific mRNA is found at the edge of the demyelinating area and extends into surrounding areas of normal-appearing white matter. Thus, in situ hybridization with myelin-specific probes appears to be a useful method for detecting the timing, intensity and location of myelin protein gene reactivation preceding remyelination. This method could be used to elucidate whether such a reactivation occurs in multiple sclerosis brain tissue. Our results suggest that in mice, glial cells react to a demyelinating process with widespread MBP mRNA synthesis which may be triggered by a diffusible factor released in the demyelinated areas. PMID:2426599

  19. Oxysterol-related-binding-protein related Protein-2 (ORP2) regulates cortisol biosynthesis and cholesterol homeostasis.

    PubMed

    Escajadillo, Tamara; Wang, Hongxia; Li, Linda; Li, Donghui; Sewer, Marion B

    2016-05-15

    Oxysterol binding protein-related protein 2 (ORP2) is a lipid binding protein that has been implicated in various cellular processes, including lipid sensing, cholesterol efflux, and endocytosis. We recently identified ORP2 as a member of a protein complex that regulates glucocorticoid biosynthesis. Herein, we examine the effect of silencing ORP2 on adrenocortical function and show that the ORP2 knockdown cells exhibit reduced amounts of multiple steroid metabolites, including progesterone, 11-deoxycortisol, and cortisol, but have increased concentrations of androgens, and estrogens. Moreover, silencing ORP2 suppresses the expression of most proteins required for cortisol production and reduces the expression of steroidogenic factor 1 (SF1). ORP2 silencing also increases cellular cholesterol, concomitant with decreased amounts of 22-hydroxycholesterol and 7-ketocholesterol, two molecules that have been shown to bind to ORP2. Further, we show that ORP2 binds to liver X receptor (LXR) and is required for nuclear LXR expression. LXR and ORP2 are recruited to the CYP11B1 promoter in response to cAMP signaling. Additionally, ORP2 is required for the expression of other LXR target genes, including ABCA1 and the LDL receptor (LDLR). In summary, we establish a novel role for ORP2 in regulating steroidogenic capacity and cholesterol homeostasis in the adrenal cortex. PMID:26992564

  20. The Influence of Tobacco Smoke on Protein and Metal Levels in the Serum of Women during Pregnancy

    PubMed Central

    Wrześniak, Marta; Kepinska, Marta; Królik, Małgorzata; Milnerowicz, Halina

    2016-01-01

    Background Tobacco smoking by pregnant women has a negative effect on fetal development and increases pregnancy risk by changing the oxidative balance and microelements level. Smoking affects the concentration, structure and function of proteins, potentially leading to various negative effects on pregnancy outcomes. Methodology/Principal Findings The influence of tobacco smoke on key protein fractions in smoking and non-smoking healthy pregnant women was determined by capillary electrophoresis (CE). Concentrations of the proteins α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin and transferrin were determined by ELISA tests. Total protein concentration was measured by the Biuret method. Smoking status was established by cotinine levels. Cadmium (Cd) and Zinc (Zn) concentrations were determined by flame atomic absorption spectrometry and the Zn/Cd ratio was calculated based on these numbers. Smoking women had a 3.7 times higher level of Cd than non-smoking women. Zn levels decreased during pregnancy for all women. The Zn/Cd ratio was three times lower in smoking women. The differences between the changes in the protein profile for smoking and non-smoking women were noted. Regarding proteins, α1-antitrypsin and α2-macroglobulin levels were lower in the non-smoking group than in the smoking group and correlated with Cd levels (r = -0.968, p = 0.032 for non-smokers; r = −0.835, p = 0.019 for smokers). Zn/Cd ratios correlated negatively with α1-, α2- and β-globulins. Conclusions/Significance Exposure to tobacco smoke increases the concentration of Cd in the blood of pregnant women and may lead to an elevated risk of pregnancy disorders. During pregnancy alter concentrations of some proteins. The correlation of Cd with proteins suggests that it is one of the causes of protein aberrations. PMID:27548057

  1. Endogenous salicylic acid levels correlate with accumulation of pathogenesis-related proteins and virus resistance in tobacco

    SciTech Connect

    Yalpani, N.; Shulaev, V.; Raskin, I. )

    1993-07-01

    Salicylic acid (SA) is hypothesized to be an endogenous regulator of local and systemic disease resistance and an inducer of pathogenesis-related (PR) proteins among plants. High levels of PR proteins have been observed in an uninoculated amphidiploid hybrid of Nicotiana glutinosa [times] N. debneyi, which is highly resistant to tobacco mosaic virus (TMV). Fluoresence, UV, and mass spectral analysis established that the levels of SA in healthy N. glutinosa [times] N. debneyi leaves were 30 times greater than in N. tabacum [open quotes]Xanthi-nc[close quotes] tobacco, which does not constitutively express PR proteins and is less resistant to TMV. Upon TMV-inoculation SA levels increased at least 70-fold leaves of Xanthi-nc but role only slightly in the hybrid. Phloem exudates of N. glutinosa [times] N. debneyi contained at least 500 times more SA than those of Xanthi-nc. SA treatment caused the appearance of PR-1 protein in Xanthi-nc but did not affect constitutively high levels of PR-1 protein in N. glutinosa [times] N. debneyi. In contrast to Xanthi-nc tobacco, TMV-inoculated N. glutinosa [times] N. debneyi kept at 32 C accumulated more than 0.5 [mu]g SA/g fresh weight, maintained high levels of PR proteins, and developed a hypersensitive response to TMV. PR proteins have previously been shown to accumulate in the lower leaves of healthy, flowering Xanthi-nc tobacco, which exhibited increased resistance to TMV. These developmentally induced increases in resistance and PR-1 proteins positively correlated with tissue levels of SA. These results affirm the regulatory role of SA in disease resistance and PR protein production. 31 refs., 9 figs., 1 tab.

  2. Influence of casein as a percentage of true protein and protein level on color and texture of milks containing 1 and 2% fat.

    PubMed

    Misawa, Noriko; Barbano, David M; Drake, MaryAnne

    2016-07-01

    Combinations of fresh liquid microfiltration retentate of skim milk, ultrafiltered retentate and permeate produced from microfiltration permeate, cream, and dried lactose monohydrate were used to produce a matrix of 20 milks. The milks contained 5 levels of casein as a percentage of true protein of about 5, 25, 50, 75, and 80% and 4 levels of true protein of 3.0, 3.76, 4.34, and 5.0% with constant lactose percentage of 5%. The experiment was replicated twice and repeated for both 1 and 2% fat content. Hunter color measurements, relative viscosity, and fat globule size distribution were measured, and a trained panel documented appearance and texture attributes on all milks. Overall, casein as a percentage of true protein had stronger effects than level of true protein on Hunter L, a, b values, relative viscosity, and fat globule size when using fresh liquid micellar casein concentrates and milk serum protein concentrates produced by a combination of microfiltration and ultrafiltration. As casein as a percentage of true protein increased, the milks became more white (higher L value), less green (lower negative a value), and less yellow (lower b value). Relative viscosity increased and d(0.9) generally decreased with increasing casein as a percentage of true protein. Panelists perceived milks with increasing casein as a percentage of true protein as more white, more opaque, and less yellow. Panelists were able to detect increased throat cling and mouthcoating with increased casein as a percentage of true protein in 2% milks, even when differences in appearance among milks were masked. PMID:27157580

  3. The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

    PubMed

    Xiong, Xin; Hao, Yan; Sun, Kan; Li, Jiaxing; Li, Xia; Mishra, Bibhudatta; Soppina, Pushpanjali; Wu, Chunlai; Hume, Richard I; Collins, Catherine A

    2012-01-01

    Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. PMID:23226106

  4. Protein carbonyl levels correlate with performance in elite field hockey players.

    PubMed

    Rosa-Lima, Frederico Luis; Lannes, Luiz; Viana-Gomes, Diego; Pierucci, Anna Paola T R; Salerno, Verônica P

    2015-07-01

    Excess and incorrectly selected exercise can degrade athletic performance from an imbalance in redox homeostasis and oxidative stress, but well-planned training and nutrition can improve antioxidant capacity. The aim of the study was to investigate how nutrient intake could influence oxidative stress and cell lesion biomarkers after 5 days of training followed by a game. Blood was collected from 10 athletes at the start of training (basal), after training (pre-game), and postgame. Their acceleration capacity also was measured pre- and postgame. Blood analysis showed an increase in lactate concentration postgame (13%) and total antioxidant capacity increased both pre-game (13.1%) and postgame (12.7%), all in comparison with basal levels. An oxidative stress marker, protein carbonyl (PC), increased 3-fold over the course of the game, which correlated with a decreased acceleration (r = 0.749). For biomarkers of tissue damage, creatine kinase and aspartate transaminase (AST) increased postgame by 150% and 75%, respectively. The AST variation had a high negative correlation with energy and carbohydrate consumption and a moderate correlation with lipid and vitamin C intake. Protein intake had a positive but moderate correlation with reduced glutathione. The observed correlations suggest that nutritional monitoring can improve exercise physiological homeostasis and that PC serves as a good biomarker for oxidative stress and performance loss. PMID:25962716

  5. Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels

    SciTech Connect

    Kise, Yoshiaki; Takenaka, Kei; Tezuka, Tohru; Yamamoto, Tadashi; Miki, Hiroaki . E-mail: miki@ims.u-tokyo.ac.jp

    2006-12-08

    Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. Here, we have investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. We next show that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, we show that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells.

  6. Correlation between erythrocyte sedimentation rate and C-reactive protein level in patients with rheumatic diseases

    PubMed Central

    Kotulska, Anna; Kopeć-Mędrek, Magdalena; Grosicka, Anida; Kubicka, Monika

    2015-01-01

    Objectives Erythrocyte sedimentation rate (ESR) and serum level of C-reactive protein (CRP) are the acute phase reactants most commonly determined in patients with rheumatic diseases. The indices are affected by different factors, but both of them are applied for evaluation of the disease activity in patients with inflammatory disorders of the musculoskeletal system. Material and methods The authors compared the results of ESR and CRP, which were carried out during routine diagnosis in 200 patients admitted to the Department of Rheumatology. Results A significant correlation between ESR and CRP was found (ESR after 1 h/CRP: correlation coefficient 0.6944, ESR after 2 h/CRP: correlation coefficient 0.6126). There was no difference in ESR or CRP between male and female patients, and patients older than 40 years had higher ESR and CRP. Conclusions The obtained results support the usefulness of both indices in the clinical practice of rheumatologists.

  7. A Theoretical Lower Bound for Selection on the Expression Levels of Proteins

    PubMed Central

    Price, Morgan N.; Arkin, Adam P.

    2016-01-01

    We use simple models of the costs and benefits of microbial gene expression to show that changing a protein’s expression away from its optimum by 2-fold should reduce fitness by at least 0.2·P, where P is the fraction the cell’s protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 106, this implies that 2-fold changes to gene expression levels are under strong selection, as Ne·s≫1, where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral. PMID:27289091

  8. Effect of vitamins, protein level and probiotics on immune response of moulted male broiler breeders.

    PubMed

    Khan, R U; Rahman, Z U; Javed, I; Muhammad, F

    2014-08-01

    This study was planned to investigate the comparative effect of vitamins C (L-ascorbic acid), E (DL-α-tocopherol acetate), probiotics, lower than normal protein level (14%) and combination of these treatments on immune response of male broiler breeders after zinc-induced moulting. One hundred and eighty birds at the age of 65 weeks were induced to moult by mixing zinc oxide (ZnO) in feed at the rate 3000 IU/kg of feed. Upon completion of moulting, birds were divided into six groups (five replicates per group) in a completely randomized design and were fed vitamin C (500 IU/kg), vitamin E (100 IU/kg), lower protein level, probiotics (50 mg/l), and a combination of these components, while one group was kept as control. After completion of moulting phase (5 weeks), the treatment effects were tested as in vitro macrophages engulfment percentage, nitric oxide (NO) production, serum antibody titres against Newcastle disease (ND) and infectious bronchitis (IB). The results showed that in vitro macrophage engulfment percentage in unopsonized conditions was significantly higher in vitamin E-supplemented group. In addition, in opsonized condition, the macrophage engulfment percentage was significantly higher in both vitamin E- and C-supplemented groups. The NO (opsonized and unopsonized) production and antibody titre against ND and IB were significantly higher in vitamin E-supplemented group. It was concluded that vitamin E is a better option for enhanced immune response in broiler breeders after zinc-induced moulting. PMID:23782414

  9. Involvement of decreased neuroglobin protein level in cognitive dysfunction induced by 1-bromopropane in rats.

    PubMed

    Guo, Ying; Yuan, Hua; Jiang, Lulu; Yang, Junlin; Zeng, Tao; Xie, Keqin; Zhang, Cuili; Zhao, Xiulan

    2015-03-10

    1-Bromopropane (1-BP) is used as a substitute for ozone-depleting solvents (ODS) in industrial applications. 1-BP could display central nervous system (CNS) neurotoxicity manifested by cognitive dysfunction. Neuroglobin (Ngb) is an endogenous neuroprotectant and is predominantly expressed in the nervous system. The present study aimed to investigate Ngb involvement in CNS neurotoxicity induced by 1-BP in rats. Male Wistar rats were randomly divided into 5 groups (n=14) and treated with 0, 100, 200, 400 and 800 mg/kg bw 1-BP, respectively, by gavage for consecutive 12 days. Rats displayed cognitive dysfunction dose-dependently through Morris water maze (MWM) test. Significant neuron loss in layer 5 of the prelimbic cortex (PL) was observed. Moreover, 1-BP decreased Ngb protein level in cerebral cortex and Ngb decrease was significantly positively correlated with cognitive dysfunction. Glutathione (GSH) content, GSH/oxidized glutathione (GSSG) ratio and glutamate cysteine ligase (GCL) activity decreased in cerebral cortex, coupled with the increase in GSSG content. GSH and GSH/GSSG ratio decrease were significantly positively correlated with cortical Ngb decrease. Additionally, levels of N-epsilon-hexanoyl-lysine (HEL) and 4-hydroxy-2-nonenal (4-HNE) modified proteins in cerebral cortex of 1-BP-treated rats increased significantly. In conclusion, it was suggested that 1-BP resulted in decreased endogenous neuroprotectant Ngb in cerebral cortex, which might play an important role in CNS neurotoxicity induced by 1-BP and that 1-BP-induced oxidative stress in cerebral cortex might partly be responsible for Ngb decrease. PMID:25557405

  10. Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

    PubMed

    Perera, Erick; Rodríguez-Viera, Leandro; Rodríguez-Casariego, Javier; Fraga, Iliana; Carrillo, Olimpia; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2012-03-01

    The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency. PMID:22323208

  11. Effects of dietary lysine/protein ratio and fat levels on growth performance and meat quality of finishing pigs.

    PubMed

    Maeda, Keisuke; Yamamoto, Fumika; Toyoshi, Masanari; Irie, Masakazu

    2014-04-01

    This study aimed to evaluate the effects of dietary lysine/protein ratio and fat levels on the growth, carcass characteristics and meat quality of finishing pigs fed feed made from food waste, including noodles and chocolate. Four dietary treatments, 2 levels of lysine/protein ratio (0.035 and 0.046) and 2 levels of fat (3.3% and 6.0%), were adapted to a 2 × 2 factorial arrangement. Each diet for the finishing pigs contained the same levels of adequate crude protein (16%) and lysine (0.58-0.75%), and similar levels of high total digestible nutrients (90.2-92.6%). In total, 32 LWD pigs with an average body weight of 57.2 kg were assigned to 4 dietary groups. The pigs were slaughtered at about 115 kg. Growth performance was not influenced by the dietary treatments. Carcass characteristics were slightly influenced by the dietary fat level. As the dietary lysine/protein ratio decreased, the marbling score of Longissimus dorsi muscle increased and the intramuscular fat (IMF) increased from 6.82% to 9.46%. Marbling score was not significantly influenced by the dietary fat level. These results indicate that IMF increased without adverse effects on growth, carcass characteristics and meat quality, when pigs were fed a diet with low lysine/protein ratio. PMID:24261827

  12. Elevated urinary level of vitamin D-binding protein as a novel biomarker for diabetic nephropathy

    PubMed Central

    TIAN, XIAO-QIN; ZHAO, LI-MIN; GE, JIA-PU; ZHANG, YAN; XU, YAN-CHENG

    2014-01-01

    Improving the early prediction and detection of diabetic nephropathy (DN) remains a great challenge in disease management. The aim of this study was to evaluate the early detection power of urinary vitamin D-binding protein (VDBP) for the diagnosis of DN. Urine samples were obtained from 45 healthy volunteers and 105 diabetic patients with normoalbuminuria (DM group), microalbuminuria (DN1 group) and macroalbuminuria (DN2 group) (n=35 per group). The VDBP expression patterns in urine from patients and controls were quantified by western blot analysis. The excretion levels of urinary VDBP were quantified with enzyme-linked immunosorbent assay. The quantification results were obtained by correcting for creatinine expression and showed that urinary VDBP levels were significantly elevated in the patients of the DN1 and DN2 groups compared with those of the DM group and normal controls (1,011.33±325.30 and 1,406.34±239.66 compared with 466.54±213.63 and 125.48±98.27 ng/mg, respectively) (P<0.001). Receiver operating characteristic analysis of urinary VDBP levels for the diagnosis of DN rendered an optimum cut-off value of 552.243 ng/mg corresponding to 92.86% sensitivity and 85.00% specificity, which also showed an area under the ROC curve of 0.966. In conclusion, the findings of the present study suggest that urinary VDBP may be a potential biomarker for the early detection and prevention of DN. Further studies are required to examine the pathogenic mechanisms of elevated VDBP levels and their role in the diagnosis of DN. PMID:24396416

  13. Diet quality is associated with circulating C-reactive protein but not irisin levels in humans

    PubMed Central

    Park, Kyung Hee; Zaichenko, Lesya; Peter, Patricia; Davis, Cynthia R.; Crowell, Judith A.; Mantzoros, Christos S.

    2015-01-01

    Objective Adherence to a healthy diet has been shown to decrease the incidence of obesity and associated comorbidities. C-reactive protein (CRP) is an established inflammatory marker and irisin was recently identified as a molecule which may play a role in energy regulation and obesity but whether diet alters irisin levels remains unknown. We aimed to investigate the association between circulating irisin, leptin, and CRP levels and dietary quantity and quality using the Alternate Healthy Eating Index (AHEI) and the Alternate Mediterranean Diet Score (aMED). Materials/Methods The study evaluated dietary data and biomarker levels of 151 participants between 2009 and 2011 (71 male vs. 80 female, over 35 years old, obese 43.7%). AHEI and aMED scores were calculated based on data derived from self-administered 110-item food-frequency questionnaires estimating usual nutrient intake over the past year. Cross-sectional associations between dietary quantity, quality, body composition by bioelectric impedance, and biomarker levels including irisin, leptin, and CRP after fasting were assessed. Results CRP, but not irisin, was negatively correlated with AHEI (r = −0.34) and aMED (r = −0.31). Irisin was positively correlated with BMI (r = 0.22), fat mass (r = 0.21), waist circumference (r = 0.24), waist-hip ratio (r = 0.20), leptin (r = 0.32), and CRP (r = 0.25). Participants with the highest AHEI scores tended to have 11.6% lower concentrations of irisin (P for trend =0.09), but they were not significant after adjustment for potential confounders. Better diet quality was associated with lower CRP concentrations (P for trend=0.02) in multivariate model. Percentage of energy from carbohydrate was inversely associated with CRP. Conclusions Unlike CRP, irisin is not associated with dietary quality or quantity. PMID:24315778

  14. 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

  15. Elevated C-reactive protein levels and metabolic syndrome in the elderly

    PubMed Central

    Zuliani, Giovanni; Volpato, Stefano; Galvani, Matteo; Blè, Alessandro; Bandinelli, Stefania; Corsi, Anna Maria; Lauretani, Fulvio; Maggio, Marcello; Guralnik, Jack M.; Fellin, Renato; Ferrucci, Luigi

    2009-01-01

    Metabolic syndrome (MS) and “low grade” systemic inflammation (LGSI) are very common findings in the older population. Although MS and LGSI have been associated in adults, it is not known what is the real contribution of MS, and its single components, to LGSI in older persons, due to the potential confounding effect of comorbidity and aging. We investigated the relationship between increased C-reactive protein (CRP) plasma levels, a marker of LGSI, and MS in 1044 older (≥65 years) community dwelling Italian individuals enrolled the InChianti study. Metabolic syndrome was defined by the NCEP-ATP III-AHA/NHLBI criteria. High sensitivity CRP (hs.CRP) levels were measured by enzyme-linked immunosorbent assay, and defined as high when >3 mg/L. The overall prevalence of MS was 31%. The prevalence of high hs.CRP was 54.5% in subjects with, and 41.3% in those without MS (p < 0.001). MS was associated with high hs.CRP levels after adjustment for age, gender, and comorbidity (OR: 1.93, 95% CI: 1.46-2.55). Compared to subjects with MS and no LGSI, individuals with MS and LGSI were characterized by higher waist circumference, BMI, and HOMA score. Multivariate logistic regression analysis confirmed the association between waist circumference and high hs.CRP levels in subjects with MS (waist circumference III vs. I tertile OR: 2.60, 95% CI: 1.79-3.77) independent of age, gender, and important confounding variables including comorbidity. Additional analyses, conducted with and without dichotomization of hs.CRP levels, confirmed the central role of waist circumference in the LGSI phenomenon, independent of gender and diagnosis of MS. We conclude that in older individuals, MS is associated with LGSI, but the association is mainly supported by a strong independent correlation between waist circumference and high hs.CRP levels. In the absence of this specific MS component, it seems that the contribution of MS to LGSI would be modest at best. PMID:18845301

  16. Protein pheromone expression levels predict and respond to the formation of social dominance networks

    PubMed Central

    Nelson, Adam C.; Cunningham, Christopher B.; Ruff, James S.; Potts, Wayne K.

    2015-01-01

    Communication signals are key regulators of social networks, and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odors of dominants and nondominants differentially influence behavior, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analyzing expression levels before, during, and after competition for reproductive resources over three days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species. PMID:25867293

  17. Elevated C-reactive protein level predicts lower gastrointestinal tract bleeding

    PubMed Central

    TOMIZAWA, MINORU; SHINOZAKI, FUMINOBU; HASEGAWA, RUMIKO; SHIRAI, YOSHINORI; MOTOYOSHI, YASUFUMI; SUGIYAMA, TAKAO; YAMAMOTO, SHIGENORI; ISHIGE, NAOKI

    2016-01-01

    Lower gastrointestinal (GI) bleeding can be caused by colorectal polyps or cancer. The aim of the present study was to identify blood test variables and medications that can predict lower GI bleeding, which would allow for appropriate colonoscopy. The medical records of patients who underwent colonoscopy from September 2014 to September 2015 were retrospectively analyzed. The selected patients included 278 men (mean age, 67.0±11.5 years) and 249 women (mean age, 69.6±12.0 years). The diagnosis, medications, and blood test variables were compared between patients with and without bleeding. Logistic regression analysis was performed to determine the factors associated with lower GI bleeding. The presence of colorectal polyp and cancer was associated with lower GI bleeding (P=0.0044) with an odds ratio of 6.71 (P=0.0148). No lower GI bleeding was observed in patients taking non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or anticoagulants. The C-reactive protein (CRP) levels were significantly higher in patients with lower GI bleeding (P=0.0227). The Hb levels were lower in patients with lower GI bleeding, however this finding was not statistically significant (P>0.05). No blood test variable was associated with lower GI bleeding. Elevated CRP was associated with lower GI bleeding, while there was no association between the medications and lower GI bleeding. PMID:27284411

  18. Neuron-Specific Enolase, S100 Calcium-Binding Protein B, and Heat Shock Protein 70 Levels in Patients With Intracranial Hemorrhage

    PubMed Central

    Alatas, Ömer Doğan; Gürger, Mehtap; Ateşçelik, Metin; Yildiz, Mustafa; Demir, Caner Feyzi; Kalayci, Mehmet; Ilhan, Nevin; Acar, Ethem

    2015-01-01

    Abstract The authors evaluated neuron-specific enolase (NSE), S100 calcium-binding protein B (S100B), and heat shock protein 70 (HSP 70) levels and their relationships with in-hospital mortality, Glasgow Coma Scale (GCS) scores, and National Institute of Health Stroke Scale (NIHSS) scores. In total, 35 patients older than 18 years were presented to our emergency department and were diagnosed with non-traumatic intracranial hemorrhage (ICH) and 32 healthy controls were included. Blood samples were drawn on days 0 and 5. S100 calcium-binding protein B and HSP levels were significantly higher in patients than in controls on days 0 and 5. Neuron-specific enolase levels were higher in patients than in controls on day 0, but there was no significant difference on day 5. S100 calcium-binding protein B was negatively correlated with GCS, whereas it was positively correlated with NIHSS and bleeding volume. There was also a negative correlation between NSE and GCS, but it was not statistically significant. In addition, no significant correlation was found in terms of bleeding volume or NIHSS. Heat shock protein 70 was negatively correlated with GCS and positively correlated with bleeding volume and NIHSS, but these results were not statistically significant. S100 calcium-binding protein B and HSP 70 levels were significantly higher in those who died compared with survivors. The areas under the curve of S100 B, NSE, and HSP 70 for mortality were 0.635, 0.477, and 0.770, respectively. Neuron-specific enolase, S100B, and HSP 70 levels are simple, inexpensive, and objective measures in cases of ICH. These tests can be used to support an assessment for screening ICH patients with clinical scoring systems, such as GCS and NIHSS. PMID:26559295

  19. Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1.

    PubMed

    Huang, Hai-Jun; Peng, Xia; Deng, Bing; Huang, Cong; Li, Jie; Qian, Yun-Guo; Gao, Qi-Shuang; Xiang, Min; Lu, Shun; Chen, Zhi-Hua; Zhan, Cai-Yao; Zhou, Li; Tao, Bi-Fei; Liu, Jie; Tan, Ben-Zhong

    2016-03-01

    Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods. PMID:25297006

  20. Baseline C-Reactive Protein Levels and Life Prognosis in Parkinson Disease

    PubMed Central

    Sawada, Hideyuki; Oeda, Tomoko; Umemura, Atsushi; Tomita, Satoshi; Kohsaka, Masayuki; Park, Kwiyoung; Yamamoto, Kenji; Sugiyama, Hiroshi

    2015-01-01

    Background C-reactive protein (CRP) is a biomarker of inflammation, and high levels of CRP correlate with vascular death. Chronic inflammation is considered to be involved in neurodegeneration, although there is no evidence linking it with the process of neurodegenerative diseases. Objective To determine the role of baseline CRP levels in the prognosis of patients with Parkinson disease (PD). Methods A cohort of 313 patients with a mean age of 69.1 and mean PD duration of 7.9 years was retrospectively followed for a mean observation time of 1,753 days. CRP was measured when patients were not diagnosed with any infections, and levels were repetitively measured to investigate a tendency of “regression to mean.” The primary outcome measure was a survival time from study enrollment to death. Results During the observation period 56 patients died. Baseline CRP was log-linearly associated with a risk of death in PD. Mean survival time was 3,149 (95% confidence interval; 3,009-3,289) days in patients with CRP ≤ 0.8mg/L (lower two thirds) and 2,620 (2,343-2,897) days in those with CRP > 0.8 mg/L (top third, p < 0.001, log-rank test). The adjusted hazard ratio (HR) per two-fold higher CRP concentration for all deaths was 1.29 (1.10-1.52), and after excluding PD-unrelated deaths, such as cancer or stroke, HR was 1.23 (1.01-1.49) (adjusted for age, sex, PD duration, modified Hohen-Yahr stages, MMSE scores, and serum albumin). Conclusions Baseline CRP concentrations were associated with the risk of death and predicted life prognosis of patients with PD. The associations were independent from PD duration, PD severity, cognitive function, ages, and nutritional conditions, suggesting the possibility that subclinical chronic inflammation is associated with a neurodegenerative process in PD. PMID:26218286

  1. Adult consumers of cranberry juice cocktail have lower C-reactive protein levels compared with nonconsumers.

    PubMed

    Duffey, Kiyah J; Sutherland, Lisa A

    2015-02-01

    Flavonoids are important bioactive plant constituents found in abundance in berries, including cranberries. Cranberry beverages have been shown to beneficially impact urinary and cardiovascular health in clinical and observational studies, but their association with anthropometric outcomes is unknown. We examined the association between cranberry juice cocktail (CJC) consumption with flavonoid intake, and cardiometabolic and anthropometric outcomes among adults in the US data for adults (≥19 years, n = 10334) were drawn from cross-sectional National Health and Nutrition Examination Survey combined 2005-2008 survey. We hypothesized that CJC consumers will have lower anthropometric measures and healthier cardiometabolic profiles, including lower cholesterol and C-reactive protein (CRP). A CJC consumer (n = 330) was defined as anyone consuming CJC for 2 nonconsecutive 24-hour dietary recalls. We used multivariate linear regression models to examine differences in anthropometric and cardiometabolic outcomes comparing CJC consumers to nonconsumers controlling for important confounders. Consumers drank an average 404 mL (14 fl oz) of CJC for 2 days and did not have higher total energy intakes compared with nonconsumers (mean [SD], 2259 [79] vs 2112 [24], respectively). In fully adjusted models, adult CJC consumers had significantly lower levels of CRP (mean [SD], -0.13 [0.05]; P = .015), results that were strengthened after further adjustment for body mass index (mean [SD], -0.98 [0.04]; P = .027). Trends toward lower weights and lower levels of cholesterol did not reach statistical significance. Intake of cranberry polyphenols may play a role in promoting anti-inflammatory markers among CJC consumers, specifically lowering CRP levels. PMID:25530012

  2. C-Reactive Protein Levels Among U.S. Adults Exposed to Parental Incarceration

    PubMed Central

    Boch, Samantha J.; Ford, Jodi L.

    2015-01-01

    Previous studies have linked childhood adversity to low-grade inflammation via C-reactive protein (CRP) levels. This study analyzed the association between low-grade inflammation and prior biological parental incarceration. Data from the National Longitudinal Study of Adolescent to Adult Health (1994–2008) were analyzed using multinomial logistic regression models. Measures included high-sensitivity (hs)-CRP (<3 mg/L = reference, 3–10 mg/L = low-grade inflammation, and >10 mg/L = acute inflammation), parent incarceration occurring in the child's lifetime, and frequency and timing of incarceration with respect to child's age (0–18 years or >18 years vs. never) of incarceration. Analyses were stratified by child's gender. Final sample sizes were n = 5,396 males and n = 6,447 females for maternal incarceration and n = 4,956 males and n = 5,860 females for paternal incarceration. In models with and without potential mediators, females whose fathers were ever incarcerated were more likely to have hs-CRP levels of 3–10 mg/L than females whose fathers were never incarcerated (adjusted odds ratio [AOR]: 1.44, 95% confidence interval [CI]: [1.09, 1.91]). Additionally, daughter's age (<18 years; AOR: 1.48, 95% CI: [1.11, 1.97]) and frequency of father's incarceration were significant (AOR: 1.24, 95% CI: [1.04, 1.49]). No mediating effects were observed. Males whose fathers were incarcerated when they were ≥ 18 years were less likely to have hs-CRP levels of 3–10 mg/L than those whose father was never incarcerated; the association was nonsignificant in the mediated model. Further investigation is needed on the physiological effects of exposure to parental incarceration and interventions to support children. PMID:25539769

  3. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    PubMed

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. PMID:26929125

  4. RESPONSE OF HEPATIC PROTEINS TO THE LOWERING OF HABITUAL DIETARY PROTEIN TO THE RECOMMENDED SAFE LEVEL OF INTAKE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plasma concentrations of albumin, HDL apolipoprotein A1 (apoA1), retinol-binding protein (RBP), transthyretin (TTR), haptoglobulin, and fibrinogen were measured, and a stable isotope infusion protocol was used to determine the fractional and absolute synthesis rates of RBP, TTR, and fibrinogen i...

  5. Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach

    PubMed Central

    Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

    2016-01-01

    Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of “cancer drivers” connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels. PMID:27412431

  6. Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach.

    PubMed

    Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

    2016-01-01

    Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of "cancer drivers" connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels. PMID:27412431

  7. Lack of association of acute phase response proteins with hormone levels and antidepressant medication in perimenopausal depression

    PubMed Central

    2014-01-01

    Background Major depression is associated with higher plasma levels of positive acute-phase proteins, as well as with lower plasma levels of negative acute-phase proteins. The aim of this study is to examine the levels of acute-phase response proteins and whether these levels are influenced by reproductive hormones and antidepressant medication in the perimenopausal depression. Methods Sixty-five women (age range: 40–58 years old) participated in this study. All women were in the perimenopausal phase. The diagnosis of depression was made through a psychiatric interview and with the aid of Hamilton Depression Rating Scale 17 (HAM-D 17). The acute-phase response proteins, such as haptoglobin (HP), transferrine (TRf), α1-antitrypsin, complement protein 3 (C3), complement protein 4 (C4) and C-reactive protein (CRP) and the reproductive hormones, for example follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2), were analyzed using standard laboratory methods. Pearson’s correlations were applied to evaluate the relationship between acute-phase proteins and hormones. Results Perimenopausal women were divided into three groups. The first group consisted of normal controls, the second one involved depressed perimenopausal women, who were taking selective serotonin reuptake inhibitors (SSRIs), and the third one included depressed women that were not treated with SSRIs. Depressed women in perimenopause, when being compared to non-depressed women, did not differ as to serum levels of acute-phase proteins. There was a positive correlation between HP and E2 in depressed perimenopausal women, who were not taking SSRIs. Conclusions The lack of association between acute-phase proteins and depressive mood mentioned in this study does not support previous findings in patients with major depression. This negative finding in perimenopausal depression indicates either the absence or a more complex nature of the interactions between acute-phase proteins

  8. Enhanced skeletal muscle protein synthesis rates in pigs treated with somatotropin requires fed amino acids levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic somatotropin (pST) treatment in pigs increases skeletal muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin alone could not account for the pST-induced increase in protein synthesis. This study...

  9. Effects of balanced dietary protein levels on egg production and egg quality parameters of individual commercial layers.

    PubMed

    Shim, M Y; Song, E; Billard, L; Aggrey, S E; Pesti, G M; Sodsee, P

    2013-10-01

    The effects of a series of balanced dietary protein levels on egg production and egg quality parameters of laying hens from 18 through 74 wk of age were investigated. One hundred forty-four pullets (Bovans) were randomly assigned to individual cages with separate feeders including 3 different protein level series of isocaloric diets. Diets were separated into 4 phases of 18-22, 23-32, 33-44, and 45-74 wk of age. The high protein (H) series contained 21.62, 19.05, 16.32, and 16.05% CP, respectively. Medium protein (M) and low protein (L) series were 2 and 4% lower in balanced dietary protein. The results clearly demonstrated that the balanced dietary protein level was a limiting factor for BW, ADFI, egg weight, hen day egg production (HDEP), and feed per kilogram of eggs. Feeding with the L series resulted in lower ADFI and HDEP (90.33% peak production) and more feed per kilogram of eggs compared with the H or M series (HDEP; 93.23 and 95.68% peak production, monthly basis). Egg weight responded in a linear manner to balanced dietary protein level (58.78, 55.94, and 52.73 g for H, M, and L, respectively). Feed intake of all hens, but especially those in the L series, increased considerably after wk 54 when the temperature of the house decreased due to winter conditions. Thus, hens fed the L series seemed particularly dependent on house temperature to maintain BW, ADFI, and HDEP. For egg quality parameters, percent yolk, Haugh units, and egg specific gravity were similar regardless of diets. Haugh units were found to be greatly affected by the variation of housing temperature (P = 0.025). Maximum performance cannot always be expected to lead to maximum profits. Contrary to the idea of a daily amino acid requirement for maximum performance, these results may be used to determine profit-maximizing levels of balanced dietary protein based on the cost of protein and returns from different possible protein levels that may be fed. PMID:24046416

  10. E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

    PubMed

    Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C; Zhang, Zhen; Young, Randee E; Verheyden, Jamie M; Sun, Xin

    2016-07-01

    The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis. PMID:27335464

  11. Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

    PubMed Central

    Furuta, Saori; Ortiz, Fausto; Zhu Sun, Xiu; Wu, Hsiao-Huei; Mason, Andrew; Momand, Jamil

    2002-01-01

    The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues. PMID:11964141

  12. 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.

  13. High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.

    PubMed

    Zhang, Xiguang; Xie, Jiasen; Sun, Yan; Xu, Huijing; Du, Tonghua; Liu, Zixuan; Chen, Jinhui; Zheng, Zhong; Liu, Keqiang; Zhang, Jizhou; Kan, Mujie; Li, Xiaokun; Xiao, Yechen

    2014-06-01

    Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 μg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development. PMID:24519456

  14. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

    1994-01-01

    Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and

  15. Influence of chlortetracycline and dietary protein level on visceral organ mass of growing beef steers.

    PubMed

    Baldwin, R L; McLeod, K R; Elsassert, T H; Kahl, S; Rumsey, T S; Streeter, M N

    2000-12-01

    Thirty-two beef steers (285 +/- 3 kg BW) were used to determine the effects of chlortetracycline and dietary protein level on visceral tissue mass, chemical composition, intestinal morphology, and proliferation rate indices. Steers were allotted randomly by weight to a factorial arrangement of dietary treatments consisting of either 10 or 13% CP diets top-dressed with a corn meal carrier (500 g/d) containing either 0 or 350 mg of chlortetracycline. After 84 d, steers were slaughtered and visceral organs removed and separated. Rinsed wet tissue mass was recorded; total RNA, total DNA, tissue DM, and tissue N content were determined; and tissue sections were prepared for immunohistochemical analysis. Thin tissue sections were evaluated to determine crypt depth and villus height as well as proliferation rate by immunohistochemical detection of the nuclear antigen Ki67. Rumen and abomasum weights and small intestinal length were greater (P < 0.04) in steers fed the 13% CP diet than in those fed the 10% CP diet on both an absolute weight basis and a percentage of empty BW. Chemical composition of the small intestinal and ruminal segments were largely unaffected by increased dietary protein. Increasing the dietary CP also increased the villus height in duodenal (P = 0.02) and the crypt depth of jejunal (P = 0.03) sections. Dietary administration of chlortetracycline decreased (P < 0.01) small intestinal weight both on absolute and empty BW bases. Nitrogen and RNA concentrations of the small intestinal segments were unaffected (P > 0.1) by dietary administration of subtherapeutic levels of chlortetracycline; however, because of increases (P < 0.05), or tendencies for an increase (P < 0.1), in the tissue content of DNA, the ratio of N to DNA was decreased (P < 0.05) or tended to be decreased (P < 0.1) in the small intestinal segments of the chlortetracycline-treated animals. The observed decrease in small intestinal epithelial mass does not appear to be due to alterations

  16. Identification of Leaf Proteins Differentially Accumulated between Wheat Cultivars Distinct in Their Levels of Drought Tolerance.

    PubMed

    Cheng, Zhiwei; Dong, Kun; Ge, Pei; Bian, Yanwei; Dong, Liwei; Deng, Xiong; Li, Xiaohui; Yan, Yueming

    2015-01-01

    The drought-tolerant 'Ningchun 47' (NC47) and drought-sensitive 'Chinese Spring' (CS) wheat (Triticum aestivum L.) cultivars were treated with different PEG6000 concentrations at the three-leaf stage. An analysis on the physiological and proteomic changes of wheat seedling in response to drought stress was performed. In total, 146 differentially accumulated protein (DAP) spots were separated and recognised using two-dimensional gel electrophoresis. In total, 101 DAP spots representing 77 unique proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These proteins were allocated to 10 groups according to putative functions, which were mainly involved in carbon metabolism (23.4%), photosynthesis/respiration (22.1%) and stress/defence/detoxification (18.2%). Some drought stress-related proteins in NC47, such as enolase, 6-phosphogluconate dehydrogenase, Oxygen-evolving enhancer protein 2, fibrillin-like protein, 2-Cys peroxiredoxin BAS1 and 70-kDa heat shock protein, were more upregulated than those in CS. Multivariate principal components analysis revealed obvious differences between the control and treatments in both NC47 and CS, while cluster analysis showed that the DAPs displayed five and six accumulation patterns in NC47 and CS, respectively. Protein-protein interaction network analysis showed that some key DAPs, such as 2-Cys peroxiredoxin BAS1, RuBisCO large subunit-binding protein, 50S ribosomal protein L1, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase isoenzyme and 70-kDa heat shock protein, with upregulated accumulation in NC47, had complex interactions with other proteins related to amino acid metabolism, carbon metabolism, energy pathway, signal transduction, stress/defence/detoxification, protein folding and nucleotide metabolism. These proteins could play important roles in drought-stress tolerance and contribute to the relatively stronger drought tolerance of NC47. PMID

  17. Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins.

    PubMed

    Black, V H; Sanjay, A; van Leyen, K; Möeller, I; Lauring, B; Kreibich, G

    2002-11-01

    Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner. PMID:12530645

  18. The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels

    SciTech Connect

    Hwang, Mi-Na; Min, Chan-Hee; Kim, Hyung Sik; Lee, Ho; Yoon, Kyong-Ah; Park, Sung Yong; Lee, Eun Sook; Yoon, Sungpil . E-mail: yoons@ncc.re.kr

    2007-08-24

    We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.

  19. Is serum or sputum eosinophil cationic protein level adequate for diagnosis of mild asthma?

    PubMed

    Khakzad, Mohammad Reza; Mirsadraee, Majid; Sankian, Mojtaba; Varasteh, Abdolreza; Meshkat, Mojtaba

    2009-09-01

    Spirometry has been used as a common diagnostic test in asthma. Most of the patients with a mild asthma have a FEV1 within normal range. Hence, other diagnostic methods are usually used. The aim of this study was to evaluate whether eosinophil Cationic Protein (ECP) could be an accurate diagnostic marker of mild asthma. In this study diagnosis of asthma was made according to internationally accepted criteria. Asthma severity was evaluated according to frequency of symptoms and FEV1.Adequate sputum samples were obtained in 50 untreated subjects. A control group of 12 normal subjects that showed PC20 more than 8 mg/dl was also examined. Sputum was induced by inhalation of hypertonic saline. Inflammatory cells in sputum smears were assessed semi-quantitatively. ECP and IgE concentrations, eosinophil (EO) percentage and ECP/EO ratio in serum and sputum were also determined. The results revealed that Cough and dyspnea were the most frequent clinical findings. Dyspnea and wheezing were the symptoms that correlated with staging of asthma. FEV1 was within normal range (more than 80% of predicted) in 22 (44%) subjects.Asthmatic patients showed significantly higher numbers of blood eosinophils (4.5+/- 3.1% vs. 1.2+/-0.2%, P=0.009), and higher levels of serum ECP than control group (3.1+/- 2.6 % and 22.6+/- 15.8 ng/ml, respectively). Sputum ECP level in asthmatics was significantly higher than non- asthmatics (55.3+/-29.8ng/mL vs. 25.0+/-24.7ng/mL, P=0.045). Regression analysis showed no significant correlation between spirometric parameters and biomarkers, the only exception was significant correlation between FEF(25-75) and serum ECP (r= 0.28, P 0.041). Regarding clinical symptoms, wheezing was significantly correlated with elevation of most of biomarkers. Since, serum and sputum ECP levels are elevated in untreated asthmatics, the ECP level could be used for accurate diagnosis of mild form of asthma in which spirometry is unremarkable. PMID:20124607

  20. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages

    PubMed Central

    Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A.; Yin, Yulong

    2015-01-01

    Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but

  1. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

    PubMed

    Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A; Yin, Yulong

    2015-01-01

    Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but

  2. In Situ Molecular Level Studies on Membrane Related Peptides and Proteins in Real Time Using Sum Frequency Generation Vibrational Spectroscopy

    PubMed Central

    Ye, Shuji; Nguyen, Khoi Tan; Le Clair, Stéphanie V.; Chen, Zhan

    2009-01-01

    Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study the molecular structures of surfaces and interfaces in different chemical environments. This review summarizes recent SFG studies on hybrid bilayer membranes and substrate-supported lipid monolayers and bilayers, the interaction between peptides/proteins and lipid monolayers/bilayers, and bilayer perturbation induced by peptides/proteins. To demonstrate the ability of SFG to determine the orientations of various secondary structures, studies on the interaction between different peptides/proteins (melittin, G proteins, almethicin, and tachyplesin I) and lipid bilayers are discussed. Molecular level details revealed by SFG in these studies show that SFG can provide a unique understanding on the interactions between a lipid monolayer/bilayer and peptides/proteins in real time, in situ and without any exogenous labeling. PMID:19306928

  3. Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations.

    PubMed Central

    Morimoto, B H; Brady, J F; Atkinson, D E

    1990-01-01

    Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate. PMID:2268294

  4. Atomic-level description of protein-lipid interactions using an accelerated membrane model.

    PubMed

    Baylon, Javier L; Vermaas, Josh V; Muller, Melanie P; Arcario, Mark J; Pogorelov, Taras V; Tajkhorshid, Emad

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26940626

  5. SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

    SciTech Connect

    Almeida, Luciana O.; Goto, Renata N.; Neto, Marinaldo P.C.; Sousa, Lucas O.; Curti, Carlos; Leopoldino, Andréia M.

    2015-03-06

    We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.

  6. Activation of TLR3 Promotes the Degeneration of Retinal Ganglion Cells by Upregulating the Protein Levels of JNK3

    PubMed Central

    Chintala, Shravan K.; Putris, Nahrain; Geno, Mason

    2015-01-01

    Purpose. To investigate whether activation of Toll-like receptor 3 (TLR3) promotes the degeneration of retinal ganglion cells (RGCs) by upregulating the protein levels of c-jun N-terminal kinase 3 (JNK3). Methods. Toll-like receptor 3-specific activator, Poly(I:C) (polyinosinic-polycytidylic acid), or PBS was injected into the vitreous humor of Thy1-YFP mice. At 24, 48, and 72 hours after treatments, degeneration of RGCs was assessed by using antibodies against brain-specific homeobox/POU domain protein 3a (Brn3a). A TLR3-specific inhibitor was injected into the vitreous humor with or without Poly(I:C). Western blot assays were performed to determine relative levels of TLR3, JNK3, pJNK3, and sterile alpha and HEAT/Armadillo motif-containing 1 (SARM1) proteins in retinal protein extracts, and immunohistochemistry assays were performed to determine their cellular localization in the retina. Mouse eyes were treated with Poly(I:C) or PBS along with MitoTracker Red, and colocalization of MitoTracker Red and JNK3 in the retinas was determined by using antibodies against JNK3. Results. Poly(I:C) activated TLR3 and upregulated its downstream target protein JNK3 but not SARM1 in the retina. Poly(I:C) activated TLR3 and upregulated JNK3 specifically in RGCs and promoted a significant degeneration of RGCs over a 72-hour time period. Toll-like receptor 3 upregulated the levels of JNK3 protein in the cytoplasm of RGCs, but not in the mitochondria. Toll-like receptor 3-specific inhibitor downregulated Poly(I:C)-mediated upregulation of JNK3 protein, and, in turn, significantly attenuated TLR3-induced degeneration of RGCs. Conclusions. Results presented in this study show that the activation of TLR3 alone promotes the degeneration of RGCs by upregulating the protein levels of JNK3. PMID:25564448

  7. Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy

    SciTech Connect

    King, Neil P.; Sheffler, William; Sawaya, Michael R.; Vollmar, Breanna S.; Sumida, John P.; André, Ingemar; Gonen, Tamir; Yeates, Todd O.; Baker, David

    2015-09-17

    We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

  8. Protein-Level Fluctuation Correlation at the Microcolony Level and Its Application to the Vibrio harveyi Quorum-Sensing Circuit

    PubMed Central

    Wang, Yufang; Tu, Kimberly C.; Ong, N.P.; Bassler, Bonnie L.; Wingreen, Ned S.

    2011-01-01

    Gene expression is stochastic, and noise that arises from the stochastic nature of biochemical reactions propagates through active regulatory links. Thus, correlations in gene-expression noise can provide information about regulatory links. We present what to our knowledge is a new approach to measure and interpret such correlated fluctuations at the level of single microcolonies, which derive from single cells. We demonstrated this approach mathematically using stochastic modeling, and applied it to experimental time-lapse fluorescence microscopy data. Specifically, we investigated the relationships among LuxO, LuxR, and the small regulatory RNA qrr4 in the model quorum-sensing bacterium Vibrio harveyi. Our results show that LuxR positively regulates the qrr4 promoter. Under our conditions, we find that qrr regulation weakly depends on total LuxO levels and that LuxO autorepression is saturated. We also find evidence that the fluctuations in LuxO levels are dominated by intrinsic noise. We furthermore propose LuxO and LuxR interact at all autoinducer levels via an unknown mechanism. Of importance, our new method of evaluating correlations at the microcolony level is unaffected by partition noise at cell division. Moreover, the method is first-order accurate and requires less effort for data analysis than single-cell-based approaches. This new correlation approach can be applied to other systems to aid analysis of gene regulatory circuits. PMID:21689539

  9. [Interest of S100B protein blood level determination in severe or moderate head injury].

    PubMed

    Bouvier, Damien

    2013-01-01

    S100B, a suffering brain marker, exhibits a different interest in traumatic brain injury (TBI) as the trauma is severe (sTBI) or mild (mTBI). Our works presented for the attribution of the SFBC 2012 price talked about both aspects. Firstly, the extent of S100B elevation has been found to be useful in predicting clinical outcome after sTBI. However, few studies were realized with jugular venous blood samples. After comparing the interest between jugular venous and arterial blood concentrations evaluation of serum S100B protein in patients with sTBI, determination of S100B concentration in jugular blood samples appears to be better than in arterial ones to predict clinical outcome after brain injury. Secondly, it's difficult to determine the indication of cranial computed tomography (CCT) for patients with mTBI. Actually, 90% of patients with mTBI have unnecessary CCT or short hospitalization for observation. Serum concentrations of S100B were found to provide useful information. We have investigated in 2 studies (1 for adult, 1 for children) whether S100B concentrations in patients with mTBI could provide additional information to improve indication of the need for an initial CCT scan or for a short hospitalization. Patients with intracerebral lesions on the CCT scan (CCT+) or with bad clinical evolution were identified with a sensitivity level of 100% and a specificity level of 30%. Adding the measurement of S-100B serum concentration to the clinical decision rules for a CCT scan or hospitalization in patients with mTBI could allow a 30% reduction in scans and in hospitalization for clinical observation. PMID:23587577

  10. Identification of Leaf Proteins Differentially Accumulated between Wheat Cultivars Distinct in Their Levels of Drought Tolerance

    PubMed Central

    Bian, Yanwei; Dong, Liwei; Deng, Xiong; Li, Xiaohui; Yan, Yueming

    2015-01-01

    The drought-tolerant ‘Ningchun 47’ (NC47) and drought-sensitive ‘Chinese Spring’ (CS) wheat (Triticum aestivum L.) cultivars were treated with different PEG6000 concentrations at the three-leaf stage. An analysis on the physiological and proteomic changes of wheat seedling in response to drought stress was performed. In total, 146 differentially accumulated protein (DAP) spots were separated and recognised using two-dimensional gel electrophoresis. In total, 101 DAP spots representing 77 unique proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These proteins were allocated to 10 groups according to putative functions, which were mainly involved in carbon metabolism (23.4%), photosynthesis/respiration (22.1%) and stress/defence/detoxification (18.2%). Some drought stress-related proteins in NC47, such as enolase, 6-phosphogluconate dehydrogenase, Oxygen-evolving enhancer protein 2, fibrillin-like protein, 2-Cys peroxiredoxin BAS1 and 70-kDa heat shock protein, were more upregulated than those in CS. Multivariate principal components analysis revealed obvious differences between the control and treatments in both NC47 and CS, while cluster analysis showed that the DAPs displayed five and six accumulation patterns in NC47 and CS, respectively. Protein–protein interaction network analysis showed that some key DAPs, such as 2-Cys peroxiredoxin BAS1, RuBisCO large subunit-binding protein, 50S ribosomal protein L1, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase isoenzyme and 70-kDa heat shock protein, with upregulated accumulation in NC47, had complex interactions with other proteins related to amino acid metabolism, carbon metabolism, energy pathway, signal transduction, stress/defence/detoxification, protein folding and nucleotide metabolism. These proteins could play important roles in drought-stress tolerance and contribute to the relatively stronger drought tolerance of NC47

  11. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

    PubMed Central

    Çelik, Haydar; Sajwan, Kamal P.; Selvanathan, Saravana P.; Marsh, Benjamin J.; Pai, Amrita V.; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z.; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A.

    2015-01-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. PMID:26149384

  12. The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

    PubMed Central

    Mortimer, Nathan T.; Moberg, Kenneth H.

    2007-01-01

    The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila melanogaster and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. Here we show that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease. PMID:17976568

  13. Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2015-10-21

    Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods. PMID:26381020

  14. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

    PubMed Central

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N.; Vainchenker, William; Solary, Eric

    2014-01-01

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. PMID:25143485

  15. Unravelling Protein-DNA Interactions at Molecular Level: A DFT and NCI Study.

    PubMed

    González, J; Baños, I; León, I; Contreras-García, J; Cocinero, E J; Lesarri, A; Fernández, J A; Millán, J

    2016-02-01

    Histone-DNA interactions were probed computationally at a molecular level, by characterizing the bimolecular clusters constituted by selected amino acid derivatives with polar (asparagine and glutamine), nonpolar (alanine, valine, and isoleucine), and charged (arginine) side chains and methylated pyrimidinic (1-methylcytosine and 1-methylthymine) and puric (9-methyladenine and 9-methylguanine) DNA bases. The computational approach combined different methodologies: a molecular mechanics (MMFFs forced field) conformational search and structural and vibrational density-functional calculations (M06-2X with double and triple-ζ Pople's basis sets). To dissect the interactions, intermolecular forces were analyzed with the Non-Covalent Interactions (NCI) analysis. The results for the 24 different clusters studied show a noticeable correlation between the calculated binding energies and the propensities for protein-DNA base interactions found in the literature. Such correlation holded even for the interaction of the selected amino acid derivatives with Watson and Crick pairs. Therefore, the balance between hydrogen bonds and van der Waals interactions (specially stacking) in the control of the final shape of the investigated amino acid-DNA base pairs seems to be well reproduced in dispersion-corrected DFT molecular models, reinforcing the idea that the specificity between the amino acids and the DNA bases play an important role in the regulation of DNA. PMID:26765058

  16. Inhibition of Fatty Acid Binding Proteins Elevates Brain Anandamide Levels and Produces Analgesia

    PubMed Central

    Kaczocha, Martin; Rebecchi, Mario J.; Ralph, Brian P.; Teng, Yu-Han Gary; Berger, William T.; Galbavy, William; Elmes, Matthew W.; Glaser, Sherrye T.; Wang, Liqun; Rizzo, Robert C.; Deutsch, Dale G.; Ojima, Iwao

    2014-01-01

    The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics. PMID:24705380

  17. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  18. Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

    PubMed Central

    Kauwe, John S. K.; Bailey, Matthew H.; Ridge, Perry G.; Perry, Rachel; Wadsworth, Mark E.; Hoyt, Kaitlyn L.; Staley, Lyndsay A.; Karch, Celeste M.; Harari, Oscar; Cruchaga, Carlos; Ainscough, Benjamin J.; Bales, Kelly; Pickering, Eve H.; Bertelsen, Sarah; Fagan, Anne M.; Holtzman, David M.; Morris, John C.; Goate, Alison M.

    2014-01-01

    Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal

  19. 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

  20. Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis.

    PubMed

    Watford, Malcolm; Wu, Guoyao

    2005-04-01

    High intracellular glutamine levels have been implicated in promoting net protein synthesis and accretion in mammalian skeletal muscle. Little is known regarding glutamine metabolism in uricotelic species but chicken breast muscle exhibits high rates of protein accretion and would be predicted to maintain high glutamine levels. However, chicken breast muscle expresses high glutaminase activity and here we report that chicken breast muscle also expresses low glutamine synthetase activity (0.07+/-0.01 U/g) when compared to leg muscle (0.50+/-0.04 U/g). Free glutamine levels were 1.38+/-0.09 and 9.69+/-0.12 nmol/mg wet weight in breast and leg muscles of fed chickens, respectively. Glutamine levels were also lower in dove breast muscle (4.82+/-0.35 nmol/mg wet weight) when compared to leg muscle (16.2+/-1.0 nmol/mg wet weight) and much lower (1.80+/-0.46 nmol/mg wet weight) in lizard leg muscle. In fed chickens, rates of fractional protein synthesis were higher in leg than in breast muscle, and starvation (48 h) resulted in a decrease in both glutamine content and rate of protein synthesis in leg muscle. Thus, although tissue-specific glutamine metabolism in uricotelic species differs markedly from that in ureotelic animals, differences in rates of skeletal muscle protein synthesis are associated with corresponding differences in intramuscular glutamine content. PMID:15763516

  1. Relationship between VEGF Gene Polymorphisms and Serum VEGF Protein Levels in Patients with Rheumatoid Arthritis

    PubMed Central

    Paradowska-Gorycka, Agnieszka; Pawlik, Andrzej; Romanowska-Prochnicka, Katarzyna; Haladyj, Ewa; Malinowski, Damian; Stypinska, Barbara; Manczak, Malgorzata; Olesinska, Marzena

    2016-01-01

    lower than the number of women with wild type allele -2578A (p = 0.006). Serum VEGF levels were significantly higher in RA patients than in control groups (both p = 0,0001). Conclusion Present findings indicated that VEGF genetic polymorphism as well as VEGF protein levels may be associated with the susceptibility to RA in the Polish population. PMID:27513931

  2. Cerebrospinal fluid neurofilament light chain protein levels in subtypes of frontotemporal dementia

    PubMed Central

    2013-01-01

    Background Frontotemporal dementia (FTD) is recognised as a clinically and morphologically heterogeneous group of interrelated neurodegenerative conditions. One of the subtypes within this disease spectrum is the behavioural variant FTD (bvFTD). This is known to be a varied disorder with a mixture of tau-positive and tau-negative underlying pathologies. The other subtypes include semantic dementia (SD), which generally exhibits tau-negative pathology, and progressive non-fluent aphasia (PNFA), which is usually tau-positive. As the clinical presentation of these subtypes may overlap, a specific diagnosis can be difficult to attain and today no specific biomarker can predict the underlying pathology. Neurofilament light chain protein (NFL), a cytoskeletal constituent of intermediate filaments, is thought to reflect neuronal and axonal death when appearing in the cerebrospinal fluid (CSF). NFL has been shown to be elevated in CSF in patients with FTD compared with AD and controls. Our hypothesis was that the levels of NFL also differ between the subtypes of FTD and may indicate the underlying pathological subtype. Methods We retrospectively analysed data from previous CSF analyses in 34 FTD cases (23 bvFTD, seven SD, four PNFA), 20 AD cases, and 26 healthy controls. A separate group of 10 neuropathologically verified and subtyped FTD cases (seven tau-negative, three tau-positive) were also analysed. Result NFL levels were significantly higher in FTD compared with both AD (p<0.001) and controls (p<0.001). The NFL levels of SD and bvFTD were significantly higher (p<0.001) compared with AD. The biomarker profiles of PNFA and AD were similar. In the neuropathologically verified FTD cases, NFL was higher in the tau-negative than in the tau-positive cases (exact p=0.017). Conclusions The marked NFL elevation in some but not all FTD cases is likely to reflect the different underlying pathologies. The highest NFL values found in the SD group as well as in the

  3. Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

    PubMed

    El Asmar, Zeina; Terrand, Jérome; Jenty, Marion; Host, Lionel; Mlih, Mohamed; Zerr, Aurélie; Justiniano, Hélène; Matz, Rachel L; Boudier, Christian; Scholler, Estelle; Garnier, Jean-Marie; Bertaccini, Diego; Thiersé, Danièle; Schaeffer, Christine; Van Dorsselaer, Alain; Herz, Joachim; Bruban, Véronique; Boucher, Philippe

    2016-03-01

    The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases. PMID:26792864

  4. Detection and identification of sub-nanogram levels of protein in a nanoLC-trypsin-MS system.

    PubMed

    Slysz, Gordon W; Lewis, Darren F; Schriemer, David C

    2006-08-01

    Proteomic workflows involving liquid-based protein separations are an alternative to gel-based protein analysis, however the trypsin digestion procedure is usually difficult to implement, particularly when processing low abundance proteins from capillary column effluent. To convert the protein to peptides for the purpose of identification, current protocols require several sample handling steps, and sample losses become an issue. In this study, we present an improved system that conducts reversed-phase protein chromatography and rapid on-line tryptic digestion requiring sub-nanogram quantities of protein. This system employs a novel mirror-gradient concept that allows for dynamic titration of the column effluent to create optimal conditions for real-time tryptic digestion. The purpose behind this development was to improve the limits of detection of the online concept, to support flow-based alternatives to gel-based proteomics and to simplify the characterization of low abundance proteins. Using test mixtures of proteins, we show that peptide mass fingerprinting with high sequence representation can be easily achieved at the 20 fmol level, with detection limits down to 5 fmol (85 pg myoglobin). Limits of identification using standard data-dependent MS/MS experiments are as low as 10 fmol. These results suggest that the nanoLC-trypsin-MS/MS system could represent an alternative to the conventional "1D-gel to MS" proteomic strategy. PMID:16889418

  5. Associations Between Common and Rare Exonic Genetic Variants and Serum Levels of 20 Cardiovascular-Related Proteins

    PubMed Central

    Solomon, Terry; Smith, Erin N.; Matsui, Hiroko; Braekkan, Sigrid K.; Wilsgaard, Tom; Njølstad, Inger; Mathiesen, Ellisiv B.; Hansen, John-Bjarne

    2016-01-01

    Background— Genetic variation can be used to study causal relationships between biomarkers and diseases. Here, we identify new common and rare genetic variants associated with cardiovascular-related protein levels (protein quantitative trait loci [pQTLs]). We functionally annotate these pQTLs, predict and experimentally confirm a novel molecular interaction, and determine which pQTLs are associated with diseases and physiological phenotypes. Methods and Results— As part of a larger case–control study of venous thromboembolism, serum levels of 51 proteins implicated in cardiovascular diseases were measured in 330 individuals from the Tromsø Study. Exonic genetic variation near each protein’s respective gene (cis) was identified using sequencing and arrays. Using single site and gene-based tests, we identified 27 genetic associations between pQTLs and the serum levels of 20 proteins: 14 associated with common variation in cis, of which 6 are novel (ie, not previously reported); 7 associations with rare variants in cis, of which 4 are novel; and 6 associations in trans. Of the 20 proteins, 15 were associated with single sites and 7 with rare variants. cis-pQTLs for kallikrein and F12 also show trans associations for proteins (uPAR, kininogen) known to be cleaved by kallikrein and with NTproBNP. We experimentally demonstrate that kallikrein can cleave proBNP (NTproBNP precursor) in vitro. Nine of the pQTLs have previously identified associations with 17 disease and physiological phenotypes. Conclusions— We have identified cis and trans genetic variation associated with the serum levels of 20 proteins and utilized these pQTLs to study molecular mechanisms underlying disease and physiological phenotypes. PMID:27329291

  6. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

    PubMed

    Narayanan, Narayanan N; Ihemere, Uzoma; Ellery, Claire; Sayre, Richard T

    2011-01-01

    Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels. PMID:21799761

  7. Residue-level global and local ensemble-ensemble comparisons of protein domains.

    PubMed

    Clark, Sarah A; Tronrud, Dale E; Karplus, P Andrew

    2015-09-01

    Many methods of protein structure generation such as NMR-based solution structure determination and template-based modeling do not produce a single model, but an ensemble of models consistent with the available information. Current strategies for comparing ensembles lose information because they use only a single representative structure. Here, we describe the ENSEMBLATOR and its novel strategy to directly compare two ensembles containing the same atoms to identify significant global and local backbone differences between them on per-atom and per-residue levels, respectively. The ENSEMBLATOR has four components: eePREP (ee for ensemble-ensemble), which selects atoms common to all models; eeCORE, which identifies atoms belonging to a cutoff-distance dependent common core; eeGLOBAL, which globally superimposes all models using the defined core atoms and calculates for each atom the two intraensemble variations, the interensemble variation, and the closest approach of members of the two ensembles; and eeLOCAL, which performs a local overlay of each dipeptide and, using a novel measure of local backbone similarity, reports the same four variations as eeGLOBAL. The combination of eeGLOBAL and eeLOCAL analyses identifies the most significant differences between ensembles. We illustrate the ENSEMBLATOR's capabilities by showing how using it to analyze NMR ensembles and to compare NMR ensembles with crystal structures provides novel insights compared to published studies. One of these studies leads us to suggest that a "consistency check" of NMR-derived ensembles may be a useful analysis step for NMR-based structure determinations in general. The ENSEMBLATOR 1.0 is available as a first generation tool to carry out ensemble-ensemble comparisons. PMID:26032515

  8. Alternations of 14-3-3 θ and β protein levels in brain during experimental sepsis.

    PubMed

    Memos, Nikolaos; Kataki, Agapi; Chatziganni, Emmy; Nikolopoulou, Marilena; Skoulakis, Euthimios; Consoulas, Christos; Zografos, George; Konstadoulakis, Manousos

    2011-09-01

    The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during se