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Sample records for 1alpha pgc-1alpha mitochondrial

  1. The transcriptional coactivator PGC-1alpha is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis.

    PubMed

    Lehman, John J; Boudina, Sihem; Banke, Natasha Hausler; Sambandam, Nandakumar; Han, Xianlin; Young, Deanna M; Leone, Teresa C; Gross, Richard W; Lewandowski, E Douglas; Abel, E Dale; Kelly, Daniel P

    2008-07-01

    High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1alpha (PGC-1alpha(-/-) mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1alpha is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1alpha(-/-) mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1alpha(-/-) mice compared with PGC-1alpha(+/+) controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1alpha(-/-) mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1alpha(-/-) fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1alpha(-/-) mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. (13)C NMR revealed that hearts from PGC-1alpha(-/-) mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during beta-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1alpha(-/-) mice relative to PGC-1alpha(+/+) following a fast. Overall, these results demonstrate that PGC-1alpha is essential for the maintenance of maximal, efficient cardiac

  2. Lithium increases PGC-1alpha expression and mitochondrial biogenesis in primary bovine aortic endothelial cells.

    PubMed

    Struewing, Ian T; Barnett, Corey D; Tang, Tao; Mao, Catherine D

    2007-06-01

    Lithium is a therapeutic agent commonly used to treat bipolar disorder and its beneficial effects are thought to be due to a combination of activation of the Wnt/beta-catenin pathway via inhibition of glycogen synthase kinase-3beta and depletion of the inositol pool via inhibition of the inositol monophosphatase-1. We demonstrated that lithium in primary endothelial cells induced an increase in mitochondrial mass leading to an increase in ATP production without any significant change in mitochondrial efficiency. This increase in mitochondrial mass was associated with an increase in the mRNA levels of mitochondrial biogenesis transcription factors: nuclear respiratory factor-1 and -2beta, as well as mitochondrial transcription factors A and B2, which lead to the coordinated upregulation of oxidative phosphorylation components encoded by either the nuclear or mitochondrial genome. These effects of lithium on mitochondrial biogenesis were independent of the inhibition of glycogen synthase kinase-3beta and independent of inositol depletion. Also, expression of the coactivator PGC-1alpha was increased, whereas expression of the coactivator PRC was not affected. Lithium treatment rapidly induced a decrease in activating Akt-Ser473 phosphorylation and inhibitory Forkhead box class O (FOXO1)-Thr24 phosphorylation, as well as an increase in activating c-AMP responsive element binding (CREB)-Ser133 phosphorylation, two mechanisms known to control PGC-1alpha expression. Together, our results show that lithium induces mitochondrial biogenesis via CREB/PGC-1alpha and FOXO1/PGC-1alpha cascades, which highlight the pleiotropic effects of lithium and reveal also novel beneficial effects via preservation of mitochondrial functions.

  3. Compensatory increases in nuclear PGC1alpha protein are primarily associated with subsarcolemmal mitochondrial adaptations in ZDF rats.

    PubMed

    Holloway, Graham P; Gurd, Brendon J; Snook, Laelie A; Lally, Jamie; Bonen, Arend

    2010-04-01

    We examined in insulin-resistant muscle if, in contrast to long-standing dogma, mitochondrial fatty acid oxidation is increased and whether this is attributed to an increased nuclear content of peroxisome proliferator-activated receptor (PPAR) gamma coactivator (PGC) 1alpha and the adaptations of specific mitochondrial subpopulations. Skeletal muscles from male control and Zucker diabetic fatty (ZDF) rats were used to determine 1) intramuscular lipid distribution, 2) subsarcolemmal and intermyofibrillar mitochondrial morphology, 3) rates of palmitate oxidation in subsarcolemmal and intermyofibrillar mitochondria, and 4) the subcellular localization of PGC1alpha. Electotransfection of PGC1alpha cDNA into lean animals tested the notion that increased nuclear PGC1alpha preferentially targeted subsarcolemmal mitochondria. Transmission electron microscope analysis revealed that in ZDF animals the number (+50%), width (+69%), and density (+57%) of subsarcolemmal mitochondria were increased (P < 0.05). In contrast, intermyofibrillar mitochondria remained largely unchanged. Rates of palmitate oxidation were approximately 40% higher (P < 0.05) in ZDF subsarcolemmal and intermyofibrillar mitochondria, potentially as a result of the increased PPAR-targeted proteins, carnitine palmitoyltransferase-I, and fatty acid translocase (FAT)/CD36. PGC1alpha mRNA and total protein were not altered in ZDF animals; however, a greater (approximately 70%; P < 0.05) amount of PGC1alpha was located in nuclei. Overexpression of PGC1alpha only increased subsarcolemmal mitochondrial oxidation rates. In ZDF animals, intramuscular lipids accumulate in the intermyofibrillar region (increased size and number), and this is primarily associated with increased oxidative capacity in subsarcolemmal mitochondria (number, size, density, and oxidation rates). These changes may result from an increased nuclear content of PGC1alpha, as under basal conditions, overexpression of PGC1alpha appears to target

  4. Impaired PGC-1alpha function in muscle in Huntington's disease.

    PubMed

    Chaturvedi, Rajnish K; Adhihetty, Peter; Shukla, Shubha; Hennessy, Thomas; Calingasan, Noel; Yang, Lichuan; Starkov, Anatoly; Kiaei, Mahmoud; Cannella, Milena; Sassone, Jenny; Ciammola, Andrea; Squitieri, Fernando; Beal, M Flint

    2009-08-15

    We investigated the role of PPAR gamma coactivator 1alpha (PGC-1alpha) in muscle dysfunction in Huntington's disease (HD). We observed reduced PGC-1alpha and target genes expression in muscle of HD transgenic mice. We produced chronic energy deprivation in HD mice by administering the catabolic stressor beta-guanidinopropionic acid (GPA), a creatine analogue that reduces ATP levels, activates AMP-activated protein kinase (AMPK), which in turn activates PGC-1alpha. Treatment with GPA resulted in increased expression of AMPK, PGC-1alpha target genes, genes for oxidative phosphorylation, electron transport chain and mitochondrial biogenesis, increased oxidative muscle fibers, numbers of mitochondria and motor performance in wild-type, but not in HD mice. In muscle biopsies from HD patients, there was decreased PGC-1alpha, PGC-1beta and oxidative fibers. Oxygen consumption, PGC-1alpha, NRF1 and response to GPA were significantly reduced in myoblasts from HD patients. Knockdown of mutant huntingtin resulted in increased PGC-1alpha expression in HD myoblast. Lastly, adenoviral-mediated delivery of PGC-1alpha resulted increased expression of PGC-1alpha and markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice. These findings show that impaired function of PGC-1alpha plays a critical role in muscle dysfunction in HD, and that treatment with agents to enhance PGC-1alpha function could exert therapeutic benefits. Furthermore, muscle may provide a readily accessible tissue in which to monitor therapeutic interventions.

  5. PGC-1alpha plays a functional role in exercise-induced mitochondrial biogenesis and angiogenesis but not fiber-type transformation in mouse skeletal muscle.

    PubMed

    Geng, Tuoyu; Li, Ping; Okutsu, Mitsuharu; Yin, Xinhe; Kwek, Jyeyi; Zhang, Mei; Yan, Zhen

    2010-03-01

    Endurance exercise stimulates peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) expression in skeletal muscle, and forced expression of PGC-1alpha changes muscle metabolism and exercise capacity in mice. However, it is unclear if PGC-1alpha is indispensible for endurance exercise-induced metabolic and contractile adaptations in skeletal muscle. In this study, we showed that endurance exercise-induced expression of mitochondrial enzymes (cytochrome oxidase IV and cytochrome c) and increases of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31)-positive endothelial cells in skeletal muscle, but not IIb-to-IIa fiber-type transformation, were significantly attenuated in muscle-specific Pgc-1alpha knockout mice. Interestingly, voluntary running effectively restored the compromised mitochondrial integrity and superoxide dismutase 2 (SOD2) protein expression in skeletal muscle in Pgc-1alpha knockout mice. Thus, PGC-1alpha plays a functional role in endurance exercise-induced mitochondrial biogenesis and angiogenesis, but not IIb-to-IIa fiber-type transformation in mouse skeletal muscle, and the improvement of mitochondrial morphology and antioxidant defense in response to endurance exercise may occur independently of PGC-1alpha function. We conclude that PGC-1alpha is required for complete skeletal muscle adaptations induced by endurance exercise in mice.

  6. Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

    SciTech Connect

    Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny; Ko, Kyung Soo; Pak, Youngmi Kim . E-mail: ymkimpak@amc.seoul.kr

    2006-06-09

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivator activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.

  7. Melatonin Improves mitochondrial function by promoting MT1/SIRT1/PGC-1 alpha-dependent mitochondrial biogenesis in cadmium-induced hepatotoxicity in vitro.

    PubMed

    Guo, Pan; Pi, Huifeng; Xu, Shangcheng; Zhang, Lei; Li, Yuming; Li, Min; Cao, Zhengwang; Tian, Li; Xie, Jia; Li, Renyan; He, Mindi; Lu, Yonghui; Liu, Chuan; Duan, Weixia; Yu, Zhengping; Zhou, Zhou

    2014-11-01

    Melatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, and it has been under clinical investigation for expanded applications. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. This study investigates the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. Consistent with this finding, Cd exposure was associated with decreased Sirtuin 1 (SIRT1) protein expression and activity, thus promoted acetylation of PGC-1 alpha, a key enzyme involved in mitochondrial biogenesis and function, although Cd did not disrupt the interaction between SIRT1 and PGC-1 alpha. However, all cadmium-induced mitochondrial oxidative injuries were efficiently attenuated by melatonin pretreatment. Moreover, Sirtinol and SIRT1 siRNA each blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT1/ PGC-1 alpha signaling. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to promote SIRT1/ PGC-1 alpha signaling. In summary, our results indicate that SIRT1 plays an essential role in the ability of moderate melatonin to stimulate PGC-1 alpha and improve mitochondrial biogenesis and function at least partially through melatonin receptors in cadmium-induced hepatotoxicity. © Published by Oxford University Press on behalf of Toxicological Sciences.

  8. Melatonin Improves Mitochondrial Function by Promoting MT1/SIRT1/PGC-1 Alpha-Dependent Mitochondrial Biogenesis in Cadmium-Induced Hepatotoxicity In Vitro

    PubMed Central

    Guo, Pan; Pi, Huifeng; Xu, Shangcheng; Zhang, Lei; Li, Yuming; Li, Min; Cao, Zhengwang; Tian, Li; Xie, Jia; Li, Renyan; He, Mindi; Lu, Yonghui; Liu, Chuan; Duan, Weixia; Yu, Zhengping; Zhou, Zhou

    2014-01-01

    Melatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, and it has been under clinical investigation for expanded applications. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. This study investigates the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10μM) for 12 h. We found that Cd stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. Consistent with this finding, Cd exposure was associated with decreased Sirtuin 1 (SIRT1) protein expression and activity, thus promoted acetylation of PGC-1 alpha, a key enzyme involved in mitochondrial biogenesis and function, although Cd did not disrupt the interaction between SIRT1 and PGC-1 alpha. However, all cadmium-induced mitochondrial oxidative injuries were efficiently attenuated by melatonin pretreatment. Moreover, Sirtinol and SIRT1 siRNA each blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT1/ PGC-1 alpha signaling. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to promote SIRT1/ PGC-1 alpha signaling. In summary, our results indicate that SIRT1 plays an essential role in the ability of moderate melatonin to stimulate PGC-1 alpha and improve mitochondrial biogenesis and function at least partially through melatonin receptors in cadmium-induced hepatotoxicity. PMID:25159133

  9. Resveratrol exerts pharmacological preconditioning by activating PGC-1alpha.

    PubMed

    Tan, Lan; Yu, Jin-Tai; Guan, Hua-Shi

    2008-11-01

    Resveratrol (RSV), a polyphenol phytoalexin abundantly found in grape skins and in wines, is currently the focus of intense research as a pharmacological preconditioning agent in kidney, heart, and brain from ischemic injury. However, the exact molecular mechanism of RSV preconditioning remains obscure. The data from current studies indicate that pharmacological preconditioning with RSV were attributed to its role as intracellular antioxidant, anti-inflammatory agent, its ability to induce nitric oxide synthase (NOS) expression, its ability to induce angiogenesis, and its ability to increases sirtuin 1 (SIRT1) activity. Peroxisome proliferators-activated receptor (PPAR) gamma co-activator-1alpha (PGC-1alpha) is a member of a family of transcription coactivators that owns mitochondrial biogenesis, antioxidation, growth factor signaling regulation, and angiogenesis activities. And, almost all the signaling pathways activated by RVS involve in PGC-1alpha activity. Moreover, it has been proofed that RVS could mediate an increase PGC-1alpha activity. These significant conditions support the hypothesis that RSV exerts pharmacological preconditioning by activating PGC-1alpha. Attempts to confirm this hypothesis will provide new directions in the study of pharmaceutical preconditioning and the development of new treatment approaches for reducing the extent of ischemia/reperfusion injury.

  10. Pyrroloquinoline quinone stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression.

    PubMed

    Chowanadisai, Winyoo; Bauerly, Kathryn A; Tchaparian, Eskouhie; Wong, Alice; Cortopassi, Gino A; Rucker, Robert B

    2010-01-01

    Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1-6 cells to 10-30 microm PQQ for 24-48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1alpha, and increased PGC-1alpha mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1alpha or CREB expression. Consistent with activation of the PGC-1alpha pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction.

  11. PGC-1 alpha regulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid.

    PubMed

    Singh, Shailendra P; Schragenheim, Joseph; Cao, Jian; Falck, John R; Abraham, Nader G; Bellner, Lars

    2016-09-01

    Obesity is a risk factor in the development of type 2 diabetes mellitus (DM2), which is associated with increased morbidity and mortality, predominantly as a result of cardiovascular complications. Increased adiposity is a systemic condition characterized by increased oxidative stress (ROS), increased inflammation, inhibition of anti-oxidant genes such as HO-1 and increased degradation of epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs attenuate mitochondrial ROS. We postulate that EETs increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which controls mitochondrial function, oxidative metabolism and induction of HO-1. Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess functional relationship between EETs, HO-1 and (PGC-1α) using an EET analogue (EET-A) and lentivirus to knock down the PPARGC1A gene. EET-A increased PGC-1α and HO-1 in cultured adipocytes and increased the expression of genes involved in thermogenesis and adipocyte browning (UCP1 and PRDM16, respectively). PGC-1α knockdown prevented EET-A-induced HO-1expression, suggesting that PGC-1α is upstream of HO-1. MRI data obtained from fat tissues showed that EET-A administration to mice on a HF diet significantly reduced total body fat content, subcutaneous and visceral fat deposits and reduced the VAT: SAT ratio. Moreover EET-A normalized the VO2 and RQ (VCO2/VO2) in mice fed a HF diet, an effect that was completely prevented in PGC-1α deficient mice. In addition, EET-A increased mitochondrial biogenesis and function as measured by OPA1, MnSOD, Mfn1, Mfn2, and SIRT3, an effect that was inhibited by knockdown of PGC-1α. Taken together, our findings show that EET-A increased PGC-1α thereby increasing mitochondrial viability, increased fusion potential thereby providing metabolic protection and increased VO2 consumption in HF-induced obesity in mice, thus demonstrating that the EET-mediated increase in HO-1

  12. Identification of novel targets for PGC-1{alpha} and histone deacetylase inhibitors in neuroblastoma cells

    SciTech Connect

    Cowell, Rita M. Talati, Pratik; Blake, Kathryn R.; Meador-Woodruff, James H.; Russell, James W.

    2009-02-06

    Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1{alpha} express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1{alpha} in neuronal cells and whether there are ways to pharmacologically target PGC-1{alpha} in neurons. Here, PGC-1{alpha} overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1{alpha} and glucose transporter 4 (GLUT4). These results suggest that PGC-1{alpha} regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1{alpha} and GLUT4 in HD and other neurological disorders.

  13. PGC-1alpha activates CYP7A1 and bile acid biosynthesis.

    PubMed

    Shin, Dong-Ju; Campos, Jose A; Gil, Gregorio; Osborne, Timothy F

    2003-12-12

    Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

  14. Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

    SciTech Connect

    Yoshioka, Toyo; Inagaki, Kenjiro; Noguchi, Tetsuya; Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya; Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji; Kasuga, Masato

    2009-04-17

    The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

  15. Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging.

    PubMed

    Wenz, Tina; Rossi, Susana G; Rotundo, Richard L; Spiegelman, Bruce M; Moraes, Carlos T

    2009-12-01

    Aging is a major risk factor for metabolic disease and loss of skeletal muscle mass and strength, a condition known as sarcopenia. Both conditions present a major health burden to the elderly population. Here, we analyzed the effect of mildly increased PGC-1alpha expression in skeletal muscle during aging. We found that transgenic MCK-PGC-1alpha animals had preserved mitochondrial function, neuromuscular junctions, and muscle integrity during aging. Increased PGC-1alpha levels in skeletal muscle prevented muscle wasting by reducing apoptosis, autophagy, and proteasome degradation. The preservation of muscle integrity and function in MCK-PGC-1alpha animals resulted in significantly improved whole-body health; both the loss of bone mineral density and the increase of systemic chronic inflammation, observed during normal aging, were prevented. Importantly, MCK-PGC-1alpha animals also showed improved metabolic responses as evident by increased insulin sensitivity and insulin signaling in aged mice. Our results highlight the importance of intact muscle function and metabolism for whole-body homeostasis and indicate that modulation of PGC-1alpha levels in skeletal muscle presents an avenue for the prevention and treatment of a group of age-related disorders.

  16. Exercise increases mitochondrial PGC-1alpha content and promotes nuclear-mitochondrial cross-talk to coordinate mitochondrial biogenesis.

    PubMed

    Safdar, Adeel; Little, Jonathan P; Stokl, Andrew J; Hettinga, Bart P; Akhtar, Mahmood; Tarnopolsky, Mark A

    2011-03-25

    Endurance exercise is known to induce metabolic adaptations in skeletal muscle via activation of the transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α). PGC-1α regulates mitochondrial biogenesis via regulating transcription of nuclear-encoded mitochondrial genes. Recently, PGC-1α has been shown to reside in mitochondria; however, the physiological consequences of mitochondrial PGC-1α remain unknown. We sought to delineate if an acute bout of endurance exercise can mediate an increase in mitochondrial PGC-1α content where it may co-activate mitochondrial transcription factor A to promote mtDNA transcription. C57Bl/6J mice (n = 12/group; ♀ = ♂) were randomly assigned to sedentary (SED), forced-endurance (END) exercise (15 m/min for 90 min), or forced endurance +3 h of recovery (END+3h) group. The END group was sacrificed immediately after exercise, whereas the SED and END+3h groups were euthanized 3 h after acute exercise. Acute exercise coordinately increased the mRNA expression of nuclear and mitochondrial DNA-encoded mitochondrial transcripts. Nuclear and mitochondrial abundance of PGC-1α in END and END+3h groups was significantly higher versus SED mice. In mitochondria, PGC-1α is in a complex with mitochondrial transcription factor A at mtDNA D-loop, and this interaction was positively modulated by exercise, similar to the increased binding of PGC-1α at the NRF-1 promoter. We conclude that in response to acute altered energy demands, PGC-1α re-localizes into nuclear and mitochondrial compartments where it functions as a transcriptional co-activator for both nuclear and mitochondrial DNA transcription factors. These results suggest that PGC-1α may dynamically facilitate nuclear-mitochondrial DNA cross-talk to promote net mitochondrial biogenesis.

  17. Mitochondrial-related gene expression profiles suggest an important role of PGC-1alpha in the compensatory mechanism of endemic dilated cardiomyopathy

    SciTech Connect

    He, Shu-Lan; Tan, Wu-Hong; Zhang, Zeng-Tie; Zhang, Feng; Qu, Cheng-Juan; Lei, Yan-Xia; Zhu, Yan-He; Yu, Han-Jie; Xiang, You-Zhang; and others

    2013-10-15

    Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios≥2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD. Highlights: • Thirty-four up-regulated genes were detected in KD versus health controls. • Forty pathways and four networks were detected in KD. • PGC-1alpha regulated energy metabolism and anti-apoptosis in KD.

  18. Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1{alpha} in rats

    SciTech Connect

    Lieber, Charles S. Leo, Maria A.; Wang Xiaolei; DeCarli, Leonore M.

    2008-05-23

    The nuclear genes, NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-{gamma} coactivator1{alpha} (PGC-1{alpha}) are regulators of energy metabolism. Here, we studied the role of alcohol consumption in expression of these sensing molecules. Alcohol significantly reduced hepatic SIRT1 mRNA by 50% and PGC-1{alpha} mRNA by 46% and it significantly inhibited the protein expression of SIRT1 and PGC-1{alpha}, while the transcription factor PPAR-{gamma} remained unchanged. However, when the lipid composition of the alcohol diet was changed by replacing long-chain triglycerides (LCT) with medium chain triglycerides (MCT), SIRT1 and PGC-1{alpha} mRNA were restored to near control levels. This study demonstrates that alcohol reduces key energy sensing proteins and that replacement of LCT by MCT affects the transcription of these genes. Since there is a pathophysiological link between SIRT1 and PGC-1{alpha} and mitochondrial energy, the implication of the study is that mitochondrial dysfunction due to alcohol abuse can be treated by dietary modifications.

  19. PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

    SciTech Connect

    Chen, Min; Wang, Yanru; Qu, Aijuan

    2010-06-11

    Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+} transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.

  20. PGC-1alpha-mediated regulation of gene expression and metabolism: implications for nutrition and exercise prescriptions.

    PubMed

    Benton, Carley R; Wright, David C; Bonen, Arend

    2008-10-01

    The discovery 10 years ago of PGC-1alpha represented a major milestone towards understanding of the molecular processes regulating energy metabolism in many tissues, including skeletal muscle. PGC-1alpha orchestrates a metabolic program regulating oxidative lipid metabolism and insulin sensitivity. This is essentially the same metabolic program that is activated by exercise and down-regulated by sedentary lifestyles and high-fat diets, as well as in cases of obesity and type 2 diabetes. The present review examines the evidence in support of the key role for PGC-1alpha regulation of substrate metabolism and mitochondrial biogenesis in skeletal muscle. Surprisingly, studies with PGC-1alpha null and transgenic mice have revealed unexpected pathologies when PGC-1alpha is completely repressed (KO animals) or is massively overexpressed. In contrast, PGC-1alpha overexpression within normal physiological limits results in marked improvements in fatty acid oxidation and insulin-stimulated glucose transport. Exercise, sedentary lifestyles, and nutritional factors can regulate PGC-1alpha expression. We speculate that optimal targeting of PGC-1alpha upregulation, whether by diet, exercise, or a combination of both, could represent effective prophylactic or therapeutic means to improve insulin sensitivity. Indeed, using modern molecular tools, it may indeed be possible to prescribe optimally individualized nutrition and exercise programs.

  1. Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle.

    PubMed

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-02-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1alpha (PGC-1alpha) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1alpha transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P < 0.05) in PGC-1alpha transcription (10- to > 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1alpha was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1alpha in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor alpha, and calcineurin Aalpha and Abeta mRNA were elevated (approximately 2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1alpha transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1alpha may coordinate the activation of metabolic genes in human muscle in response to exercise.

  2. PGC-1alpha downstream transcription factors NRF-1 and TFAM are genetic modifiers of Huntington disease

    PubMed Central

    2011-01-01

    Background Huntington disease (HD) is an inherited neurodegenerative disease caused by an abnormal expansion of a CAG repeat in the huntingtin HTT (HD) gene. The primary genetic determinant of the age at onset (AO) is the length of the HTT CAG repeat; however, the remaining genetic contribution to the AO of HD has largely not been elucidated. Recent studies showed that impaired functioning of the peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1alpha) contributes to mitochondrial dysfunction and appears to play an important role in HD pathogenesis. Further genetic evidence for involvement of PGC-1alpha in HD pathogenesis was generated by the findings that sequence variations in the PPARGC1A gene encoding PGC-1alpha exert modifying effects on the AO in HD. In this study, we hypothesised that polymorphisms in PGC-1alpha downstream targets might also contribute to the variation in the AO. Results In over 400 German HD patients, polymorphisms in the nuclear respiratory factor 1 gene, NRF-1, and the mitochondrial transcription factor A, encoded by TFAM showed nominally significant association with AO of HD. When combining these results with the previously described modifiers rs7665116 in PPARGC1A and C7028T in the cytochrome c oxidase subunit I (CO1, mt haplogroup H) in a multivariable model, a substantial proportion of the variation in AO can be explained by the joint effect of significant modifiers and their interactions, respectively. Conclusions These results underscore that impairment of mitochondrial function plays a critical role in the pathogenesis of HD and that upstream transcriptional activators of PGC-1alpha may be useful targets in the treatment of HD. PMID:21595933

  3. Partnership of PGC-1alpha and HNF4alpha in the regulation of lipoprotein metabolism.

    PubMed

    Rhee, James; Ge, Hongfei; Yang, Wenli; Fan, Melina; Handschin, Christoph; Cooper, Marcus; Lin, Jiandie; Li, Cai; Spiegelman, Bruce M

    2006-05-26

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator involved in several aspects of energy metabolism. It is induced or activated under different stimuli in a highly tissue-specific manner and subsequently partners with certain transcription factors in those tissues to execute various biological programs. In the fasted liver, PGC-1alpha is induced and interacts with hepatocyte nuclear factor 4alpha (HNF4alpha) and other transcription factors to activate gluconeogenesis and increase hepatic glucose output. Given the broad spectrum of liver genes responsive to HNF4alpha, we sought to determine those that were specifically targeted by the combination of PGC-1alpha and HNF4alpha. Coexpression of these two molecules in murine stem cells reveals a high induction of mRNA for apolipoproteins A-IV and C-II. Forced expression of PGC-1alpha in mouse and human hepatoma cells increases the mRNA of a subset of apolipoproteins implicated in very low density lipoprotein and triglyceride metabolism, including apolipoproteins A-IV, C-II, and C-III. Coactivation of the apoC-III/A-IV promoter region by PGC-1alpha occurs through a highly conserved HNF4alpha response element, the loss of which completely abolishes activation by PGC-1alpha and HNF4alpha. Adenoviral infusion of PGC-1alpha into live mice increases hepatic expression of apolipoproteins A-IV, C-II, and C-III and increases serum and very low density lipoprotein triglyceride levels. Conversely, knock down of PGC-1alpha in vivo causes a decrease in both apolipoprotein expression and serum triglyceride levels. These data point to a crucial role for the PGC-1alpha/HNF4alpha partnership in hepatic lipoprotein metabolism.

  4. Mitochondrial and sarcoplasmic protein changes in hearts from copper-deficient rats: up-regulation of PGC-1alpha transcript and protein as a cause for mitochondrial biogenesis in copper deficiency.

    PubMed

    Medeiros, Denis M; Jiang, Yu; Klaahsen, Darcey; Lin, Dingbo

    2009-10-01

    Changes in mitochondrial and sarcoplasmic proteins using proteinomics and Western blotting in hearts from copper-deficient rats were explored in this study. Also, key enzymes that are involved in cardiac energy metabolism via glycolysis and fatty acid oxidation and related transcription factors were determined. Rats were fed one of two diets: a copper-adequate diet containing 6 mg Cu/kg diet or a diet with less than 1 mg Cu/kg diet for 5 weeks. Copper deficiency was confirmed by low liver copper levels, decreased hematocrit levels and cardiac hypertrophy. Proteinomic data revealed that of the more than 50 proteins identified from the mitochondrial fraction of heart tissue, six were significantly down-regulated and nine were up-regulated. The proteins that were decreased were beta enolase 3, carbonic anhydrase 2, aldose reductase 1, glutathione peroxidase, muscle creatine kinase and mitochondrial aconitase 2. The proteins that were up-regulated were isocitrate dehydrogenase, dihydrolipoamide dehydrogenase, transferrin, subunit d of ATP synthase, transthyretin, preproapolipoprotein A-1, GRP 75, alpha-B crystalline and heat shock protein alpha. Follow-up Western blots on rate-limiting enzymes in glycolysis (phosphofructose kinase), fatty acid oxidation (medium chain acyl dehydrogenase, peroxisome proliferator-actvator receptor-alpha or PPARalpha) and gluconeogenesis (phosphoenolpyruvate carboxykinase) did not reveal changes in metabolic enzymes. However, a significant increase in peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha protein, as well as the transcript, which increased 2.5-fold, was observed. It would appear that increased mitochondrial biogenesis known to occur in copper deficiency hearts is caused by an increased expression in the master regulator of mitochondrial biogenesis, PGC-1alpha.

  5. Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction.

    PubMed

    Puigserver, Pere; Rhee, James; Donovan, Jerry; Walkey, Christopher J; Yoon, J Cliff; Oriente, Francesco; Kitamura, Yukari; Altomonte, Jennifer; Dong, Hengjiang; Accili, Domenico; Spiegelman, Bruce M

    2003-05-29

    Hepatic gluconeogenesis is absolutely required for survival during prolonged fasting or starvation, but is inappropriately activated in diabetes mellitus. Glucocorticoids and glucagon have strong gluconeogenic actions on the liver. In contrast, insulin suppresses hepatic gluconeogenesis. Two components known to have important physiological roles in this process are the forkhead transcription factor FOXO1 (also known as FKHR) and peroxisome proliferative activated receptor-gamma co-activator 1 (PGC-1alpha; also known as PPARGC1), a transcriptional co-activator; whether and how these factors collaborate has not been clear. Using wild-type and mutant alleles of FOXO1, here we show that PGC-1alpha binds and co-activates FOXO1 in a manner inhibited by Akt-mediated phosphorylation. Furthermore, FOXO1 function is required for the robust activation of gluconeogenic gene expression in hepatic cells and in mouse liver by PGC-1alpha. Insulin suppresses gluconeogenesis stimulated by PGC-1alpha but co-expression of a mutant allele of FOXO1 insensitive to insulin completely reverses this suppression in hepatocytes or transgenic mice. We conclude that FOXO1 and PGC-1alpha interact in the execution of a programme of powerful, insulin-regulated gluconeogenesis.

  6. Structural and Biochemical Basis for the Binding Selectivity of Peroxisome Proliferator-activated Receptor [gamma] to PGC-1[alpha

    SciTech Connect

    Li, Yong; Kovach, Amanda; Suino-Powell, Kelly; Martynowski, Dariusz; Xu, H. Eric

    2008-07-23

    The functional interaction between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and its coactivator PGC-1{alpha} is crucial for the normal physiology of PPAR{gamma} and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPAR{gamma} ligand-binding domain bound to rosiglitazone and to a large PGC-1{alpha} fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1{alpha} and the PPAR{gamma} coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif is the most potent among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1{alpha} is capable of binding to PPAR{gamma}. Our studies reveal that the strong interaction of PGC-1{alpha} and PPAR{gamma} is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1{alpha} indicate that the first PGC-1{alpha} motif is necessary and sufficient for PGC-1{alpha} to coactivate PPAR{gamma} in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPAR{gamma} and PGC-1{alpha} in glucose homeostasis and adipocyte differentiation.

  7. Coordinated balancing of muscle oxidative metabolism through PGC-1{alpha} increases metabolic flexibility and preserves insulin sensitivity

    SciTech Connect

    Summermatter, Serge; Santos, Gesa

    2011-04-29

    Highlights: {yields} PGC-1{alpha} enhances muscle oxidative capacity. {yields} PGC-1{alpha} promotes concomitantly positive and negative regulators of lipid oxidation. {yields} Regulator abundance enhances metabolic flexibility and balances oxidative metabolism. {yields} Balanced oxidation prevents detrimental acylcarnitine and ROS generation. {yields} Absence of detrimental metabolites preserves insulin sensitivity -- Abstract: The peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) enhances oxidative metabolism in skeletal muscle. Excessive lipid oxidation and electron transport chain activity can, however, lead to the accumulation of harmful metabolites and impair glucose homeostasis. Here, we investigated the effect of over-expression of PGC-1{alpha} on metabolic control and generation of insulin desensitizing agents in extensor digitorum longus (EDL), a muscle that exhibits low levels of PGC-1{alpha} in the untrained state and minimally relies on oxidative metabolism. We demonstrate that PGC-1{alpha} induces a strictly balanced substrate oxidation in EDL by concomitantly promoting the transcription of activators and inhibitors of lipid oxidation. Moreover, we show that PGC-1{alpha} enhances the potential to uncouple oxidative phosphorylation. Thereby, PGC-1{alpha} boosts elevated, yet tightly regulated oxidative metabolism devoid of side products that are detrimental for glucose homeostasis. Accordingly, PI3K activity, an early phase marker for insulin resistance, is preserved in EDL muscle. Our findings suggest that PGC-1{alpha} coordinately coactivates the simultaneous transcription of gene clusters implicated in the positive and negative regulation of oxidative metabolism and thereby increases metabolic flexibility. Thus, in mice fed a normal chow diet, over-expression of PGC-1{alpha} does not alter insulin sensitivity and the metabolic adaptations elicited by PGC-1{alpha} mimic the beneficial effects of endurance training

  8. Coactivator PGC-1{alpha} regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

    SciTech Connect

    Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki; Viitala, Pirkko; Lang, Matti A.; Pelkonen, Olavi; Hakkola, Jukka

    2008-10-01

    The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1{alpha} and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1{alpha} expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1{alpha} expression vector demonstrated that PGC-1{alpha} is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4{alpha} response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1{alpha} binds, together with HNF-4{alpha}, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1{alpha} mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4{alpha}. This strongly suggests that PGC-1{alpha} is the major factor mediating the fasting response of CYP2A5.

  9. Communication between the ERRalpha homodimer interface and the PGC-1alpha binding surface via the helix 8-9 loop.

    PubMed

    Greschik, Holger; Althage, Magnus; Flaig, Ralf; Sato, Yoshiteru; Chavant, Virginie; Peluso-Iltis, Carole; Choulier, Laurence; Cronet, Philippe; Rochel, Natacha; Schüle, Roland; Strömstedt, Per-Erik; Moras, Dino

    2008-07-18

    Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-alpha (ERalpha), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-alpha (ERRalpha) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1alpha. Surprisingly, in a previous structural study, the ERalpha LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRalpha LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRalpha LBD in complex with a PGC-1alpha box3 peptide. In this structure, residues N-terminal of the PGC-1alpha LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRalpha LBD. Interaction studies using wild-type and mutant PGC-1alpha and ERRalpha showed that these contacts are functionally relevant and are required for efficient ERRalpha/PGC-1alpha interaction. Furthermore, a structure comparison between ERRalpha and ERalpha and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRalpha the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1alpha binding to ERRalpha monomers and homodimers.

  10. Molecular mechanisms underlying the development of endothermy in birds (Gallus gallus): a new role of PGC-1 alpha?

    PubMed

    Walter, Isabel; Seebacher, Frank

    2007-12-01

    In endotherms, plasticity of internal heat production in response to environmental variability is an important component of thermoregulation. During embryogenesis endotherms cannot regulate their body temperature metabolically and are therefore similar to ectotherms. The transition from ectothermy to endothermy occurs by the development of metabolic capacity during embryogenesis. Here we test the hypothesis that the development of metabolism during embryogenesis in birds is under transcriptional control and that metabolic capacity is upregulated in colder environments. The peroxisome proliferator-activated receptor-gamma (PPAR gamma) coactivator-1 alpha (PGC-1 alpha) is the major metabolic regulator in mammals. PGC-1 alpha and its target PPAR gamma were significantly elevated during development in pectoral muscle and liver of chickens (Gallus gallus) compared with adults. However, the timing of upregulation of PGC-1 alpha and PPAR gamma was not in synchrony. In cool incubation temperatures (35 degrees C) both PGC-1 alpha and PPAR gamma gene expression was increased in liver but not in skeletal muscle, compared with a 38 degrees C incubation treatment. Cytochrome c oxidase and citrate synthase enzyme activities and ATP synthase gene expression increased during embryonic development in liver and muscle, and there was a significant effect of incubation temperature on these parameters. Our findings suggest that PGC-1 alpha might be important for establishing endothermic metabolic capacity during embryogenesis in birds.

  11. The region of CQQQKPQRRP of PGC-1{alpha} interacts with the DNA-binding complex of FXR/RXR{alpha}

    SciTech Connect

    Kanaya, Eiko; Jingami, Hisato . E-mail: jingami@mfour.med.kyoto-u.ac.jp

    2006-04-14

    PGC-1{alpha} co-activates transcription by several nuclear receptors. To study the interaction among PGC-1{alpha}, RXR{alpha}/FXR, and DNA, we performed electrophoresis mobility shift assays. The RXR{alpha}/FXR proteins specifically bound to DNA containing the IR-1 sequence in the absence of ligand. When the fusion protein of GST-PGC-1{alpha} was added to the mixture of RXR{alpha}/FXR/DNA, the ligand-influenced retardation of the mobility was observed. The ligand for RXR{alpha} (9-cis-retinoic acid) was necessary for this retardation, whereas, the ligand for FXR, chenodeoxycholic acid, barely had an effect. The results obtained using truncated PGC-1{alpha} proteins suggested that two regions are necessary for PGC-1{alpha} to interact with the DNA-binding complex of RXR{alpha}/FXR. One is the region of the second leucine-rich motif, and the other is that of the amino acid sequence CQQQKPQRRP, present between the second and third leucine-rich motifs. The results obtained with the SPQSS mutation for KPQRR suggested that the basic amino acids are important for the interaction.

  12. The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

    SciTech Connect

    Xu, Wei; Guo, Ting; Zhang, Yan; Jiang, Xiaohong; Zhang, Yongxian; Zen, Ke; Yu, Bo; Zhang, Chen-Yu

    2011-05-01

    Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.

  13. Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

    SciTech Connect

    Qin, Weiping; Pan, Jiangping; Wu, Yong; Bauman, William A.; Cardozo, Christopher

    2010-12-17

    Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis, REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius

  14. Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha.

    PubMed

    Dietrich, Christoph G; Martin, Ina V; Porn, Anne C; Voigt, Sebastian; Gartung, Carsten; Trautwein, Christian; Geier, Andreas

    2007-09-01

    Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.

  15. Dissociation between PGC-1alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet.

    PubMed

    Higashida, Kazuhiko; Higuchi, Mitsuru; Terada, Shin

    2009-12-01

    It has recently been reported that a 4-wk high-fat diet gradually increases skeletal muscle peroxisome proliferator activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha) protein content, which has been suggested to regulate GLUT-4 gene transcription. However, it has not been reported that a high-fat diet enhances GLUT-4 mRNA expression and protein content in skeletal muscle, suggesting that an increase in PGC-1alpha protein content is not sufficient to induce muscle GLUT-4 biogenesis in a high-fat fed animal. Therefore, we first evaluated the relationship between PGC-1alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet for 4 wk. The PGC-1alpha protein content in rat epitrochlearis muscle significantly increased by twofold after the 4-wk high-fat diet feeding. However, the high-fat diet had no effect on GLUT-4 protein content and induced a 30% decrease in GLUT-4 mRNA expression in rat skeletal muscle (p<0.05). To clarify the mechanism by which a high-fat diet downregulates GLUT-4 mRNA expression, we next examined the effect of PPARdelta activation, which is known to occur in response to a high-fat diet, on GLUT-4 mRNA expression in L6 myotubes. Incubation with 500 nM GW501516 (PPARdelta activator) for 24 h significantly decreased GLUT-4 mRNA in L6 myotubes. Taken together, these findings suggest that a high-fat diet downregulates GLUT-4 mRNA, possibly through the activation of PPARdelta, despite an increase in PGC-1alpha protein content in rat skeletal muscle, and that a posttranscriptional regulatory mechanism maintains GLUT-4 protein content in skeletal muscle of rats fed a high-fat diet.

  16. Aqueous Extract of Paris polyphylla (AEPP) Inhibits Ovarian Cancer via Suppression of Peroxisome Proliferator-Activated Receptor-Gamma Coactivator (PGC)-1alpha.

    PubMed

    Wang, Chia-Woei; Tai, Cheng-Jeng; Choong, Chen-Yen; Lin, Yu-Chun; Lee, Bao-Hong; Shi, Yeu-Ching; Tai, Chen-Jei

    2016-06-03

    Chemotherapy, a major approach was used in carcinoma treatment, always involves the development of drug resistance as well as side-effects that affect the quality of patients' lives. An association between epithelial-mesenchymal transition (EMT) and chemotherapy resistance was established recently. We demonstrate in this paper that the aqueous extract of Paris polyphylla (AEPP)-a traditional Chinese medicine-can be used in various cancer types for suppression of carcinogenesis. We evaluated the suppressions of EMT and mitochondrial activity by AEPP treatment in a high-glucose (HG) induced-human ovarian carcinoma cell line (OVCAR-3 cells). The mitochondrial morphology was investigated using MitoTracker Deep Red FM staining. Our results indicated that AEPP reduced the viability of OVCAR-3 cells considerably through induction of apoptosis. However, this inhibitory potential of AEPP was attenuated by HG induction in OVCAR-3 cells. The levels of estrogen-related receptor (ERR)-alpha activator and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha were elevated by HG induction, but were suppressed by AEPP treatment. Down-regulations of cell survival and EMT were oberved in OVCAR-3 cells through suppression of PGC-1alpha by AEPP treatment. These results were confirmed through PGC-1alpha knockdown and overexpression in OVCAR-3 cells. Thus, AEPP can be beneficial for treating ovarian cancer and has potential for development of an integrative cancer therapy against ovarian cancer proliferation, metastasis, and migration.

  17. Possible roles of myostatin and PGC-1alpha in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: expression of myostatin and PGC-1alpha in chicks exposed to cold.

    PubMed

    Ijiri, Daichi; Kanai, Yukio; Hirabayashi, Miho

    2009-07-01

    This study examined the hypothesis that myostatin and PGC-1alpha are involved in the increase in skeletal muscle mass and transformation of fiber type in cold-exposed chicks. One-week-old chicks were exposed to acute (24h) or long-term (8d) cold at 4 degrees C or kept warm at 30 degrees C. Acute cold exposure induced a significant increase in the skeletal muscle weight and the ratio of slow- to fast-fiber specific troponin I expression (sTnI/fTnI), accompanied by a significant decrease in lactate dehydrogenase activity. Expression of myostatin mRNA in the muscle was significantly lower in cold-exposed chicks than in the controls, whereas PGC-1alpha mRNA expression was significantly enhanced. These changes in the gene expression rapidly returned to the levels of the control chicks after the end of cold exposure, whereas the changes in fiber type and enzymatic activity were not resumed within 24h after removal of cold exposure. On the other hand, long-term exposure to cold resulted in a remarkable increase in skeletal muscle weight, accompanied by a significant increase in the ratio of sTnI/fTnI and the enzymatic activities of cytochrome oxidase and lactate dehydrogenase. However, the expression level of myostatin mRNA in cold-exposed chicks was not different from that in their age-matched control chicks and that of PGC-1alpha mRNA was significantly lower than in the controls. These results indicate that myostatin and PGC-1alpha expression in the skeletal muscle rapidly change in response to acute cold, suggesting the possibility that these two genes could be involved in the increase in muscle mass and transformation of fiber type, respectively, at the initial stage of adaptation in cold-exposed chicks.

  18. Multiple Binding Modes between HNF4[alpha] and the LXXLL Motifs of PGC-1[alpha] Lead to Full Activation

    SciTech Connect

    Rha, Geun Bae; Wu, Guangteng; Shoelson, Steven E.; Chi, Young-In

    2010-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4{alpha} interacts, peroxisome proliferation-activated receptor {gamma} (PPAR{gamma}) coactivator 1{alpha} (PGC-1{alpha}) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1{alpha} recruitment, we have determined the crystal structure of HNF4{alpha} in complex with a fragment of PGC-1{alpha} containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4{alpha} toward the LXXLL motifs of PGC-1{alpha} could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.

  19. Rb and p107 regulate preadipocyte differentiation into white versus brown fat through repression of PGC-1alpha.

    PubMed

    Scimè, Anthony; Grenier, Guillaume; Huh, Michael S; Gillespie, Mark A; Bevilacqua, Lisa; Harper, Mary-Ellen; Rudnicki, Michael A

    2005-11-01

    The Rb family, Rb, p107, and p130, play important roles in cell cycle control and cellular differentiation, and Rb has been suggested to regulate adipocyte differentiation. We report here that mice lacking p107 displayed a uniform replacement of white adipose tissue (WAT) with brown adipose tissue (BAT). Mutant WAT depots contained mutilocular adipocytes that expressed elevated levels of PGC-1alpha and UCP-1 typical of BAT. WAT from p107-/- mice contained markedly elevated numbers of adipogenic precursors that displayed downregulated expression of pRb. Consistent with the hypothesis that pRb is required for adult adipocyte differentiation, Cre-mediated deletion of Rb in adult primary preadipocytes blocked their differentiation into white adipocytes. Importantly, pRb was observed to bind the PGC-1alpha promoter and repress transcription. Therefore, p107 and pRb regulate PGC-1alpha expression to control the switch between white and brown adipocyte differentiation from a common pool of presumptive adult progenitors in fat tissue.

  20. PGC-1alpha Down-Regulation Affects the Antioxidant Response in Friedreich's Ataxia

    PubMed Central

    Marmolino, Daniele; Manto, Mario; Acquaviva, Fabio; Vergara, Paola; Ravella, Ajay; Monticelli, Antonella; Pandolfo, Massimo

    2010-01-01

    Background Cells from individuals with Friedreich's ataxia (FRDA) show reduced activities of antioxidant enzymes and cannot up-regulate their expression when exposed to oxidative stress. This blunted antioxidant response may play a central role in the pathogenesis. We previously reported that Peroxisome Proliferator Activated Receptor Gamma (PPARγ) Coactivator 1-alpha (PGC-1α), a transcriptional master regulator of mitochondrial biogenesis and antioxidant responses, is down-regulated in most cell types from FRDA patients and animal models. Methodology/Principal Findings We used primary fibroblasts from FRDA patients and the knock in-knock out animal model for the disease (KIKO mouse) to determine basal superoxide dismutase 2 (SOD2) levels and the response to oxidative stress induced by the addition of hydrogen peroxide. We measured the same parameters after pharmacological stimulation of PGC-1α. Compared to control cells, PGC-1α and SOD2 levels were decreased in FRDA cells and did not change after addition of hydrogen peroxide. PGC-1α direct silencing with siRNA in control fibroblasts led to a similar loss of SOD2 response to oxidative stress as observed in FRDA fibroblasts. PGC-1α activation with the PPARγ agonist (Pioglitazone) or with a cAMP-dependent protein kinase (AMPK) agonist (AICAR) restored normal SOD2 induction. Treatment of the KIKO mice with Pioglitazone significantly up-regulates SOD2 in cerebellum and spinal cord. Conclusions/Significance PGC-1α down-regulation is likely to contribute to the blunted antioxidant response observed in cells from FRDA patients. This response can be restored by AMPK and PPARγ agonists, suggesting a potential therapeutic approach for FRDA. PMID:20383327

  1. Analysis of PGC-1{alpha} variants Gly482Ser and Thr612Met concerning their PPAR{gamma}2-coactivation function

    SciTech Connect

    Nitz, Inke . E-mail: initz@molnut.uni-kiel.de; Ewert, Agnes; Klapper, Maja; Doering, Frank

    2007-02-09

    Peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) is a cofactor involved in adaptive thermogenesis, fatty acid oxidation, and gluconeogenesis. Dysfunctions of this protein are likely to contribute to the development of obesity and the metabolic syndrome. This is in part but not definitely confirmed by results of population studies. The aim of this study was to investigate if common genetic variants rs8192678 (Gly482Ser) and rs3736265 (Thr612Met) in the PGC-1{alpha} gene lead to a functional consequence in cofactor activity using peroxisome proliferator-activated receptor-{gamma} 2 (PPAR{gamma}2) as interacting transcription factor. Reporter gene assays in HepG2 cells with wildtype and mutant proteins of both PGC1{alpha} and PPAR{gamma}2 (Pro12Ala, rs1801282) using the acyl-CoA-binding protein (ACBP) promoter showed no difference in coactivator activity. This is First study implicating that the Gly482Ser and Thr612Met polymorphisms in PGC-1{alpha} and Pro12Ala polymorphism in PPAR{gamma}2 do not affect the functional integrity of these proteins.

  2. Association of PGC-1alpha polymorphisms with age of onset and risk of Parkinson's disease.

    PubMed

    Clark, Joanne; Reddy, Sonika; Zheng, Kangni; Betensky, Rebecca A; Simon, David K

    2011-05-19

    Peroxisome proliferator-activated receptor-γ co-activator (PGC)-1α is a transcriptional co-activator of antioxidant genes and a master regulator of mitochondrial biogenesis. Parkinson's disease (PD) is associated with oxidative stress and mitochondrial dysfunction and recent work suggests a role for PGC-1α. We hypothesized that the rs8192678 PGC-1α single nucleotide polymorphism (SNP) may influence risk or age of onset of PD. The A10398G mitochondrial SNP has been inversely associated with risk of PD in some studies. In the current study we analyzed whether rs8192678 or other PGC-1α SNPs affect PD risk or age of onset, singularly or in association with the A10398G SNP. Genomic DNA samples from 378 PD patients and 173 age-matched controls were analyzed by multiplexed probe sequencing, followed by statistical analyses of the association of each SNP, alone or in combination, with risk or age of onset of PD. Adjustments were made for age of onset being less than the age of sampling, and for the observed dependence between these two ages. The PD samples were obtained as two separate cohorts, therefore statistical methods accounted for different sampling methods between the two cohorts, and data were analyzed using Cox regression adjusted for sampling in the risk set definition and in the model. The rs8192678 PGC-1α SNP was not associated with the risk of PD. However, an association of the PGC-1α rs8192678 GG variant with longevity was seen in control subjects (p=0.019). Exploratory studies indicated that the CC variant of rs6821591 was associated with risk of early onset PD (p=0.029), with PD age of onset (p=0.047), and with longevity (p=0.022). The rs2970848 GG allele was associated with risk of late onset PD (p=0.027). These data reveal possible associations of the PGC-1α SNPs rs6821591 and rs2970848 with risk or age of onset of PD, and of the PGC-1α rs8192678 GG and the rs6821591 CC variants with longevity. If replicated in other datasets, these findings may

  3. The microRNA miR-696 regulates PGC-1{alpha} in mouse skeletal muscle in response to physical activity.

    PubMed

    Aoi, Wataru; Naito, Yuji; Mizushima, Katsura; Takanami, Yoshikazu; Kawai, Yukari; Ichikawa, Hiroshi; Yoshikawa, Toshikazu

    2010-04-01

    MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional gene regulation that have been shown to be involved in growth, development, function, and stress responses of various organs. The purpose of this study was to identify the miRNA response to physical activity, which was related to functions such as nutrient metabolism, although the miRNAs involved are currently unknown. C57BL/6 mice were divided into exercise and control groups. The exercise group performed running exercise, with a gradual increase of the load over 4 wk. On the other hand, to examine the effect of muscle inactivity, the unilateral hindlimbs of other mice were fixed in a cast for 5 days. Microarray analysis for miRNA in gastrocnemius revealed that miR-696 was markedly affected by both exercise and immobilization, showing opposite responses to these two interventions. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which was increased by exercise and decreased by immobilization in the protein level, was predicted as a target regulated by miR-696. In cultured myocytes, intracellular miR-696 variation led to negative regulation of PGC-1alpha protein along with the expression of mRNAs for downstream genes. In addition, we found decreases in the biogenesis of mitochondria and fatty acid oxidation in miR-696-overexpressing myocytes compared with normal control myocytes. These observations demonstrate that miR-696 is a physical activity-dependent miRNA involved in the translational regulation of PGC-1alpha and skeletal muscle metabolism in mice.

  4. Peroxisome proliferator-activated receptor gamma coactivator-1alpha enhances antiproliferative activity of 5'-deoxy-5-fluorouridine in cancer cells through induction of uridine phosphorylase.

    PubMed

    Kong, Xingxing; Fan, Heng; Liu, Xiaojun; Wang, Rui; Liang, Jichao; Gupta, Nishith; Chen, Yong; Fang, Fude; Chang, Yongsheng

    2009-10-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is capable of coactivating several nuclear receptors and transcription factors that participate in the regulation of multiple metabolic processes, including gluconeogenesis, mitochondrial biogenesis, and adaptive thermogenesis. Uridine phosphorylase (UPase) catalyzes the reversible conversion of uridine into uracil and contributes to the antineoplastic activity of 5'-deoxy-5-fluorouridine (5'-DFUR) and homeostasis of uridine levels in plasma and tissues. This study demonstrates uridine phosphorylase as a novel target gene of PGC-1alpha, which induces the transcription and enzymatic activity of UPase in various cancer cells and thus augments their susceptibility to 5'-DFUR. PGC-1alpha-induced activation of UPase expression occurs at its transcription level that is mediated by an estrogen-related receptor (ERR) binding site (-1078 to -1070 base pairs) mapped in the promoter region of UPase gene. Our mutational studies using luciferase reporter construct together with electrophoretic mobility shift assays confirm the binding of ERR to PGC-1alpha-responsive element. Moreover, the inhibition of PGC-1alpha/ERRalpha-dependent signaling by 3-[4-(2,4-bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT790) compromises the ability of PGC-1alpha to induce the transcript of UPase, indicating PGC-1alpha-dependent and ERRalpha-mediated up-regulation of UPase. Finally, the overexpression of PGC-1alpha sensitizes breast and colon cancer cells to growth inhibition by 5'-DFUR presumably by inducing apoptosis in tumor cells and XCT790 can inhibit the process. Taken together, our results corroborate the regulatory function of PGC-1alpha in uridine homeostasis and imply its links with the energy metabolism. The mechanistic elucidation of this association between both cellular pathways should advance the clinical use of 5-fluorouracil

  5. Statins enhance peroxisome proliferator-activated receptor gamma coactivator-1alpha activity to regulate energy metabolism.

    PubMed

    Wang, Wenxian; Wong, Chi-Wai

    2010-03-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) serves as an inducible coactivator for a number of transcription factors to control energy metabolism. Insulin signaling through Akt kinase has been demonstrated to phosphorylate PGC-1alpha at serine 571 and downregulate its activity in the liver. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that reduce cholesterol synthesis in the liver. In this study, we found that statins reduced the active form of Akt and enhanced PGC-1alpha activity. Specifically, statins failed to activate an S571A mutant of PGC-1alpha. The activation of PGC-1alpha by statins selectively enhanced the expression of energy metabolizing enzymes and regulators including peroxisome proliferator-activated receptor alpha, acyl-CoA oxidase, carnitine palmitoyl transferase-1A, and pyruvate dehydrogenase kinase 4. Importantly, a constitutively active form of Akt partially reduced the statin-enhanced gene expression. Our study thus provides a plausible mechanistic explanation for the hypolipidemic effect of statin through elevating the rate of beta-oxidation and mitochondrial Kreb's cycle capacity to enhance fatty acid utilization while reducing the rate of glycolysis.

  6. Relationship between Sirt1 expression and mitochondrial proteins during conditions of chronic muscle use and disuse.

    PubMed

    Chabi, Beatrice; Adhihetty, Peter J; O'Leary, Michael F N; Menzies, Keir J; Hood, David A

    2009-12-01

    Sirt1 is a NAD(+)-dependent histone deacetylase that interacts with the regulatory protein of mitochondrial biogenesis PGC-1alpha and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins, and PGC-1alpha existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days, 10 Hz; 3 h/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1alpha levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70-90% in soleus and plantaris muscles of running animals, whereas Sirt1 activity remained unchanged. In chronically stimulated muscle, cytochrome c was increased by 30% compared with nonstimulated muscle, whereas Sirt1 activity was increased modestly by 20-25%. In contrast, in denervated muscle, these markers of mitochondrial content were decreased by 30-50% compared with the control muscle, whereas Sirt1 activity was increased by 75-80%. Our data suggest that Sirt1 and PGC-1alpha expression are independently regulated and that, although Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse.

  7. Maternal low protein diets decrease skeletal muscle growth, PGC-1alpha mRNA expression and mitochondrial oxidative respiration and increase obesity and insulin resistance in obesity prone Sprague-Dawley rats

    USDA-ARS?s Scientific Manuscript database

    Malnutrition during the fetal growth period followed by postnatal catch-up growth results in obesity and the development of type 2 diabetes (T2D). To determine whether a prenatal low protein diet followed by postnatal high fat diet increases propensity for obesity and T2D in offspring, obese-prone f...

  8. Estrogen-related receptor {alpha} is essential for the expression of antioxidant protection genes and mitochondrial function

    SciTech Connect

    Rangwala, Shamina M. . E-mail: shamina.rangwala@novartis.com; Li, Xiaoyan; Lindsley, Loren; Wang, Xiaomei; Shaughnessy, Stacey; Daniels, Thomas G.; Szustakowski, Joseph; Nirmala, N.R.; Wu, Zhidan; Stevenson, Susan C.

    2007-05-25

    Estrogen-related receptor {alpha} (ERR{alpha}) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERR{alpha} null mice. Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) stimulated mitochondrial gene expression program in control cells, but not in the ERR{alpha} null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1{alpha} levels was dependent on ERR{alpha}. Furthermore, we found that the PGC-1{alpha}-mediated induction of estrogen-related receptor {gamma} and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERR{alpha}. Basal levels of NRF-2 were decreased in the absence of ERR{alpha}. The absence of ERR{alpha} resulted in a decrease in citrate synthase enzyme activity in response to PGC-1{alpha} overexpression. Our results indicate an essential role for ERR{alpha} as a key regulator of oxidative metabolism.

  9. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha activation of CYP7A1 during food restriction and diabetes is still inhibited by small heterodimer partner.

    PubMed

    Shin, Dong-Ju; Osborne, Timothy F

    2008-05-30

    Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic pathway of hepatic bile acid biosynthesis from cholesterol. During fasting and in type I diabetes, elevated levels of peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) induce expression of the Cyp7A1 gene and overexpression of PGC-1alpha in hepatoma cells stimulates bile acid synthesis. Using Ad-PGC-1alpha-RNA interference to induce acute disruption of PGC-1alpha in mice, here we show that PGC-1alpha is necessary for fasting-mediated induction of CYP7A1. Co-immunoprecipitation and promoter activation studies reveal that the induction of CYP7A1 is mediated by direct interaction between PGC-1alpha and the AF2 domain of liver receptor homolog-1 (LRH-1). In contrast, the very similar PGC-1beta could not substitute for PGC-1alpha. We also show that transactivation of PGC-1alpha and LRH-1 is repressed by the small heterodimer partner (SHP). Treatment of mice with GW4064, a synthetic agonist for farnesoid X receptor, induced SHP expression and decreased both the recruitment of PGC-1alpha to the Cyp7A1 promoter and the fasting-induced expression of CYP7A1 mRNA. These data suggest that PGC-1alpha is an important co-activator for LRH-1 and that SHP targets the interaction between LRH-1 and PGC-1alpha to inhibit CYP7A1 expression. Overall, these studies provide further evidence for the important role of PGC-1alpha in bile acid homeostasis and suggest that pharmacological targeting of farnesoid X receptor in vivo can be used to reverse the increase in CYP7A1 associated with adverse metabolic conditions.

  10. Maintenance of Glucose Homeostasis Through Acetylation of the Metabolic Transcriptional Coactivator PGC1-alpha

    DTIC Science & Technology

    2011-02-01

    acetylation. HEK293 cells were transfected with the indicated plasmids and treated with 10 mM nicotinamide for 16 hr. PGC-1a acet- ylation levels were...40 mM sodium bu- tyrate, 20 mM nicotinamide , 4 mM DTT, and 1 mM PMSF) with 1 nmol [14C]- acetyl-CoA (55 mCi/mmol). After incubation at 30ºC for 10...Krebs, H.A. (1967). The redox state of free nicotinamide -adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103, 514–527

  11. Maintenance of Glucose Homeostasis through Acetylation of the Metabolic Transcriptional Coactivator PGC-1alpha

    DTIC Science & Technology

    2008-02-01

    GCN5 in cells using adenoviruses that encode both proteins. 12 hours before harvesting, cells were treated with nicotinamide (a SIRT1 deacetylase...Hepatic SIRT1 . Proc. Natl. Acad. Sci. USA. 2007 104(31) 12861-12866. C. Lerin, T.J. Kelly, W. Haas, S.P. Gygi, and P. Puigserver. GCN5-Mediated...Puigserver. Metabolic Adaptations Through PGC-1α and SIRT1 Pathways. FEBS Letters. 582: 46-53. 2008 - University of Utah. Department of

  12. Maintenance of Glucose Homeostasis through Acetylation of the Metabolic Transcriptional Coactivator PGC1-alpha

    DTIC Science & Technology

    2009-02-01

    highlight that PGC-1α chemical acetylation is directly controlled by two enzymes: GCN5 and SIRT1 ; this strengths the possibility to use small...acetylated through GCN5 acetyltransferase activity, however under low nutrient conditions Sirt1 deacetylase will keep PGC-1α de-acetylated in an active form...acetylated by GCN5, we decided to use R13 because it did not respond to low glucose levels or Sirt1 activators. We think that the additional acetylation

  13. Dual modulation of both lipid oxidation and synthesis by peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta in cultured myotubes.

    PubMed

    Espinoza, Daniel O; Boros, Laszlo G; Crunkhorn, Sarah; Gami, Hiral; Patti, Mary-Elizabeth

    2010-04-01

    The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family is a key regulator of mitochondrial function, and reduced mRNA expression may contribute to muscle lipid accumulation in obesity and type 2 diabetes. To characterize the effects of PGC-1 on lipid metabolism, we overexpressed PGC-1alpha and PGC-1beta in C2C12 myotubes using adenoviral vectors. Both PGC-1alpha and -1beta increased palmitate oxidation [31% (P<0.01) and 26% (P<0.05), respectively] despite reductions in cellular uptake [by 6% (P<0.05) and 21% (P<0.001)]. Moreover, PGC-1alpha and -1beta increased mRNA expression of genes regulating both lipid oxidation (e.g., CPT1b and ACADL/M) and synthesis (FAS, CS, ACC1/2, and DGAT1). To determine the net effect, we assessed lipid composition in PGC-1-expressing cells. Total lipid content decreased by 42% in palmitate-loaded serum-starved cells overexpressing PGC-1alpha (P<0.05). In contrast, in serum-replete cells, total lipid content was not significantly altered, but fatty acids C14:0, C16:0, C18:0, and C18:1 were increased 2- to 4-fold for PGC-1alpha/beta (P<0.05). Stable isotope-based dynamic metabolic profiling in serum-replete cells labeled with (13)C substrates revealed both increased de novo fatty acid synthesis from glucose and increased fatty acid synthesis by chain elongation with either PGC-1alpha or -1beta expression. These results indicate that PGC-1 can promote both lipid oxidation and synthesis, with net balance determined by the nutrient/hormonal environment.-Espinoza, D. O., Boros, L. G., Crunkhorn, S., Gami, H., Patti, M.-E. Dual Modulation of both lipid oxidation and synthesis by peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta in cultured myotubes.

  14. Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle.

    PubMed

    Garnier, Anne; Fortin, Dominique; Zoll, Joffrey; N'Guessan, Benoit; Mettauer, Bertrand; Lampert, Eliane; Veksler, Vladimir; Ventura-Clapier, Renée

    2005-01-01

    We examined the transcriptional signaling cascade involved in the changes of mitochondrial biogenesis and mitochondrial function of skeletal muscle and of the exercise capacity of humans in response to long-term physical activity and chronic heart failure (CHF). Biopsy samples of vastus lateralis muscle were obtained from 18 healthy subjects with different fitness levels (assessed by maximal oxygen uptake, VO2 peak). We compared 9 sedentary subjects with 10 CHF patients undergoing transplantation. Muscle oxidative capacity was measured in permeabilized fibers (Vmax). Transcript levels of target genes were quantified by RT-PCR. In healthy subjects, VO2 peak was linearly related to Vmax (P<0.01) and to the gene expression of mitochondrial proteins and of the coactivator PGC-1alpha and its downstream transcription factors. A coordinate increase in PGC-1alpha and mRNA levels of proteins involved in degradation, fusion, and fission of mitochondria was observed associated with calcineurin activation. Despite decreased VO2 peak, in CHF patients skeletal muscles showed preserved Vmax in accordance with preserved markers and transcription factors of mitochondrial biogenesis and dynamics, with no calcineurin activation. The results provide strong support for a central role for PGC-1alpha and calcineurin activation in mitochondrial biogenesis in healthy and diseased human skeletal muscles.

  15. Myogenic basic helix-loop-helix proteins regulate the expression of peroxisomal proliferator activated receptor-gamma coactivator-1alpha.

    PubMed

    Chang, Ju Hui; Lin, Kwang Huei; Shih, Chung Hsuan; Chang, Yu Jung; Chi, Hsiang Chung; Chen, Shen Liang

    2006-06-01

    Peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of the PGC-1alpha gene in either a cell or an animal has been shown to promote fast to slow fiber-type switch. The expression of PGC-1alpha in muscle is regulated by myocyte enhancer factor 2 and Forkhead in rhabdomyosarcoma, two transcription factors implicated in terminal muscle differentiation. In this study we found that PGC-1alpha expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus-mediated MyoD overexpression in C3H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1alpha expression in vivo. Our transient transfection results also show that myogenic basic helix-loop-helix (bHLH) proteins, especially MyoD, can activate PGC-1alpha expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1alpha promoter were localized to a short region (-49 to approximately +2) adjacent to the transcription start site, which contains two putative E boxes. Mutation of either site significantly reduced MyoD-mediated transactivation in the cells, suggesting that both sites are required for myogenic bHLH protein-mediated activation. However, only one site, the E2 box, was directly bound by glutathione-S-transferase-MyoD protein in EMSAs. Our results indicate that myogenic bHLH proteins not only are involved in lineage determination and terminal differentiation, but also are directly implicated in activation of the key fiber-type and metabolic switch gene, PGC-1alpha.

  16. p21{sup WAF1/CIP1} deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

    SciTech Connect

    Kim, Ae Jeong; Jee, Hye Jin; Song, Naree; Kim, Minjee; Jeong, Seon-Young; Yun, Jeanho

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer p21{sup -/-} HCT116 cells exhibited an increase in mitochondrial mass. Black-Right-Pointing-Pointer The expression levels of PGC-1{alpha} and AMPK were upregulated in p21{sup -/-} HCT116 cells. Black-Right-Pointing-Pointer The proliferation of p21{sup -/-} HCT116 cells in galactose medium was significantly impaired. Black-Right-Pointing-Pointer p21 may play a role in maintaining proper mitochondrial mass and respiratory function. -- Abstract: p21{sup WAF1/CIP1} is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21{sup -/-} HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53{sup -/-} cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1{alpha} and TFAM and AMPK activity were also elevated in p21{sup -/-} cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1{alpha} axis. However, the increase in mitochondrial biogenesis in p21{sup -/-} cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21{sup -/-} cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

  17. Mitochondrial biogenesis and turnover.

    PubMed

    Diaz, Francisca; Moraes, Carlos T

    2008-07-01

    Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. The mechanisms associated with these events have been intensively studied in the last 20 years and our understanding of their details is much improved. Mitochondrial biogenesis requires the participation of calcium signaling that activates a series of calcium-dependent protein kinases that in turn activate transcription factors and coactivators such as PGC-1alpha that regulates the expression of genes coding for mitochondrial components. In addition, mitochondrial biogenesis involves the balance of mitochondrial fission-fusion. Mitochondrial malfunction or defects in any of the many pathways involved in mitochondrial biogenesis can lead to degenerative diseases and possibly play an important part in aging.

  18. Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

    SciTech Connect

    Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa; Babu, Satish; Pal, Amit; Khare, Drirh; Godbole, Madan M.

    2010-07-02

    Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1{alpha}, NRF-1{alpha} and Tfam. Also, we for the first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.

  19. Rice bran extract improves mitochondrial dysfunction in brains of aged NMRI mice.

    PubMed

    Hagl, Stephanie; Berressem, Dirk; Grewal, Rehka; Sus, Nadine; Frank, Jan; Eckert, Gunter P

    2016-01-01

    Aging represents a major risk factor for neurodegenerative diseases such as Alzheimer's disease. Mitochondria are significantly involved in both the aging process and neurodegeneration. One strategy to protect the brain and to prevent neurodegeneration is a healthy lifestyle including a diet rich in antioxidants and polyphenols. Rice bran extract (RBE) contains various antioxidants including natural vitamin E forms (tocopherols and tocotrienols) and gamma-oryzanol. In this work, we examined the effects of a stabilized RBE on mitochondrial function in 18-month-old Naval Medical Research Institute mice (340 mg/kg body weight/day), which received the extract for 3 weeks via oral gavage. Mitochondrial parameters were measured using high-resolution respirometry (Oroboros Oxygraph-2k), Western blot analysis, and photometric methods in dissociated brain cells, isolated mitochondria, and brain homogenate. Vitamin E concentrations in blood plasma and brain tissue were measured using HPLC with fluorescence detection. Aging leads to decreased mitochondrial function (decreased mitochondrial respiration and ATP production) and decreased protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1alpha). RBE administration increased alpha-tocopherol concentrations in the brain and compensated for age-related mitochondrial dysfunction by increasing mitochondrial respiration, membrane potential, PGC1alpha protein expression, and citrate synthase activity. Furthermore, resistance of brain cells to sodium nitroprusside-induced nitrosative stress was improved. According to these results, RBE is a promising candidate nutraceutical for the prevention of age-related neurodegenerative diseases.

  20. A high-fat diet coordinately downregulates genes required for mitochondrial oxidative phosphorylation in skeletal muscle.

    PubMed

    Sparks, Lauren M; Xie, Hui; Koza, Robert A; Mynatt, Randall; Hulver, Matthew W; Bray, George A; Smith, Steven R

    2005-07-01

    Obesity and type 2 diabetes have been associated with a high-fat diet (HFD) and reduced mitochondrial mass and function. We hypothesized a HFD may affect expression of genes involved in mitochondrial function and biogenesis. To test this hypothesis, we fed 10 insulin-sensitive males an isoenergetic HFD for 3 days with muscle biopsies before and after intervention. Oligonucleotide microarray analysis revealed 297 genes were differentially regulated by the HFD (Bonferonni adjusted P < 0.001). Six genes involved in oxidative phosphorylation (OXPHOS) decreased. Four were members of mitochondrial complex I: NDUFB3, NDUFB5, NDUFS1, and NDUFV1; one was SDHB in complex II and a mitochondrial carrier protein SLC25A12. Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1) alpha and PGC1beta mRNA were decreased by -20%, P < 0.01, and -25%, P < 0.01, respectively. In a separate experiment, we fed C57Bl/6J mice a HFD for 3 weeks and found that the same OXPHOS and PGC1 mRNAs were downregulated by approximately 90%, cytochrome C and PGC1alpha protein by approximately 40%. Combined, these results suggest a mechanism whereby HFD downregulates genes necessary for OXPHOS and mitochondrial biogenesis. These changes mimic those observed in diabetes and insulin resistance and, if sustained, may result in mitochondrial dysfunction in the prediabetic/insulin-resistant state.

  1. Alcohol alters hepatic FoxO1, p53, and mitochondrial SIRT5 deacetylation function

    SciTech Connect

    Lieber, Charles S. Leo, Maria Anna; Wang, Xiaolei; DeCarli, Leonore M.

    2008-08-22

    Chronic alcohol consumption affects the gene expression of a NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-{gamma} coactivator1{alpha} (PGC-1{alpha}). Our aim was to verify that it also alters the forkhead (FoxO1) and p53 transcription factor proteins, critical in the hepatic response to oxidative stress and regulated by SIRT1 through its deacetylating capacity. Accordingly, rats were pair-fed the Lieber-DeCarli alcohol-containing liquid diets for 28 days. Alcohol increased hepatic mRNA expression of FoxO1 (p = 0.003) and p53 (p = 0.001) while corresponding protein levels remained unchanged. However phospho-FoxO1 and phospho-Akt (protein kinase) were both decreased by alcohol consumption (p = 0.04 and p = 0.02, respectively) while hepatic p53 was found hyperacetylated (p = 0.017). Furthermore, mitochondrial SIRT5 was reduced (p = 0.0025), and PGC-1{alpha} hyperacetylated (p = 0.027), establishing their role in protein modification. Thus, alcohol consumption disrupts nuclear-mitochondrial interactions by post-translation protein modifications, which contribute to alteration of mitochondrial biogenesis through the newly discovered reduction of SIRT5.

  2. A practical model of low-volume high-intensity interval training induces mitochondrial biogenesis in human skeletal muscle: potential mechanisms.

    PubMed

    Little, Jonathan P; Safdar, Adeel; Wilkin, Geoffrey P; Tarnopolsky, Mark A; Gibala, Martin J

    2010-03-15

    High-intensity interval training (HIT) induces skeletal muscle metabolic and performance adaptations that resemble traditional endurance training despite a low total exercise volume. Most HIT studies have employed 'all out', variable-load exercise interventions (e.g. repeated Wingate tests) that may not be safe, practical and/or well tolerated by certain individuals. Our purpose was to determine the performance, metabolic and molecular adaptations to a more practical model of low-volume HIT. Seven men (21 + or - 0.4 years, V(O2peak) = 46 + or - 2 ml kg(-1) min(-1)) performed six training sessions over 2 weeks. Each session consisted of 8-12 x 60 s intervals at approximately 100% of peak power output elicited during a ramp V(O2) peak test (355 + or - 10 W) separated by 75 s of recovery. Training increased exercise capacity, as assessed by significant improvements on both 50 kJ and 750 kJ cycling time trials (P < 0.05 for both). Skeletal muscle (vastus lateralis) biopsy samples obtained before and after training revealed increased maximal activity of citrate synthase (CS) and cytochrome c oxidase (COX) as well as total protein content of CS, COX subunits II and IV, and the mitochondrial transcription factor A (Tfam) (P < 0.05 for all). Nuclear abundance of peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) was approximately 25% higher after training (P < 0.05), but total PGC-1alpha protein content remained unchanged. Total SIRT1 content, a proposed activator of PGC-1alpha and mitochondrial biogenesis, was increased by approximately 56% following training (P < 0.05). Training also increased resting muscle glycogen and total GLUT4 protein content (both P < 0.05). This study demonstrates that a practical model of low volume HIT is a potent stimulus for increasing skeletal muscle mitochondrial capacity and improving exercise performance. The results also suggest that increases in SIRT1, nuclear PGC-1alpha, and Tfam may be involved in

  3. Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.

    PubMed

    Yui, Rika; Iketani, Satoru; Mikami, Tetsuo; Kubo, Tomohiko

    2003-04-01

    We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.

  4. Effect of thyroid hormone on mitochondrial properties and oxidative stress in cells from patients with mtDNA defects.

    PubMed

    Menzies, Keir J; Robinson, Brian H; Hood, David A

    2009-02-01

    Mitochondrial (mt)DNA mutations contribute to various disease states characterized by low ATP production. In contrast, thyroid hormone [3,3',5-triiodothyronine (T(3))] induces mitochondrial biogenesis and enhances ATP generation within cells. To evaluate the role of T(3)-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leigh's syndrome and one with hypertrophic cardiomyopathy. Compared with control cells, patient fibroblasts displayed similar levels of mitochondrial mass, peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), mitochondrial transcription factor A (Tfam), and uncoupling protein 2 (UCP2) protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold elevation in mitochondrial membrane potential, and 30% less complex V activity compared with control cells. Patient cells also displayed 20-25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with control cells. After T(3) treatment of patient cells, ROS production was decreased by 40%, cytoplasmic Ca2+ was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1alpha, Tfam, or UCP2 in either T(3)-treated patient or control cells. However, T(3) restored the mitochondrial membrane potential, complex V activity, and levels of MnSOD to normal values in patient cells and elevated MnSOD levels by 21% in control cells. These results suggest that T(3) acts to reduce cellular oxidative stress, which may help attenuate ROS-mediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects.

  5. Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance.

    PubMed

    Davis, J Mark; Murphy, E Angela; Carmichael, Martin D; Davis, Ben

    2009-04-01

    Quercetin is one of a broad group of natural polyphenolic flavonoid substances that are being investigated for their widespread health benefits. These benefits have generally been ascribed to its combination of antioxidant and anti-inflammatory activity, but recent in vitro evidence suggests that improved mitochondrial biogenesis could play an important role. In addition, the in vivo effects of quercetin on mitochondrial biogenesis exercise tolerance are unknown. We examined the effects of 7 days of quercetin feedings in mice on markers of mitochondrial biogenesis in skeletal muscle and brain, and on endurance exercise tolerance. Mice were randomly assigned to one of the following three treatment groups: placebo, 12.5 mg/kg quercetin, or 25 mg/kg quercetin. Following 7 days of treatment, mice were killed, and soleus muscle and brain were analyzed for mRNA expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha) and sirtuin 1 (SIRT1), and mitochondrial DNA (mtDNA) and cytochrome c. Additional mice underwent a treadmill performance run to fatigue or were placed in voluntary activity wheel cages, and their voluntary activity (distance, time, and peak speed) was recorded. Quercetin increased mRNA expression of PGC-1alpha and SIRT1 (P < 0.05), mtDNA (P < 0.05) and cytochrome c concentration (P < 0.05). These changes in markers of mitochondrial biogenesis were associated with an increase in both maximal endurance capacity (P < 0.05) and voluntary wheel-running activity (P < 0.05). These benefits of querectin on fitness without exercise training may have important implications for enhancement of athletic and military performance and may also extend to prevention and/or treatment of chronic diseases.

  6. Successive bouts of cycling stimulates genes associated with mitochondrial biogenesis.

    PubMed

    Dumke, Charles L; Mark Davis, J; Angela Murphy, E; Nieman, David C; Carmichael, Martin D; Quindry, John C; Travis Triplett, N; Utter, Alan C; Gross Gowin, Sarah J; Henson, Dru A; McAnulty, Steven R; McAnulty, Lisa S

    2009-11-01

    Exercise increases mRNA for genes involved in mitochondrial biogenesis and oxidative enzyme capacity. However, little is known about how these genes respond to consecutive bouts of prolonged exercise. We examined the effects of 3 h of intensive cycling performed on three consecutive days on the mRNA associated with mitochondrial biogenesis in trained human subjects. Forty trained cyclists were tested for VO(2max) (54.7 +/- 1.1 ml kg(-1) min(-1)). The subjects cycled at 57% watts(max) for 3 h using their own bicycles on CompuTrainer Pro Model trainers (RacerMate, Seattle, WA) on three consecutive days. Muscle biopsies were obtained from the vastus lateralis pre- and post-exercise on days one and three. Muscle samples were analyzed for mRNA content of peroxisome proliferator receptor gamma coactivator-1 alpha (PGC-1alpha), sirtuin 1 (Sirt-1), cytochrome c, and citrate synthase. Data were analyzed using a 2 (time) x 2 (day) repeated measures ANOVA. Of the mRNA analyzed, the following increased from pre to post 3 h rides: cytochrome c (P = 0.006), citrate synthase (P = 0.03), PGC-1alpha (P < 0.001), and Sirt-1 (P = 0.005). The following mRNA showed significant effects from days one to three: cytochrome c (P < 0.001) and citrate synthase (P = 0.01). These data show that exhaustive cycling performed on three consecutive days resulted in both acute and chronic stimuli for mRNA associated with mitochondrial biogenesis in already trained subjects. This is the first study to illustrate an increase in sirtuin-1 mRNA with acute and chronic exercise. These data contribute to the understanding of mRNA expression during both acute and successive bouts of prolonged exercise.

  7. Melatonin prevents mitochondrial dysfunction and insulin resistance in rat skeletal muscle.

    PubMed

    Teodoro, Bruno G; Baraldi, Flavia G; Sampaio, Igor H; Bomfim, Lucas H M; Queiroz, André L; Passos, Madla A; Carneiro, Everardo M; Alberici, Luciane C; Gomis, Ramon; Amaral, Fernanda G; Cipolla-Neto, José; Araújo, Michel B; Lima, Tanes; Akira Uyemura, Sérgio; Silveira, Leonardo R; Vieira, Elaine

    2014-09-01

    Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin-stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin-resistant cells. Melatonin treatment increases CAMKII and p-CREB but had no effect on p-AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin-induced increase in p-AKT in palmitic acid-treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p-AKT, mitochondrial respiration, and p-CREB and PGC-1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB-PGC-1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.

  8. Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents.

    PubMed

    Turner, Nigel; Bruce, Clinton R; Beale, Susan M; Hoehn, Kyle L; So, Trina; Rolph, Michael S; Cooney, Gregory J

    2007-08-01

    A reduced capacity for mitochondrial fatty acid oxidation in skeletal muscle has been proposed as a major factor leading to the accumulation of intramuscular lipids and their subsequent deleterious effects on insulin action. Here, we examine markers of mitochondrial fatty acid oxidative capacity in rodent models of insulin resistance associated with an oversupply of lipids. C57BL/6J mice were fed a high-fat diet for either 5 or 20 weeks. Several markers of muscle mitochondrial fatty acid oxidative capacity were measured, including (14)C-palmitate oxidation, palmitoyl-CoA oxidation in isolated mitochondria, oxidative enzyme activity (citrate synthase, beta-hydroxyacyl CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and carnitine palmitoyl-transferase 1), and expression of proteins involved in mitochondrial metabolism. Enzyme activity and mitochondrial protein expression were also examined in muscle from other rodent models of insulin resistance. Compared with standard diet-fed controls, muscle from fat-fed mice displayed elevated palmitate oxidation rate (5 weeks +23%, P < 0.05, and 20 weeks +29%, P < 0.05) and increased palmitoyl-CoA oxidation in isolated mitochondria (20 weeks +49%, P < 0.01). Furthermore, oxidative enzyme activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, uncoupling protein (UCP) 3, and mitochondrial respiratory chain subunits were significantly elevated in fat-fed animals. A similar pattern was present in muscle of fat-fed rats, obese Zucker rats, and db/db mice, with increases observed for oxidative enzyme activity and expression of PGC-1alpha, UCP3, and subunits of the mitochondrial respiratory chain. These findings suggest that high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance in rodents by decreasing muscle mitochondrial fatty acid oxidative capacity.

  9. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  10. Effect of peroxisome proliferator-activated receptors-gamma and co-activator-1alpha genetic polymorphisms on plasma adiponectin levels and susceptibility of non-alcoholic fatty liver disease in Chinese people.

    PubMed

    Hui, Yang; Yu-Yuan, Li; Yu-Qiang, Nie; Wei-Hong, Sha; Yan-Lei, Du; Xiao-Bo, Lai; Yong-Jian, Zhou

    2008-03-01

    Peroxisome proliferator-activated receptors-gamma (PPAR-gamma) and its co-activator-1alpha (PGC-1alpha) are involved in the regulation of lipid and glucose metabolisms. This study aimed to investigate the genetic polymorphisms of PPAR-gamma and PGC-1alpha in Chinese people and their influence on plasma adiponectin levels and non-alcoholic fatty liver disease (NAFLD) susceptibility. Ninety-six patients with NAFLD and 96 healthy controls were included. The single nucleotide polymorphisms (SNPs) of C161T PPAR-gammaand Gly482Ser PGC-1alpha genes were analysed by polymerase chain reaction and restriction fragment length polymorphism. The CC, CT and TT genotypic distributions of the NAFLD group were significantly different from those of controls (55.2, 39.6, 5.2 vs. 74.0, 25.0, 1.0%; P=0.015). The allelic frequencies of C and T were also different between the two groups (P=0.004). As for the PGC-1alpha gene, there was no difference of the genotypic and allelic frequencies between the two groups (P>0.05). In NAFLD patients, the plasma adiponectin concentrations were lower in the PPAR-gamma CT/TT genotypes compared with those in the CC genotype group (3.0+/-0.6 vs. 4.3+/-0.9, P=0.02). Multivariate logistic regression analysis showed that CT/TT genotypes of PPAR-gamma, TG, waist hip ratio, hypoadiponectinaemia and homoeostasis model assessment (HOMA)-IR were the risk factors for NAFLD. SNPs in the PPAR-gamma, but not PGC-1alpha, gene are associated with NAFLD susceptibility possibly through the adiponectin pathway.

  11. Muscle-specific differences in the response of mitochondrial proteins to beta-GPA feeding: an evaluation of potential mechanisms.

    PubMed

    Williams, Deon B; Sutherland, Lindsey N; Bomhof, Marc R; Basaraba, Susan A U; Thrush, A Brianne; Dyck, David J; Field, Catherine J; Wright, David C

    2009-06-01

    Beta-Guanadinopropionic acid (beta-GPA) feeding leads to reductions in skeletal muscle phosphagen concentrations and has been used as a tool with which to study the effects of energy charge on skeletal muscle metabolism. Supplementing standard rodent diets with beta-GPA leads to increases in mitochondrial enzyme content in fast but not slow-twitch muscles from male rats. Given this apparent discrepancy between muscle types we used beta-GPA feeding as a model to study signaling pathways involved in mitochondrial biogenesis. We hypothesized that beta-GPA feeding would result in a preferential activation of p38 MAPK and AMPK signaling and reductions in RIP140 protein content in triceps but not soleus muscle. Despite similar reductions in high-energy phosphate concentrations, 6 wk of beta-GPA feeding led to increases in mitochondrial proteins in triceps but not soleus muscles. Differences in the response of mitochondrial proteins to beta-GPA feeding did not seem to be related to a differential activation of p38 MAPK and AMPK signaling pathways or discrepancies in the induction of PPARgamma coactivator (PGC)-1alpha and -1beta. The protein content and expression of the nuclear corepressor RIP140 was reduced in triceps but not soleus muscle. Collectively our results indicate that chronic reductions in high-energy phosphates lead to the activation of p38 MAPK and AMPK signaling and increases in the expression of PGC-1alpha and -1beta in both soleus and triceps muscles. The lack of an effect of beta-GPA feeding on mitochondrial proteins in the soleus muscles could be related to a fiber type-specific effect of beta-GPA on RIP140 protein content.

  12. A novel partial agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride accumulation, and potentiates insulin signaling in vitro.

    PubMed

    Burgermeister, Elke; Schnoebelen, Astride; Flament, Angele; Benz, Jörg; Stihle, Martine; Gsell, Bernard; Rufer, Arne; Ruf, Armin; Kuhn, Bernd; Märki, Hans Peter; Mizrahi, Jacques; Sebokova, Elena; Niesor, Eric; Meyer, Markus

    2006-04-01

    Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.

  13. Functional interaction of hepatic nuclear factor-4 and peroxisome proliferator-activated receptor-gamma coactivator 1alpha in CYP7A1 regulation is inhibited by a key lipogenic activator, sterol regulatory element-binding protein-1c.

    PubMed

    Ponugoti, Bhaskar; Fang, Sungsoon; Kemper, Jongsook Kim

    2007-11-01

    Insulin inhibits transcription of cholesterol 7alpha-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1alpha for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1alpha transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1alpha-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1alpha was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1alpha. This mechanism may be relevant to known repression of many other HNF-4 target genes upon

  14. Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Ding, Xunshan; Lichti, Kristin; Kim, Insook; Gonzalez, Frank J; Staudinger, Jeff L

    2006-09-08

    Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.

  15. Coactivation of liver receptor homologue-1 by peroxisome proliferator-activated receptor gamma coactivator-1alpha on aromatase promoter II and its inhibition by activated retinoid X receptor suggest a novel target for breast-specific antiestrogen therapy.

    PubMed

    Safi, Rachid; Kovacic, Agnes; Gaillard, Stéphanie; Murata, Yoko; Simpson, Evan R; McDonnell, Donald P; Clyne, Colin D

    2005-12-15

    Aromatase inhibitors target the production of estrogen in breast adipose tissue, but in doing so, also decrease estrogen formation in bone and other sites, giving rise to deleterious side effects, such as bone loss and arthralgia. Thus, it would be clinically useful to selectively inhibit aromatase production in breast. In this regard, we have determined that the orphan nuclear receptor liver receptor homologue-1 (LRH-1) is a specific transcriptional activator of aromatase gene expression in human breast preadipocytes but not in other tissues of postmenopausal women. In this study, we show that the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is a physiologically relevant modulator of LRH-1, and that its transcriptional activity can be inhibited effectively using receptor-interacting peptide antagonists that prevent PGC-1alpha recruitment. Interestingly, we note that all of these peptides also interact in an agonist-dependent manner with retinoid X receptor alpha (RXRalpha), suggesting that these two receptors may compete for limiting cofactors within target cells. In support of this hypothesis, we show that 9-cis-retinoic acid, acting through RXR, inhibits both the basal and PGC-1alpha-induced transcriptional activity of LRH-1. The importance of this finding was confirmed by showing that LRH-1-dependent, PGC-1alpha-stimulated regulation of aromatase gene expression in primary human breast preadipocytes was effectively suppressed by RXR agonists. We infer from these data that LRH-1 is a bona fide target whose inhibition would selectively block aromatase expression in breast, while sparing other sites of expression.

  16. Long-term fasting decreases mitochondrial avian UCP-mediated oxygen consumption in hypometabolic king penguins.

    PubMed

    Rey, Benjamin; Halsey, Lewis G; Dolmazon, Virginie; Rouanet, Jean-Louis; Roussel, Damien; Handrich, Yves; Butler, Patrick J; Duchamp, Claude

    2008-07-01

    In endotherms, regulation of the degree of mitochondrial coupling affects cell metabolic efficiency. Thus it may be a key contributor to minimizing metabolic rate during long periods of fasting. The aim of the present study was to investigate whether variation in mitochondrial avian uncoupling proteins (avUCP), as putative regulators of mitochondrial oxidative phosphorylation, may contribute to the ability of king penguins (Aptenodytes patagonicus) to withstand fasting for several weeks. After 20 days of fasting, king penguins showed a reduced rate of whole animal oxygen consumption (Vo2; -33%) at rest, together with a reduced abundance of avUCP and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-alpha) mRNA in pectoralis muscle (-54%, -36%, respectively). These parameters were restored after the birds had been refed for 3 days. Furthermore, in recently fed, but not in fasted penguins, isolated muscle mitochondria showed a guanosine diphosphate-inhibited, fatty acid plus superoxide-activated respiration, indicating the presence of a functional UCP. It was calculated that variation in mitochondrial UCP-dependent respiration in vitro may contribute to nearly 20% of the difference in resting Vo2 between fed or refed penguins and fasted penguins measured in vivo. These results suggest that the lowering of avUCP activity during periods of long-term energetic restriction may contribute to the reduction in metabolic rate and hence the ability of king penguins to face prolonged periods of fasting.

  17. Large-scale chemical dissection of mitochondrial function.

    PubMed

    Wagner, Bridget K; Kitami, Toshimori; Gilbert, Tamara J; Peck, David; Ramanathan, Arvind; Schreiber, Stuart L; Golub, Todd R; Mootha, Vamsi K

    2008-03-01

    Mitochondrial oxidative phosphorylation (OXPHOS) is under the control of both mitochondrial (mtDNA) and nuclear genomes and is central to energy homeostasis. To investigate how its function and regulation are integrated within cells, we systematically combined four cell-based assays of OXPHOS physiology with multiplexed measurements of nuclear and mtDNA gene expression across 2,490 small-molecule perturbations in cultured muscle. Mining the resulting compendium revealed, first, that protein synthesis inhibitors can decouple coordination of nuclear and mtDNA transcription; second, that a subset of HMG-CoA reductase inhibitors, combined with propranolol, can cause mitochondrial toxicity, yielding potential clues about the etiology of statin myopathy; and, third, that structurally diverse microtubule inhibitors stimulate OXPHOS transcription while suppressing reactive oxygen species, via a transcriptional mechanism involving PGC-1alpha and ERRalpha, and thus may be useful in treating age-associated degenerative disorders. Our screening compendium can be used as a discovery tool both for understanding mitochondrial biology and toxicity and for identifying novel therapeutics.

  18. Dietary whey protein stimulates mitochondrial activity and decreases oxidative stress in mouse female brain.

    PubMed

    Shertzer, Howard G; Krishan, Mansi; Genter, Mary Beth

    2013-08-26

    In humans and experimental animals, protein-enriched diets are beneficial for weight management, muscle development, managing early stage insulin resistance and overall health. Previous studies have shown that in mice consuming a high fat diet, whey protein isolate (WPI) reduced hepatosteatosis and insulin resistance due in part to an increase in basal metabolic rate. In the current study, we examined the ability of WPI to increase energy metabolism in mouse brain. Female C57BL/6J mice were fed a normal AIN-93M diet for 12 weeks, with (WPI group) or without (Control group) 100g WPI/L drinking water. In WPI mice compared to controls, the oxidative stress biomarkers malondialdehyde and 4-hydroxyalkenals were 40% lower in brain homogenates, and the production of hydrogen peroxide and superoxide were 25-35% less in brain mitochondria. Brain mitochondria from WPI mice remained coupled, and exhibited higher rates of respiration with proportionately greater levels of cytochromes a+a3 and c+c1. These results suggested that WPI treatment increased the number or improved the function of brain mitochondria. qRT-PCR revealed that the gene encoding a master regulator of mitochondrial activity and biogenesis, Pgc-1alpha (peroxisome proliferator-activated receptor-gamma coactivator-1alpha) was elevated 2.2-fold, as were the PGC-1alpha downstream genes, Tfam (mitochondrial transcription factor A), Gabpa/Nrf-2a (GA-binding protein alpha/nuclear respiratory factor-2a), and Cox-6a1 (cytochrome oxidase-6a1). Each of these genes had twice the levels of transcript in brain tissue from WPI mice, relative to controls. There was no change in the expression of the housekeeping gene B2mg (beta-2 microglobulin). We conclude that dietary whey protein decreases oxidative stress and increases mitochondrial activity in mouse brain. Dietary supplementation with WPI may be a useful clinical intervention to treat conditions associated with oxidative stress or diminished mitochondrial activity in the

  19. Triacylglycerol-induced impairment in mitochondrial biogenesis and function in J774.2 and mouse peritoneal macrophage foam cells.

    PubMed

    Aronis, Anna; Aharoni-Simon, Michal; Madar, Zecharia; Tirosh, Oren

    2009-12-01

    The aim of this study was to detect mitochondrial alterations in J774.2 macrophages and mouse peritoneal macrophages (MPM) foam cells. J774.2 and MPM cells were exposed to triacylglycerol (TG) emulsion (1 mg/ml) for induction of fat accumulation. Impairment of mitochondrial function was reflected by reduced cellular ATP production and decreased expression of subunits of mitochondrial complexes I and III. The expression of subunit IV of complex IV remained unchanged, however, the content of its precursor in cells increased. Inhibitors of mitochondrial complexes, rotenone (0.1 microM) and myxothiazol (25 nM), protected the viability in TG-loaded macrophages. The exposure to TG caused downregulation of PPARgamma coactivator (PGC)-1alpha and nuclear respiratory factor (NRF)-1. Activation of peroxisome proliferator-activated receptors attenuated reactive oxygen species production in the foam cells. Treatment with antioxidant N-acetylcysteine (NAC) prevented lipid-mediated mitochondrial and cellular damage. In conclusion, this study demonstrates the important role of mitochondrial biogenesis dysfunction in TG-induced lipotoxicity in macrophages.

  20. Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model.

    PubMed

    Keeney, Paula M; Quigley, Caitlin K; Dunham, Lisa D; Papageorge, Christina M; Iyer, Shilpa; Thomas, Ravindar R; Schwarz, Kathleen M; Trimmer, Patricia A; Khan, Shaharyar M; Portell, Francisco R; Bergquist, Kristen E; Bennett, James P

    2009-08-01

    Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection") that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD = PTD + MLS = "mitochondrial transduction domain") binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM + mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM + mtDNA complex increased expression of PGC-1alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled

  1. Effect of every other day feeding on mitochondrial free radical production and oxidative stress in mouse liver.

    PubMed

    Caro, Pilar; Gómez, José; López-Torres, Mónica; Sánchez, Inés; Naudi, Alba; Portero-Otín, Manuel; Pamplona, Reinald; Barja, Gustavo

    2008-06-01

    It is known that dietary restriction (DR) increases maximum longevity in rodents, but the mechanisms involved remain unknown. Among the possible mechanisms, several lines of evidence support the idea that decreases in mitochondrial oxidative stress and in insulin signaling are involved but it is not known if they are interconnected. It has been reported that when C57BL/6 mice are maintained on an every other day (EOD) feeding their overall food intake is only slightly decreased and plasma insulin-like growth factor (IGF)-1 is even somewhat increased. In spite of this, their maximum longevity is increased, analogously to what occurs in classic DR. Thus, this model dissociates the increase in longevity from the decrease in IGF-1 observed in classic DR. Based on these facts, we have studied the effect of EOD DR on the rate of mitochondrial reactive oxygen species (ROS) production, oxygen consumption, and the percent free radical leak (FRL) of well-coupled liver mitochondria, the marker of mtDNA oxidative damage 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG), the content of complexes I to IV of the respiratory chain, the apoptosis inducing factor (AIF), PGC1-alpha, UCP2, five different markers of oxidative damage to proteins and the full fatty acid composition on C57BL/6 mice liver. It was found that EOD DR decreased ROS production in complex I but not in complex III without changes in oxygen consumption. As a result, FRL was decreased in complex I. Oxidative damage to mtDNA (8-oxodG) and protein oxidation, glycoxidation and lipoxidation were also lower in the EOD restricted group in comparison with the control one while the degree of fatty acid unsaturation was held constant. The EOD group also showed decreases in AIF, PGC1-alpha, and UCP2. These results support the possibility that EOD DR increases maximum life span at least in part through decreases in mitochondrial oxidative stress which are independent from insulin/IGF-1-like signaling.

  2. HIF-1alpha activation by a redox-sensitive pathway mediates cyanide-induced BNIP3 upregulation and mitochondrial-dependent cell death.

    PubMed

    Zhang, L; Li, L; Liu, H; Prabhakaran, K; Zhang, X; Borowitz, J L; Isom, G E

    2007-07-01

    Cyanide produces degeneration of the nervous system in which different modes of cell death are activated in the vulnerable brain areas. In brain, the mechanism underlying the cell death is not clear. In this study, an immortalized dopaminergic cell line was used to characterize the cell death signaling cascade activated by cyanide. Cyanide-treated cells exhibited a time- and concentration-dependent apoptosis that was caspase independent. Cyanide induced a rapid surge of intracellular reactive oxygen species (ROS) generation, followed by p38 mitogen-activated protein kinase (MAPK) activation and nuclear accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). Activation of p38 MAPK and HIF-1alpha accumulation were attenuated by N-acetyl-L-cysteine (antioxidant), catalase (hydrogen peroxide scavenger), or a selective p38 MAPK inhibitor (SB203580). Cyanide activated the hypoxia response element (HRE) promoter, which was also blocked by the antioxidants and SB203580. HRE activation was followed by increased BNIP3 gene transcription, as reflected by elevated BNIP3 mRNA and protein levels. BNIP3 upregulation was reduced by selective RNAi knockdown of HIF-1alpha. Overexpression of BNIP3 produced mitochondrial dysfunction (reduced membrane potential), caspase-independent apoptosis, and sensitization of the cells to cyanide-induced toxicity. Expression of a dominant-negative mutant or RNAi knockdown of BNIP3 protected the cells from cyanide. It was concluded that cyanide activated the HIF-1alpha-mediated pathway of BNIP3 induction through a redox-sensitive process. Increased BNIP3 expression then served as an initiator of mitochondrial-mediated death.

  3. Effect of bezafibrate treatment on late-onset mitochondrial myopathy in mice.

    PubMed

    Yatsuga, Shuichi; Suomalainen, Anu

    2012-02-01

    Mitochondrial dysfunction is an important cause of metabolic disorders of children and adults, with no effective therapy options. Recently, induction of mitochondrial biogenesis, by transgenic overexpression of PGC1-alpha [peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1-alpha], was reported to delay progression of early-onset cytochrome-c-oxidase (COX) deficiency in skeletal muscle of two mouse models: a muscle-specific knock-out of COX10 (COX10-mKO) and a constitutive knock-out of Surf1 (Surf1-KO). A pan-PPAR agonist, bezafibrate, could similarly delay myopathy progression in COX10-mKOs, but not in SURF1-KOs. We asked whether bezafibrate affected disease progression in late-onset adult-type mitochondrial myopathy mice. These 'Deletor mice' express a dominant patient mutation in Twinkle-helicase, leading to accumulation of multiple mtDNA deletions and subsequent progressive respiratory chain (RC) deficiency with COX-negative muscle fibers at 12 months of age. The primary and secondary molecular findings in Deletor mice mimic closely those in patients with Twinkle myopathy. We applied 0.5% bezafibrate diet to Deletors for 22 weeks, starting at disease manifestation, mimicking patient treatment after diagnosis. Bezafibrate delayed significantly the accumulation of COX-negative fibers and multiple mtDNA deletions. However, mitochondrial biogenesis was not induced: mitochondrial DNA copy number, transcript and RC protein amounts decreased in both Deletors and wild-type mice. Furthermore, bezafibrate induced severe lipid oxidation effects, with hepatomegaly and loss of adipose tissue, the mechanism involving lipid mobilization by high hepatic expression of FGF21 cytokine. However, as bezafibrate has been tolerated well by humans, the beneficial muscle findings in Deletor mice support consideration of bezafibrate trials on adult patients with mitochondrial myopathy.

  4. Mechanisms of calcium-induced mitochondrial biogenesis and GLUT4 synthesis.

    PubMed

    Wright, David C

    2007-10-01

    Regularly performed aerobic exercise leads to increases in skeletal muscle mitochondria and glucose transporter 4 (GLUT4) protein content, resulting in an enhanced capacity to oxidize substrates and improvements in insulin- and contraction-mediated glucose uptake. Although the specific mechanisms governing these adaptive responses have not been fully elucidated, accumulating evidence suggests that the increase in cytosolic Ca2+ that occurs with each wave of sacrolemmal depolarization is a key component of these processes. Treating L6 muscle cells with agents that increase Ca2+ without causing reductions in ~P or the activation of 5'-AMP-activated protein kinase leads to increases in GLUT4 and mitochondrial protein contents. This effect is likely controlled through calcium/calmodulin-dependent protein kinase (CaMK), since KN93, a specific CaMK inhibitor, blocks these adaptive responses. Recent findings provide evidence that the activation of p38 mitogen-activated protein kinase (MAPK) is involved in the pathway through which Ca2+/CaMK mediates mitochondrial and GLUT4 biogenesis. p38 MAPK initiates GLUT4 and mitochondrial biogenesis through the activation of transcription factors and transcriptional coactivators such as myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha). Subsequent increases in the content of these proteins further enhance Ca2+-induced GLUT4 and mitochondrial biogenesis. Since decreases in mitochondrial and GLUT4 contents are associated with skeletal muscle insulin resistance, an understanding of the mechanisms by which these processes can be normalized will aid in the prevention and treatment of type 2 diabetes.

  5. The mating type-specific homeodomain genes SXI1 alpha and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans.

    PubMed

    Yan, Zhun; Hull, Christina M; Sun, Sheng; Heitman, Joseph; Xu, Jianping

    2007-03-01

    In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type alpha (MATalpha) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type alpha (MATalpha)-specific gene SXI1a, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATalpha parent, and both the MATa and the MATalpha parents. In addition, progeny from same-sex mating between MATalpha strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1alpha and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.

  6. Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice.

    PubMed

    Toh, Shen Yon; Gong, Jingyi; Du, Guoli; Li, John Zhong; Yang, Shuqun; Ye, Jing; Yao, Huilan; Zhang, Yinxin; Xue, Bofu; Li, Qing; Yang, Hongyuan; Wen, Zilong; Li, Peng

    2008-08-06

    Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.

  7. HIF-1alpha Activation by a Redox-Sensitive Pathway Mediates Cyanide-induced BNIP3 Upregulation and Mitochondrial Dependent Cell Death

    PubMed Central

    Zhang, L.; Li, L.; Liu, H.; Prabhakaran, K.; Zhang, X.; Borowitz, J. L.; Isom, G. E.

    2007-01-01

    Cyanide produces degeneration of the nervous system in which different modes of cell death are activated in the vulnerable brain areas. In brain, the mechanism underlying the cell death is not clear. In this study, an immortalized dopaminergic cell line was used to characterize the cell death signaling cascade activated by cyanide. Cyanide-treated cells exhibited a time- and concentration-dependent apoptosis that was caspase-independent. Cyanide induced a rapid surge of intracellular reactive oxygen species (ROS) generation, followed by p38 mitogen-activated protein kinase (MAPK) activation and nuclear accumulation of hypoxia-inducible factor-1α (HIF-1α). Activation of p38 MAPK and HIF-1α accumulation were attenuated by N-acetyl-L-cysteine (antioxidant), catalase (hydrogen peroxide scavenger) or a selective p38 MAPK inhibitor (SB203580). Cyanide activated the hypoxia response element (HRE) promoter, which was also blocked by the antioxidants and SB203580. HRE activation was followed by increased BNIP3 gene transcription, as reflected by elevated BNIP3 mRNA and protein levels. BNIP3 upregulation was reduced by selective RNAi knockdown of HIF-1α. Overexpression of BNIP3 produced mitochondrial dysfunction (reduced membrane potential), caspase-independent apoptosis, and sensitization of the cells to cyanide-induced toxicity. Expression of a dominant negative mutant or RNAi knockdown of BNIP3 protected the cells from cyanide. It was concluded that cyanide activated the HIF-1α-mediated pathway of BNIP3 induction through a redox-sensitive process. Increased BNIP3 expression then served as an initiator of mitochondrial-mediated death. PMID:17561100

  8. Regulation by exercise of skeletal muscle content of mitochondria and GLUT4.

    PubMed

    Holloszy, J O

    2008-12-01

    Endurance exercise-training results in an increase in the size and number of mitochondria in the skeletal muscles that are involved in the exercise. In early studies of this phenomenon, long-term training programs of progressively increasing intensity and duration were used. These studies gave the impression that the adaptive increase in mitochondria is a slow process. Recent advances in the understanding of how mitochondrial biogenesis is regulated, have made it possible to study the mechanisms by which exercise regulates mitochondrial biogenesis. These studies have shown that a single bout of exercise induces a rapid increase in mitochondrial biogenesis that is mediated both by activation and by increased expression of a transcription coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha). PGC-1alpha docks on and coactivates transcription factors that regulate expression of nuclear genes that encode mitochondrial proteins and also of the nuclear gene that encodes mitochondrial transcription factor A (TFAM). TFAM regulates mitochondrial DNA transcription. Thus, PGC-1alpha regulates the coordinated expression of mitochondrial proteins encoded in both nuclear and mitochondrial genes. In addition to an increase in mitochondrial biogenesis, exercise induces an increase in the GLUT4 isoform of the glucose transporter. This increase in GLUT4 occurs in parallel with, and is mediated by, the same signals and some of the same transcription factors as the increase in mitochondrial biogenesis. Two signals generated during exercise, the increase in cytosolic Ca(2+) and the decrease in high energy phosphates, mediate the activation and increased expression of PGC-1alpha. The purpose of this article is to present an overview of what is known regarding these phenomena.

  9. The nuclear receptor ERRalpha is required for the bioenergetic and functional adaptation to cardiac pressure overload.

    PubMed

    Huss, Janice M; Imahashi, Ken-ichi; Dufour, Catherine R; Weinheimer, Carla J; Courtois, Michael; Kovacs, Atilla; Giguère, Vincent; Murphy, Elizabeth; Kelly, Daniel P

    2007-07-01

    Downregulation and functional deactivation of the transcriptional coactivator PGC-1alpha has been implicated in heart failure pathogenesis. We hypothesized that the estrogen-related receptor alpha (ERRalpha), which recruits PGC-1alpha to metabolic target genes in heart, exerts protective effects in the context of stressors known to cause heart failure. ERRalpha(-/-) mice subjected to left ventricular (LV) pressure overload developed signatures of heart failure including chamber dilatation and reduced LV fractional shortening. (31)P-NMR studies revealed abnormal phosphocreatine depletion in ERRalpha(-/-) hearts subjected to hemodynamic stress, indicative of a defect in ATP reserve. Mitochondrial respiration studies demonstrated reduced maximal ATP synthesis rates in ERRalpha(-/-) hearts. Cardiac ERRalpha target genes involved in energy substrate oxidation, ATP synthesis, and phosphate transfer were downregulated in ERRalpha(-/-) mice at baseline or with pressure overload. These results demonstrate that the nuclear receptor ERRalpha is required for the adaptive bioenergetic response to hemodynamic stressors known to cause heart failure.

  10. Induction of the nuclear factor HIF-1{alpha} in acetaminophen toxicity: Evidence for oxidative stress

    SciTech Connect

    James, Laura P. . E-mail: jameslaurap@uams.edu; Donahower, Brian; Burke, Angela S.; McCullough, Sandra; Hinson, Jack A.

    2006-04-28

    Hypoxia inducible factor (HIF) controls the transcription of genes involved in angiogenesis, erythropoiesis, glycolysis, and cell survival. HIF-1{alpha} levels are a critical determinant of HIF activity. The induction of HIF-1{alpha} was examined in the livers of mice treated with a toxic dose of APAP (300 mg/kg IP) and sacrificed at 1, 2, 4, 8, and 12 h. HIF-1{alpha} was induced at 1-12 h and induction occurred prior to the onset of toxicity. Pre-treatment of mice with N-acetylcysteine (1200 mg/kg IP) prevented toxicity and HIF-1{alpha} induction. In further studies, hepatocyte suspensions were incubated with APAP (1 mM) in the presence of an oxygen atmosphere. HIF-1{alpha} was induced at 1 h, prior to the onset of toxicity. Inclusion of cyclosporine A (10 {mu}M), an inhibitor of mitochondrial permeability transition, oxidative stress, and toxicity, prevented the induction of HIF-1{alpha}. Thus, HIF-1{alpha} is induced before APAP toxicity and can occur under non-hypoxic conditions. The data suggest a role for oxidative stress in the induction of HIF-1{alpha} in APAP toxicity.

  11. The effect of transcutaneous application of carbon dioxide (CO{sub 2}) on skeletal muscle

    SciTech Connect

    Oe, Keisuke; Ueha, Takeshi; Sakai, Yoshitada; Niikura, Takahiro; Lee, Sang Yang; Koh, Akihiro; Hasegawa, Takumi; Tanaka, Masaya; Miwa, Masahiko; Kurosaka, Masahiro

    2011-04-01

    Highlights: {yields} PGC-1{alpha} is up-regulated as a result of exercise such as mitochondrial biogenesis and muscle fiber-type switching, and up-regulation of VEGF. {yields} We demonstrated transcutaneous application of CO{sub 2} up-regulated the gene expression of PGC-1{alpha}, SIRT1 and VEGF, and instance of muscle fiber switching. {yields} Transcutaneous application of CO{sub 2} may cause similar effect to aerobic exercise in skeletal muscle. -- Abstract: In Europe, carbon dioxide therapy has been used for cardiac disease and skin problems for a long time. However there have been few reports investigating the effects of carbon dioxide therapy on skeletal muscle. Peroxisome proliferators-activated receptor (PPAR)-gamma coactivator-1 (PGC-1{alpha}) is up-regulated as a result of exercise and mediates known responses to exercise, such as mitochondrial biogenesis and muscle fiber-type switching, and neovascularization via up-regulation of vascular endothelial growth factor (VEGF). It is also known that silent mating type information regulation 2 homologs 1 (SIRT1) enhances PGC-1{alpha}-mediated muscle fiber-type switching. Previously, we demonstrated transcutaneous application of CO{sub 2} increased blood flow and a partial increase of O{sub 2} pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO{sub 2} to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO{sub 2} application caused: (1) the gene expression of PGC-1{alpha}, silent mating type information regulation 2 homologs 1 (SIRT1) and VEGF, and increased the number of mitochondria, as proven by real-time PCR and immunohistochemistry, (2) muscle fiber switching in the TA muscle, as proven by isolation of myosin heavy chain and ATPase staining. Our results suggest the transcutaneous application of CO{sub 2} may have therapeutic potential for muscular strength recovery resulting from disuse

  12. RETRACTED: Bezafibrate improves mitochondrial function in the CNS of a mouse model of mitochondrial encephalopathy.

    PubMed

    Noe, Natalie; Dillon, Lloye; Lellek, Veronika; Diaz, Francisca; Hida, Aline; Moraes, Carlos T; Wenz, Tina

    2013-09-01

    This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This retraction was suggested by the University of Cologne Investigation committee and seconded by the authors who the journal was able to contact (Wenz, T., Dillon, L., Diaz, F., Hida, A., and Moraes, C.T.). Following an investigation of the last author, Dr. Tina Wenz, by the University of Cologne, Germany, the university determined that data presented in this article have been inappropriately manipulated https://www.portal.uni-koeln.de/9015.html?&tx_news_pi1%5Bnews%5D=4335&tx_news_pi1%5Bcontroller%5D=News&tx_news_pi1%5Baction%5D=detail&cHash=1deb8399d7f796d65ca9f6ae4764a1ce. Specifically, western blot images in Figure 5F (tubulin in cortex), 2F (COXI in hippocampus) and 3B (Sod2 in hippocampus) were re-used from an earlier article published elsewhere [Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging" Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, and Moraes CT. Proc Natl Acad Sci U S A. 2009;106:20405-10, doi: 10.1073/pnas.0911570106] representing different experimental findings. Therefore, whether or not the main conclusions are still valid, the authors request retraction of this publication because the scientific integrity of the study was compromised. The authors sincerely apologize to the scientific community. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Chronic AMP-activated protein kinase activation and a high-fat diet have an additive effect on mitochondria in rat skeletal muscle.

    PubMed

    Fillmore, Natasha; Jacobs, Daniel L; Mills, David B; Winder, William W; Hancock, Chad R

    2010-08-01

    Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMP-activated protein kinase (AMPK), calcium, and circulating free fatty acids (FFAs). Chronic treatment with either 5-aminoimidazole-4-carboxamide riboside (AICAR), a chemical activator of AMPK, or increasing circulating FFAs with a high-fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high-fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high-fat diet (HF), AICAR injections (AICAR), or a high-fat diet and AICAR injections (HF + AICAR) for 6 wk. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on long-chain acyl-CoA dehydrogenase, cytochrome c, and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein, as well as on citrate synthase and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) activity. In contrast, no additive effect was noted in the soleus, and in the red quadriceps only beta-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high-fat diet-induced posttranscriptional increase in PGC-1alpha protein and AMPK-mediated increase in PGC-1alpha protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high-fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

  14. Gene expression in breast muscle and duodenum from low and high feed efficient broilers.

    PubMed

    Ojano-Dirain, C; Toyomizu, M; Wing, T; Cooper, M; Bottje, W G

    2007-02-01

    This study was conducted to evaluate messenger RNA (mRNA) expression of genes that are involved in energy metabolism and mitochondrial biogenesis: avian adenine nucleotide translocator (avANT), cytochrome oxidase III (COX III), inducible nitric oxide synthase (iNOS), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), avian PPAR-gamma coactivator-1alpha (avPGC-1alpha), and avian uncoupling protein in breast muscle and duodenum of broilers with low and high feed efficiency (FE). Total RNA was extracted from snap-frozen tissues from male broilers with low (0.55 +/- 0.01) and high (0.72 +/- 0.01) FE (n = 8 per group). Total RNA was reverse-transcribed using oligo(dT), random primers, or both followed by real-time reverse transcription-PCR. Protein oxidation, measured as protein carbonyls, was also evaluated in duodenal mucosa. Protein carbonyls were higher in low FE mucosa in tissue homogenate and mitochondrial fraction. The mRNA expression of iNOS and PPAR-gamma in the duodenum was lower in the low FE broilers, with no differences in avANT, COX III, and avPGC-1alpha. In contrast, expression of avANT and COX III mRNA in breast muscle was lower in low FE broilers with no differences in iNOS, PPAR-gamma, and avPGC-1alpha. The avian uncoupling protein in breast muscle was higher in low FE birds (P = 0.068). These results indicate that there are differences in the expression of mRNA encoding for mitochondrial transcription factors and proteins in breast muscle and duodenal tissue between low and high FE birds. The differences that were observed may also reflect inherent metabolic and gene regulation differences between tissues.

  15. Gene expression in skeletal muscle of coronary artery disease patients after concentric and eccentric endurance training.

    PubMed

    Zoll, J; Steiner, R; Meyer, K; Vogt, M; Hoppeler, H; Flück, M

    2006-03-01

    Low-intensity concentric (CET) and eccentric (EET) endurance-type training induce specific structural adaptations in skeletal muscle. We evaluated to which extent steady-state adaptations in transcript levels are involved in the compensatory alterations of muscle mitochondria and myofibrils with CET versus EET at a matched metabolic exercise intensity of medicated, stable coronary patients (CAD). Biopsies were obtained from vastus lateralis muscle before and after 8 weeks of CET (n=6) or EET (n=6). Transcript levels for factors involved in mitochondrial biogenesis (PGC-1alpha, Tfam), mitochondrial function (COX-1, COX-4), control of contractile phenotype (MyHC I, IIa, IIx) as well as mechanical stress marker (IGF-I) were quantified using an reverse-transcriptase polymerase chain reaction approach. After 8 weeks of EET, a reduction of the COX-4 mRNA level by 41% and a tendency for a drop in Tfam transcript concentration (-33%, P=0.06) was noted. This down-regulation corresponded to a drop in total mitochondrial volume density. MyHC-IIa transcript levels were specifically decreased after EET, and MyHC-I mRNA showed a trend towards a reduction (P=0.08). Total fiber cross-sectional area was not altered. After CET and EET, the IGF-I mRNA level was significantly increased. The PGC-1alpha significantly correlated with Tfam, and both PGC-1alpha and Tfam significantly correlated with COX-1 and COX-4 mRNAs. Post-hoc analysis identified significant interactions between the concurrent medication and muscular transcript levels as well as fiber size. Our findings support the concept that specific transcriptional adaptations mediate the divergent mitochondrial response of muscle cells to endurance training under different load condition and indicate a mismatch of processes related to muscle hypertrophy in medicated CAD patients.

  16. Uncoupling protein-1 and related messenger ribonucleic acids in human epicardial and other adipose tissues: epicardial fat functioning as brown fat.

    PubMed

    Sacks, Harold S; Fain, John N; Holman, Ben; Cheema, Paramjeet; Chary, Aron; Parks, Frank; Karas, James; Optican, Robert; Bahouth, Suleiman W; Garrett, Edward; Wolf, Rodney Y; Carter, Russell A; Robbins, Todd; Wolford, David; Samaha, Joseph

    2009-09-01

    Uncoupling protein-1 (UCP-1) is the inner mitochondrial membrane protein that is a specific marker for and mediator of nonshivering thermogenesis in brown adipocytes. This study was performed to better understand the putative thermogenic function of human epicardial fat. We measured the expression of UCP-1 and brown adipocyte differentiation transcription factors PR-domain-missing 16 (PRDM16) and peroxisome-proliferator-activated receptor gamma co-activator-1 alpha (PGC-1 alpha) in epicardial, substernal, and sc thoracic, abdominal, and leg fat. The study was conducted at a tertiary care hospital cardiac center. Forty-four patients had coronary artery bypass surgery, and six had heart valve replacement. Fat samples were taken at open heart surgery. UCP-1 expression was 5-fold higher in epicardial fat than substernal fat and barely detectable in sc fat. Epicardial fat UCP-1 expression decreased with age, increased with body mass index, was similar in women and men and patients on and not on statin therapy, and showed no relationship to epicardial fat volume or waist circumference. UCP-1 expression was similar in patients without and with severe coronary atherosclerosis and metabolic syndrome or type 2 diabetes. PRDM16 and PGC-1 alpha expression was 2-fold greater in epicardial than sc fat. Epicardial fat UCP-1, PRDM16, and PGC1-alpha mRNAs were similar in diabetics treated with thiazolidinediones compared to diabetics not treated with thiazolidinediones. Because UCP-1 is expressed at high levels in epicardial fat as compared to other fat depots, the possibility should be considered that epicardial fat functions like brown fat to defend the myocardium and coronary vessels against hypothermia. This process could be blunted in the elderly.

  17. Acetaminophen hepatotoxicity and HIF-1{alpha} induction in acetaminophen toxicity in mice occurs without hypoxia

    SciTech Connect

    Chaudhuri, Shubhra; McCullough, Sandra S.; Hennings, Leah; Letzig, Lynda; Simpson, Pippa M.; Hinson, Jack A.; James, Laura P.

    2011-05-01

    HIF-1{alpha} is a nuclear factor important in the transcription of genes controlling angiogenesis including vascular endothelial growth factor (VEGF). Both hypoxia and oxidative stress are known mechanisms for the induction of HIF-1{alpha}. Oxidative stress and mitochondrial permeability transition (MPT) are mechanistically important in acetaminophen (APAP) toxicity in the mouse. MPT may occur as a result of oxidative stress and leads to a large increase in oxidative stress. We previously reported the induction of HIF-1{alpha} in mice with APAP toxicity and have shown that VEGF is important in hepatocyte regeneration following APAP toxicity. The following study was performed to examine the relative contribution of hypoxia versus oxidative stress to the induction of HIF-1{alpha} in APAP toxicity in the mouse. Time course studies using the hypoxia marker pimonidazole showed no staining for pimonidazole at 1 or 2 h in B6C3F1 mice treated with APAP. Staining for pimonidazole was present in the midzonal to periportal regions at 4, 8, 24 and 48 h and no staining was observed in centrilobular hepatocytes, the sites of the toxicity. Subsequent studies with the MPT inhibitor cyclosporine A showed that cyclosporine A (CYC; 10 mg/kg) reduced HIF-1{alpha} induction in APAP treated mice at 1 and 4 h and did not inhibit the metabolism of APAP (depletion of hepatic non-protein sulfhydryls and hepatic protein adduct levels). The data suggest that HIF-1{alpha} induction in the early stages of APAP toxicity is secondary to oxidative stress via a mechanism involving MPT. In addition, APAP toxicity is not mediated by a hypoxia mechanism.

  18. Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences.

    PubMed

    Stenglein, S A; Rodriguero, M S; Chandler, E; Jennings, P; Salerno, G L; Nicholson, P

    2010-01-01

    A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations. Copyright © 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  19. [Effect of dominant negative HIF-1alpha (dn HIF-1alpha) on biological characteristics of uterine cervix cancer cells].

    PubMed

    Tang, Bin-Zhi; Zhao, Feng-Yan; Wei, Ting; Mu, De-Zhi; Mao, Meng; Fu, Qiang; Zhang, Lin; Qu, Yia

    2008-05-01

    To explore the effect of dominant negative HIF-1alpha (dn HIF-1alpha) on biological characteristics of uterine cervix cancer cell SiHa and elucidate the related mechanism. pcDNA3. 1-dn HIF-1alpha was transfected into SiHa cells. The expression of HIF-1alpha and VEGF protein were detected by immunocytochemical method and Western Blotting. The growth proliferation of cells was surveyed by the MTT assay and cell apoptosis was detected through TUNEL after treated with CoCl2, meanwhile the results were compared with the group transfected with mock plasmid and untransfected group. After successfully transfected with relevant plasmid, there's no obvious difference of expression of HIF-1alpha among dn HIF-1alpha group, pcDNA3. 1 group, and untransfected group, however the expression of VEGF of dn HIF-1alpha group was significantly lower than that of the others (P < 0. 05). The proliferation ability of dn HIF-1alpha group was obviously lower than that of the other two (P < 0.05), whether it was under normoxia or chemical hypoxia induced by CoCl2. The characteristic apoptotic morphology was most significantly apparent in dn HIF-1alpha group among these three (P < 0.05). Domain negative HIF-1alpha can inhibit the proliferation of uterine cervix cancer cell and accelerate its apoptosis under hypoxia induced by CoCl2, as well as decrease the expression of VEGF protein. The implications of all this were that the domain negative HIF-1alpha may play an important role in the therapy of uterine cervix cancer.

  20. CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

    SciTech Connect

    Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion; Gahl, Anja; Scheeder, Martin R.L.; Cardoso, M. Cristina; Leonhardt, Heinrich; Geary, Nori; Langhans, Wolfgang; Leonhardt, Monika . E-mail: monika.leonhardt@inw.agrl.ethz.ch

    2005-12-16

    To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.

  1. Estrogen-related receptor alpha modulates the expression of adipogenesis-related genes during adipocyte differentiation.

    PubMed

    Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Yagi, Ken; Okazaki, Yasushi; Inoue, Satoshi

    2007-07-06

    Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERRalpha in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERRalpha and ERRalpha-related transcriptional coactivators, peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) and PGC-1beta, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERRalpha-specific siRNA results in mRNA down-regulation of fatty acid binding protein 4, PPARgamma, and PGC-1alpha in 3T3-L1 cells in the adipogenesis medium. ERRalpha and PGC-1beta mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERRalpha in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERRalpha may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.

  2. Structure and expression of elongation factor 1 alpha in tomato.

    PubMed Central

    Pokalsky, A R; Hiatt, W R; Ridge, N; Rasmussen, R; Houck, C M; Shewmaker, C K

    1989-01-01

    A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues. Images PMID:2748335

  3. Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

    PubMed

    Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

    2017-09-29

    Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK

  4. Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

    SciTech Connect

    Ikeda, Shin-ichi; Kizaki, Takako; Haga, Shukoh; Ohno, Hideki; Takemasa, Tohru

    2008-04-04

    Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1{alpha} and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1{alpha} protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1{alpha} expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1{alpha} and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.

  5. Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: involvement of muscarinic receptors and HIF-1 alpha pathway.

    PubMed

    Zhu, Xu; Zhou, Wei; Cui, Yongyao; Zhu, Liang; Li, Juan; Feng, Xuemei; Shao, Biyun; Qi, Hong; Zheng, Jun; Wang, Hao; Chen, Hongzhuan

    2010-04-01

    The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl(2))-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1 alpha (HIF-1 alpha), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 microM CoCl(2) for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 microM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 microM pilocarpine could significantly prevent CoCl(2)-induced HIF-1 alpha translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1 alpha, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1 alpha pathway. The findings suggest that HIF-1 alpha pathway as a "master switch" may be used as a therapeutic target in the cholinergic treatment of glaucoma.

  6. Expression and modulation of IL-1 alpha in murine keratinocytes

    SciTech Connect

    Ansel, J.C.; Luger, T.A.; Lowry, D.; Perry, P.; Roop, D.R.; Mountz, J.D.

    1988-04-01

    Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low (Ca2+) tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high (Ca2+) media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low (Ca2+) conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes.

  7. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    SciTech Connect

    Shlomai, Amir; Shaul, Yosef

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  8. Antioxidant supplementation enhances the exercise-induced increase in mitochondrial uncoupling protein 3 and endothelial nitric oxide synthase mRNA content in human skeletal muscle.

    PubMed

    Hellsten, Ylva; Nielsen, Jens J; Lykkesfeldt, Jens; Bruhn, Maria; Silveira, Leonardo; Pilegaard, Henriette; Bangsbo, Jens

    2007-08-01

    The effects of acute exercise on the mRNA content of selected genes were examined during control conditions and after oral intake of antioxidants. In addition, to provide evidence for formation of reactive oxygen species (ROS) in human skeletal muscle during exercise, cytochrome c reduction was measured in microdialysate from the muscle. For the study on the effects of antioxidants on mRNA content, seven healthy, habitually active, male subjects participated in a double-blinded experimental design in which they, on one occasion, received a placebo and, on another, a mixture of antioxidants containing 1500 mg vitamin C, 120 mg coenzyme Q, and 345 mg alpha-tocopherol every day for 7 days before the experiment. On the experimental day the subjects cycled for 90 min and muscle biopsies were taken preexercise and at 1, 3, and 5 h after exercise. Exercise induced an increase in the eNOS, UCP3, PGC-1alpha, VEGF, Hsp72, and HO-1 mRNA content (p < 0.001), whereas there was no change in the Hsc70 mRNA level. Prior antioxidant treatment further enhanced (p < 0.05) the eNOS and UCP3 mRNA content after exercise. Moreover, the overall level of Hsc70 mRNA tended (p = 0.07) to be higher after antioxidant treatment. In another group of healthy male subjects, cytochrome c reduction was determined in microdialysate from the thigh muscle at rest and during knee extensor exercise to determine ROS formation. There was a significant increase in cytochrome c reduction with exercise both at 14 ( approximately 25%) and at 30 W ( approximately 50%). The data show that ROS are formed within skeletal muscle during exercise and that oral intake of antioxidants can enhance the exercise-induced adaptive mRNA responses of eNOS and UCP3.

  9. Reduced adiposity in bitter melon (Momordica charantia)-fed rats is associated with increased lipid oxidative enzyme activities and uncoupling protein expression.

    PubMed

    Chan, Laureen L Y; Chen, Qixuan; Go, Adi G G; Lam, Emily K Y; Li, Edmund T S

    2005-11-01

    To further explore the antiobesity effect of freeze-dried bitter melon (BM) juice, activities of mitochondrial lipid oxidative enzymes as well as the expression of uncoupling proteins and their transcription coactivator peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1alpha) were determined in diet-induced obese (DIO) rats. Rats were fed high-fat (HF) diets to induce obesity, and the effect of BM was assessed at doses of 0.75, 1.0, or 1.25% (wt:wt). In a dose-response experiment, BM-supplemented rats had lower energy efficiency (g weight gained/kJ consumed), visceral fat mass, serum glucose, and insulin resistance index, but higher plasma norepinephrine than unsupplemented rats (P < 0.05). Hepatic and skeletal muscle triglyceride concentrations were lower in supplemented HF diet-fed rats than in unsupplemented HF diet-fed rats (P < 0.05). An HF diet supplemented with BM elevated activities of hepatic and muscle mitochondrial carnitine palmitoyl transferase-I (CPT-I) and acyl-CoA dehydrogenase (AD) (P < 0.05). In another experiment, BM (1.0 g/100 g) lowered visceral fat mass but increased serum adiponectin concentration in HF diet-fed rats (P < 0.05). In the final study, rats were fed the HF diet with 0, 1.0 or 1.25% BM. Both groups of BM-supplemented rats had higher uncoupling protein 1 in brown adipose tissue (P < 0.05) and uncoupling protein 3 in red gastrocnemius muscle (P < 0.05), measured by Western blotting and RT-PCR, than the controls. The expression of the transcription coactivator PGC-1alpha in both tissues was also significantly elevated in the BM-supplemented rats (P < 0.05). The present results suggest that decreased adiposity in BM-supplemented rats may result from lower metabolic efficiency, a consequence of increased lipid oxidation and mitochondrial uncoupling.

  10. Similar expression of oxidative genes after interval and continuous exercise.

    PubMed

    Wang, Li; Psilander, Niklas; Tonkonogi, Michail; Ding, Shuzhe; Sahlin, Kent

    2009-12-01

    There is a debate whether interval or traditional endurance training is the most effective stimulus of mitochondrial biogenesis. Here, we compared the effects of acute interval exercise (IE) or continuous exercise (CE) on the muscle messenger RNA (mRNA) content for several genes involved in mitochondrial biogenesis and lipid metabolism. Nine sedentary subjects cycled for 90 min with two protocols: CE (at 67% VO2max) and IE (12 s at 120% and 18 s at 20% of VO2max). The duration of exercise and work performed with CE and IE was identical. Muscle biopsies were taken before and 3 h after exercise. There were no significant differences between the two exercise protocols in the increases in VO2 and HR, the reduction in muscle glycogen (35%-40% with both protocols) or the changes in blood metabolites (lactate, glucose, and fatty acids). The mRNA content for major regulators of mitochondrial biogenesis [peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1alpha (PGC-1alpha), PGC-1-related coactivator, PPARbeta/delta] and of lipid metabolism [pyruvate dehydrogenase kinase isozyme 4 (PDK4)] increased after exercise, but there was no significant difference between IE and CE. However, the mRNA content for several downstream targets of PGC-1alpha increased significantly only after CE, and mRNA content for nuclear respiratory factor 2 was significantly higher after CE (P < 0.025 vs IE). The present findings demonstrate that, when the duration of exercise and work performed is the same, IE and CE influence the transcription of genes involved in oxidative metabolism in a similar manner.

  11. Characterization of yeast EF-1 alpha: non-conservation of post-translational modifications.

    PubMed

    Cavallius, J; Zoll, W; Chakraburtty, K; Merrick, W C

    1993-04-21

    Elongation factor 1 alpha (EF-1 alpha) is an abundant cellular protein and its amino-acid sequence has been inferred from numerous organisms, including bacteria, archaebacteria, plants and animals. In large measure, it would appear that the overall structure has probably been maintained given the 33% identity and 56% similarity of Escherichia coli EF-Tu with human EF-1 alpha. Chemical sequencing of EF-Tu and EF-1 alpha has revealed that these proteins are post-translationally modified. In order to assess the possible function of these modifications, we have chemically sequenced the EF-1 alpha from the lower eukaryote Saccharomyces cerevisiae (yeast). To our surprise, the methylation pattern of yeast EF-1 alpha was quite different from either rabbit or brine shrimp EF-1 alpha with only the trimethyllysine at position 79 conserved although the yeast protein is 81% identical to rabbit EF-1 alpha. A dimethyllysine was observed at position 316 which corresponds to a trimethyllysine in brine shrimp and rabbit EF-1 alpha. The other positions in yeast EF-1 alpha which were methylated were unrelated to the other six possible positions for modification observed in brine shrimp or rabbit EF-1 alpha. In addition, the unique glyceryl-phosphorylethanolamine observed in mammalian EF-1 alpha and suspected in brine shrimp EF-1 alpha was not found in yeast EF-1 alpha.

  12. Changes in skeletal muscle mitochondria in response to the development of type 2 diabetes or prevention by daily wheel running in hyperphagic OLETF rats.

    PubMed

    Rector, R Scott; Uptergrove, Grace M; Borengasser, Sarah J; Mikus, Catherine R; Morris, E Matthew; Naples, Scott P; Laye, Matthew J; Laughlin, M Harold; Booth, Frank W; Ibdah, Jamal A; Thyfault, John P

    2010-06-01

    The temporal changes in skeletal muscle mitochondrial content and lipid metabolism that precede type 2 diabetes are largely unknown. Here we examined skeletal muscle mitochondrial fatty acid oxidation (MitoFAOX) and markers of mitochondrial gene expression and protein content in sedentary 20- and 40-wk-old hyperphagic, obese Otsuka Long-Evans Tokushima fatty (OLETF-SED) rats. Changes in OLETF-SED rats were compared with two groups of rats who maintained insulin sensitivity: age-matched OLETF rats given access to voluntary running wheels (OLETF-EX) and sedentary, nonobese Long-Evans Tokushima Otsuka (LETO-SED) rats. As expected, glucose tolerance tests revealed insulin resistance at 20 wk that progressed to type 2 diabetes at 40 wk in the OLETF-SED, whereas both the OLETF-EX and LETO-SED maintained whole body insulin sensitivity. At 40 wk, complete MitoFAOX (to CO(2)), beta-hydroxyacyl-CoA dehydrogenase activity, and citrate synthase activity did not differ between OLETF-SED and LETO-SED but were significantly (P < 0.05) higher in OLETF-EX compared with OLETF-SED rats. Genes controlling skeletal muscle MitoFAOX (PGC-1alpha, PPARdelta, mtTFA, cytochrome c) were not different between OLETF-SED and LETO-SED at any age. Compared with the OLETF-SED, the OLETF-EX rats had significantly (P < 0.05) higher skeletal muscle PGC-1alpha, cytochrome c, and mtTFA mRNA levels at 20 and 40 wk and PPARdelta at 40 wk; however, protein content for each of these markers did not differ between groups at 40 wk. Limited changes in skeletal muscle mitochondria were observed during the transition from insulin resistance to type 2 diabetes in the hyperphagic OLETF rat. However, diabetes prevention through increased physical activity appears to be mediated in part through maintenance of skeletal muscle mitochondrial function.

  13. Garlic-derived diallyl disulfide modulates peroxisome proliferator activated receptor gamma co-activator 1 alpha in neuroblastoma cells.

    PubMed

    Pagliei, Beatrice; Aquilano, Katia; Baldelli, Sara; Ciriolo, Maria R

    2013-02-01

    The peroxisome proliferator activated receptor gamma co-activator 1 alpha (PGC1α) is an inducible transcriptional co-activator with direct function in the induction of mitochondrial biogenesis. In the present report we show that, in SH-SY5Y neuroblastoma cells, garlic-derived diallyl disulfide (DADS) is able to increase PGC1α expression in a ROS-dependent manner and to induce mitochondrial biogenesis at early stage of treatment that precede cell cycle arrest and apoptosis outcome. In particular, we demonstrate that DADS elicits: i) the increase of PGC1α within nuclear compartment; ii) the decrease of PGC1α non-active acetylated form; iii) the induction of nuclear-encoded mitochondrial genes such as TFAM and TFBM1. We also show an accumulation of PGC1α within mitochondria along with an increased association with the regulatory D-Loop region of mtDNA and a concomitant augmented expression of mitochondrial RNA. Such events are related to a prompt elevation of mitochondrial mass, as assessed by evaluating the content of mtDNA. We show that the induction of mitochondrial biogenesis is directed to dampen the cytotoxic effect of DADS. Indeed, PGC1α overexpression or down-regulation prevents or exacerbates mtDNA loss and apoptosis. Overall the data highlight an anti-apoptotic role of PGC1α-mediated mitochondrial biogenesis in neuroblatoma cells and suggest PGC1α as a potential target for enhancing the effectiveness of therapy in aggressive neuroblastoma with high drug-resistance. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Toxic effects of cobalt in primary cultures of mouse astrocytes. Similarities with hypoxia and role of HIF-1alpha.

    PubMed

    Karovic, Olga; Tonazzini, Ilaria; Rebola, Nelson; Edström, Erik; Lövdahl, Cecilia; Fredholm, Bertil B; Daré, Elisabetta

    2007-03-01

    Cobalt is suspected to cause memory deficit in humans and was reported to induce neurotoxicity in animal models. We have studied the effects of cobalt in primary cultures of mouse astrocytes. CoCl(2) (0.2-0.8mM) caused dose-dependent ATP depletion, apoptosis (cell shrinkage, phosphatidylserine externalization and chromatin rearrangements) and secondary necrosis. The mitochondria appeared to be a main target of cobalt toxicity, as shown by the loss of mitochondrial membrane potential (DeltaPsi(m)) and release from the mitochondria of apoptogenic factors, e.g. apoptosis inducing factor (AIF). Pre-treatment with bongkrekic acid reduced ATP depletion, implicating the involvement of the mitochondrial permeability transition (MPT) pore. Cobalt increased the generation of oxygen radicals, but antioxidants did not prevent toxicity. There was also an impaired response to ATP stimulation, evaluated as a lower raise in intracellular calcium. Similarly to hypoxia and dymethyloxallyl glycine (DMOG), cobalt triggered stabilization of the alpha-subunit of hypoxia-inducible factor HIF-1 (HIF-1alpha). This early event was followed by an increased expression of HIF-1 regulated genes, e.g. stress protein HO-1, pro-apoptotic factor Nip3 and iNOS. Although all of the three stimuli activated the HIF-1alpha pathway and decreased ATP levels, the downstream effects were different. DMOG only inhibited cell proliferation, whereas the other two conditions caused cell death by apoptosis and necrosis. This points to cobalt and hypoxia not only inducing HIF-1alpha regulated genes but also affecting similarly other cellular functions, including metabolism.

  15. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  16. [Construction of recombinant baculovirus vectors started by EF1alpha].

    PubMed

    Gao, Dongni; Jin, Liying; Ge, Jingping; Wang, Kun; An, Qi; Ping, Wenxiang; Lou, Zhuangwei

    2014-06-04

    To improve the transduction efficiency of baculovirus and exogenous gene expression level, we chose a mammalian cell-specific promoter-human extension factor 1alpha promoter (EF1-alpha), used virus pseudotyped tools--truncated vessicular stomatitis virus protein G (VSV-GED), added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs). We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE. The recombinant transfer vectors pWK-eGFP, pWK-ITR-eGFP and pWK (-)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1alpha promoter. Constructed recombinant plasmid transfected Sf9 insect cells, and observed the expression of green fluorescent protein by using the inverted fluorescence microscope. The fluorescence expression rate of BV-WK-eGFP, BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control, ITRs can effectively extend the expression time of eGFP, the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BV-WK (-) -eGFP. The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP, BV-WK-ITR-eGFP was obviously shorten in OL cells, and reduced 24 hours compared to the negative control BV-WK (-) -eGFP, transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK (-) -eGFP, respectively. The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus, WPRE could enhance the expression efficiency of the exogenous gene significantly, and ITRs extend the expression time. The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine.

  17. Hydroxytyrosol promotes mitochondrial biogenesis and mitochondrial function in 3T3-L1 adipocytes.

    PubMed

    Hao, Jiejie; Shen, Weili; Yu, Guangli; Jia, Haiqun; Li, Xuesen; Feng, Zhihui; Wang, Ying; Weber, Peter; Wertz, Karin; Sharman, Edward; Liu, Jiankang

    2010-07-01

    Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1-10 micromol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1 alpha, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1 alpha by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1 alpha activation signaling pathway demonstrated that HT is an activator of 5'AMP-activated protein kinase and also up-regulates gene expression of PPAR alpha, CPT-1 and PPAR gamma. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis. (c) 2010 Elsevier Inc. All rights reserved.

  18. Cardiac overexpression of insulin-like growth factor 1 attenuates chronic alcohol intake-induced myocardial contractile dysfunction but not hypertrophy: Roles of Akt, mTOR, GSK3beta, and PTEN.

    PubMed

    Zhang, Bingfang; Turdi, Subat; Li, Quan; Lopez, Faye L; Eason, Anna R; Anversa, Piero; Ren, Jun

    2010-10-15

    Chronic alcohol intake leads to the development of alcoholic cardiomyopathy manifested by cardiac hypertrophy and contractile dysfunction. This study was designed to examine the effects of transgenic overexpression of insulin-like growth factor 1 (IGF-1) on alcohol-induced cardiac contractile dysfunction. Wild-type FVB and cardiac-specific IGF-1 mice were placed on a 4% alcohol or control diet for 16weeks. Cardiac geometry and mechanical function were evaluated by echocardiography and cardiomyocyte and intracellular Ca(2+) properties. Histological analyses for cardiac fibrosis and apoptosis were evaluated by Masson trichrome staining and TUNEL assay, respectively. Expression and phosphorylation of Cu/Zn superoxide dismutase (SOD1), Ca(2+) handling proteins, and key signaling molecules for survival including Akt, mTOR, GSK3beta, Foxo3a, and the negative regulator of Akt, phosphatase and tensin homolog on chromosome 10 (PTEN), as well as mitochondrial proteins UCP-2 and PGC1alpha, were evaluated by Western blot analysis. Chronic alcohol intake led to cardiac hypertrophy, interstitial fibrosis, reduced mitochondrial number, compromised cardiac contractile function and intracellular Ca(2+) handling, decreased SOD1 expression, elevated superoxide production, and overt apoptosis, all of which, with the exception of cardiac hypertrophy, were abrogated by the IGF-1 transgene. Immunoblotting data showed reduced phosphorylation of Akt, mTOR, GSK3beta, and Foxo3a; upregulated Foxo3a and PTEN; and dampened SERCA2a, PGC1alpha, and UCP-2 after alcohol intake. All these alcohol-induced changes in survival and mitochondrial proteins were alleviated by IGF-1. Taken together, these data favor a beneficial role for IGF-1 in alcohol-induced myocardial contractile dysfunction independent of cardiac hypertrophy. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Multiple roles for the active zone protein RIM1alpha in late stages of neurotransmitter release.

    PubMed

    Calakos, Nicole; Schoch, Susanne; Südhof, Thomas C; Malenka, Robert C

    2004-06-24

    The active zone protein RIM1alpha interacts with multiple active zone and synaptic vesicle proteins and is implicated in short- and long-term synaptic plasticity, but it is unclear how RIM1alpha's biochemical interactions translate into physiological functions. To address this question, we analyzed synaptic transmission in autaptic neurons cultured from RIM1alpha-/- mice. Deletion of RIM1alpha causes a large reduction in the readily releasable pool of vesicles, alters short-term plasticity, and changes the properties of evoked asynchronous release. Lack of RIM1alpha, however, had no effect on synapse formation, spontaneous release, overall Ca2+ sensitivity of release, or synaptic vesicle recycling. These results suggest that RIM1alpha modulates sequential steps in synaptic vesicle exocytosis through serial protein-protein interactions and that this modulation is the basis for RIM1alpha's role in synaptic plasticity. Copyright 2004 Cell Press

  20. Estrogen-related receptor {alpha} modulates the expression of adipogenesis-related genes during adipocyte differentiation

    SciTech Connect

    Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Yagi, Ken; Okazaki, Yasushi; Inoue, Satoshi . E-mail: INOUE-GER@h.u-tokyo.ac.jp

    2007-07-06

    Estrogen-related receptor {alpha} (ERR{alpha}) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERR{alpha} in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERR{alpha} and ERR{alpha}-related transcriptional coactivators, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) and PGC-1{beta}, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERR{alpha}-specific siRNA results in mRNA down-regulation of fatty acid binding protein 4, PPAR{gamma}, and PGC-1{alpha} in 3T3-L1 cells in the adipogenesis medium. ERR{alpha} and PGC-1{beta} mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERR{alpha} in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERR{alpha} may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.

  1. Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects.

    PubMed

    Danforth, B N; Ji, S

    1998-03-01

    We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.

  2. Gefitinib circumvents hypoxia-induced drug resistance by the modulation of HIF-1alpha.

    PubMed

    Rho, Jin Kyung; Choi, Yun Jung; Lee, Jin Kyung; Ryoo, Baek-Yeol; Na, Im Ii; Yang, Sung Hyun; Kim, Cheol Hyeon; Yoo, Young Do; Lee, Jae Cheol

    2009-03-01

    Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcriptional factor which is activated by hypoxia and associated with cell survival, proliferation and drug resistance. Recent studies have shown that the down-stream molecules of the epidermal growth factor receptor (EGFR) signal are involved in the hypoxia-dependent or -independent HIF-1alpha protein accumulation. Thus, we hypothesized that an EGFR-TK inhibitor, gefitinib, might circumvent the hypoxia-induced drug resistance via the regulation of HIF-1alpha expression. In our data, treatment of gefitinib suppressed induced HIF-1alpha by hypoxia. This action of gefitinib was caused by reduced protein stability without any change in the level of HIF-1alpha mRNA. The effect of gefitinib on down-regulation of HIF-1alpha was reversed by transfection of constitutively active form of Akt. The cellular response to gefitinib was similar in both normoxia and hypoxia condition. However, the response to conventional chemotherapeutic drugs decreased >50% under hypoxia condition and they did not change HIF-1alpha expression. In addition, the suppression of HIF-1alpha using siRNA overcame partially hypoxia-induced drug resistance. In conclusion, gefitinib was able to circumvent hypoxia-induced drug resistance suggesting that the effective suppression of HIF-1alpha by the inhibition of EGFR-Akt pathway may overcome the hypoxia-induced drug resistance.

  3. Regulation of protein kinase CK1alphaLS by dephosphorylation in response to hydrogen peroxide.

    PubMed

    Bedri, Shahinaz; Cizek, Stephanie M; Rastarhuyeva, Iryna; Stone, James R

    2007-10-15

    Low levels of hydrogen peroxide (H(2)O(2)) are mitogenic to mammalian cells and stimulate the hyperphosphorylation of heterogeneous nuclear ribonucleoprotein C (hnRNP-C) by protein kinase CK1alpha. However, the mechanisms by which CK1alpha is regulated have been unclear. Here it is demonstrated that low levels of H(2)O(2) stimulate the rapid dephosphorylation of CK1alphaLS, a nuclear splice form of CK1alpha. Furthermore, it is demonstrated that either treatment of endothelial cells with H(2)O(2), or dephosphorylation of CK1alphaLS in vitro enhances the association of CK1alphaLS with hnRNP-C. In addition, dephosphorylation of CK1alphaLS in vitro enhances the kinase's ability to phosphorylate hnRNP-C. While CK1alpha appears to be present in all metazoans, analysis of CK1alpha genomic sequences from several species reveals that the alternatively spliced nuclear localizing L-insert is unique to vertebrates, as is the case for hnRNP-C. These observations indicate that CK1alphaLS and hnRNP-C represent conserved components of a vertebrate-specific H(2)O(2)-responsive nuclear signaling pathway.

  4. Assessing functional divergence in EF-1alpha and its paralogs in eukaryotes and archaebacteria.

    PubMed

    Inagaki, Yuji; Blouin, Christian; Susko, Edward; Roger, Andrew J

    2003-07-15

    A number of methods have recently been published that use phylogenetic information extracted from large multiple sequence alignments to detect sites that have changed properties in related protein families. In this study we use such methods to assess functional divergence between eukaryotic EF-1alpha (eEF-1alpha), archaebacterial EF-1alpha (aEF-1alpha) and two eukaryote-specific EF-1alpha paralogs-eukaryotic release factor 3 (eRF3) and Hsp70 subfamily B suppressor 1 (HBS1). Overall, the evolutionary modes of aEF-1alpha, HBS1 and eRF3 appear to significantly differ from that of eEF-1alpha. However, functionally divergent (FD) sites detected between aEF-1alpha and eEF-1alpha only weakly overlap with sites implicated as putative EF-1beta or aminoacyl-tRNA (aa-tRNA) binding residues in EF-1alpha, as expected based on the shared ancestral primary translational functions of these two orthologs. In contrast, FD sites detected between eEF-1alpha and its paralogs significantly overlap with the putative EF-1beta and/or aa-tRNA binding sites in EF-1alpha. In eRF3 and HBS1, these sites appear to be released from functional constraints, indicating that they bind neither eEF-1beta nor aa-tRNA. These results are consistent with experimental observations that eRF3 does not bind to aa-tRNA, but do not support the 'EF-1alpha-like' function recently proposed for HBS1. We re-assess the available genetic data for HBS1 in light of our analyses, and propose that this protein may function in stop codon-independent peptide release.

  5. Variable patterns in the molecular evolution of the hypoxia-inducible factor-1 alpha (HIF-1alpha) gene in teleost fishes and mammals.

    PubMed

    Rytkönen, Kalle T; Ryynänen, Heikki J; Nikinmaa, Mikko; Primmer, Craig R

    2008-08-15

    The hypoxia-inducible factor-1 alpha (HIF-1alpha) protein is the major regulator of oxygen-dependent gene expression and a member of the bHLH-PAS family of transcription factors. In this study we compared and contrasted the rate and mode of HIF-1alpha molecular evolution between teleost fishes and mammals, as well as within teleost fishes and mammals. Various likelihood methods for estimating codon substitutions were used to detect different modes of selection. Although the overall evolutionary rate in teleost HIF-1alpha was at least twice as fast as in mammalian HIF-1alpha, the crucial interaction domains were observed to be under stringent negative selection in all vertebrates. Relaxed negative selection on less crucial regions of teleost HIF-1alpha compared to mammalian HIF-1alpha was detected, but no evidence for positive selection that was supported by all methods was found. We suggest that the relaxed selective constraints in teleost HIF-1alpha may be associated with the variable environmental oxygen levels to which teleosts have been exposed during their evolutionary history. However, in teleosts the positions with partial support for positive selection were not found in the vicinity of the HIF-1alpha domains which confer the oxygen sensitivity, but in the bHLH-PAS domain responsible for DNA binding and dimerization. The pattern of selection in the bHLH-PAS domain has some similarities with the patterns observed in the adaptive evolution of the homeodomain of Hox genes and may be typical in the molecular evolution of transcription factors.

  6. High-fat diets cause insulin resistance despite an increase in muscle mitochondria.

    PubMed

    Hancock, Chad R; Han, Dong-Ho; Chen, May; Terada, Shin; Yasuda, Toshihiro; Wright, David C; Holloszy, John O

    2008-06-03

    It has been hypothesized that insulin resistance is mediated by a deficiency of mitochondria in skeletal muscle. In keeping with this hypothesis, high-fat diets that cause insulin resistance have been reported to result in a decrease in muscle mitochondria. In contrast, we found that feeding rats high-fat diets that cause muscle insulin resistance results in a concomitant gradual increase in muscle mitochondria. This adaptation appears to be mediated by activation of peroxisome proliferator-activated receptor (PPAR)delta by fatty acids, which results in a gradual, posttranscriptionally regulated increase in PPAR gamma coactivator 1alpha (PGC-1alpha) protein expression. Similarly, overexpression of PPARdelta results in a large increase in PGC-1alpha protein in the absence of any increase in PGC-1alpha mRNA. We interpret our findings as evidence that raising free fatty acids results in an increase in mitochondria by activating PPARdelta, which mediates a posttranscriptional increase in PGC-1alpha. Our findings argue against the concept that insulin resistance is mediated by a deficiency of muscle mitochondria.

  7. Pyrithione-zinc Prevents UVB-induced Epidermal Hyperplasia by Inducing HIF-1alpha.

    PubMed

    Cho, Young-Suk; Lee, Kyung-Hoon; Park, Jong-Wan

    2010-04-01

    Epidermal keratinocytes overgrow in response to ultraviolet-B (UVB), which may be associated with skin photoaging and cancer development. Recently, we found that HIF-1alpha controls the keratinocyte cell cycle and thereby contributes to epidermal homeostasis. A further study demonstrated that HIF-1alpha is down-regulated by UVB and that this process is involved in UVB-induced skin hyperplasia. Therefore, we hypothesized that the forced expression of HIF-1alpha in keratinocytes would prevent UVB-induced keratinocyte overgrowth. Among several agents known to induce HIF-1alpha, pyrithione-zinc (Py-Zn) overcame the UVB suppression of HIF-1alpha in cultured keratinocytes. Mechanistically, Py-Zn blocked the degradation of HIF-1alpha protein in keratinocytes, while it did not affect the synthesis of HIF-1alpha. Moreover, the p21 cell cycle inhibitor was down-regulated after UVB exposure, but was robustly induced by Py-Zn. In mice repeatedly irradiated with UVB, the epidermis became hyperplastic and HIF-1alpha disappeared from nuclei of epidermal keratinocytes. However, a cream containing Py-Zn effectively prevented the skin thickening and up-regulated HIF-1alpha to the normal level. These results suggest that Py-Zn is a potential agent to prevent UVB-induced photoaging and skin cancer development. This work also provides insight into a molecular target for treatment of UVB-induced skin diseases.

  8. Protein synthesis elongation factor EF-1 alpha expression and longevity in Drosophila melanogaster.

    PubMed Central

    Shikama, N; Ackermann, R; Brack, C

    1994-01-01

    It has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in elongation factor EF-1 alpha. Transgenic Drosophila melanogaster flies carrying an additional copy of the EF-1 alpha gene under control of a heat-inducible promoter have an extended lifespan, further indicating that the EF-1 alpha gene may play an important role in determining longevity. To test this hypothesis, we have quantitated EF-1 alpha mRNA, EF-1 alpha protein, and the EF-1 alpha complex-formation activity in these transgenic flies. Furthermore, we have tested whether the transgene construct is functional--i.e., whether transgenic mRNA is induced when flies are grown at higher temperature. The results show that although there is a clear difference in mean lifespan between the EF-1 alpha transgenic (E) flies and the control transgenic (C) flies, E flies do not express more EF-1 alpha protein or mRNA than C flies kept at the same experimental conditions. Although the transgene can be induced when E flies are heat-shocked at 37 degrees C, transgenic mRNA is not detectable in E flies aged at 29 degrees C. In both lines, the loss in catalytic activity with age is the same. We conclude that the E flies examined here do not live longer because of overexpressing the EF-1 alpha gene. Images PMID:8183891

  9. Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

  10. EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells.

    PubMed Central

    Clore, A. M.; Dannenhoffer, J. M.; Larkins, B. A.

    1996-01-01

    By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei. PMID:12239373

  11. Interaction between HP1{alpha} and replication proteins in mammalian cells

    SciTech Connect

    Auth, Tanja . E-mail: tauth@uni-bonn.de; Kunkel, Elisabeth; Grummt, Friedrich . E-mail: grummt@biozentrum.uni-wuerzburg.de

    2006-10-15

    HP1 is an essential heterochromatin-associated protein known to play an important role in the organization of heterochromatin as well as in the transcriptional regulation of heterochromatic and euchromatic genes both in repression and activation. Using the yeast two-hybrid system and immunoprecipitation, we report here that murine HP1{alpha} interacts with the preRC proteins ORC1, ORC2 and CDC6. Immunofluorescence staining and EGFP/DsRed fusion proteins revealed a colocalization of HP1{alpha} with ORC1, ORC2 and CDC6 in heterochromatin, supporting the notion that ORC and probably CDC6 play an important role in murine HP1{alpha} function. Besides that, we also observed a colocalization of HP1{alpha} with {gamma}-tubulin suggesting a centrosomal localization of HP1{alpha} in murine cells. To gain insight into HP1{alpha} function, we applied the RNAi technique. Depletion of HP1{alpha} leads to a slow down of cell proliferation, an aberrant cell cycle progression as well as to multinucleated cells with insufficiently organized microtubule. These results together indicate that HP1{alpha} exerts functions in mitosis and cytokinesis.

  12. Castration Therapy of Prostate Cancer Results in Downregulation of HIF-1{alpha} Levels

    SciTech Connect

    Al-Ubaidi, Firas L.T.; Schultz, Niklas; Egevad, Lars; Granfors, Torvald; Helleday, Thomas

    2012-03-01

    Background and Purpose: Neoadjuvant androgen deprivation in combination with radiotherapy of prostate cancer is used to improve radioresponsiveness and local tumor control. Currently, the underlying mechanism is not well understood. Because hypoxia causes resistance to radiotherapy, we wanted to test whether castration affects the degree of hypoxia in prostate cancer. Methods and Materials: In 14 patients with locally advanced prostate cancer, six to 12 prostatic needle core biopsy specimens were taken prior to castration therapy. Bilateral orchidectomy was performed in 7 patients, and 7 were treated with a GnRH-agonist (leuprorelin). After castrationm two to four prostatic core biopsy specimens were taken, and the level of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in cancer was determined by immunofluorescence. Results: Among biopsy specimens taken before castration, strong HIF-1{alpha} expression (mean intensity above 30) was shown in 5 patients, weak expression (mean intensity 10-30) in 3 patients, and background levels of HIF-1{alpha} (mean intensity 0-10) in 6 patients. Downregulation of HIF-1{alpha} expression after castration was observed in all 5 patients with strong HIF-1{alpha} precastration expression. HIF-1{alpha} expression was also reduced in 2 of 3 patients with weak HIF-1{alpha} precastration expression. Conclusions: Our data suggest that neoadjuvant castration decreases tumor cell hypoxia in prostate cancer, which may explain increased radiosensitivity after castration.

  13. Fruit flies with additional expression of the elongation factor EF-1 alpha live longer.

    PubMed Central

    Shepherd, J C; Walldorf, U; Hug, P; Gehring, W J

    1989-01-01

    In Drosophila melanogaster, the decrease in protein synthesis that accompanies aging is preceded by a decrease in elongation factor EF-1 alpha protein and mRNA. Here we show that Drosophila transformed with a P-element vector containing an EF-1 alpha gene under control of hsp70 regulatory sequences have a longer life-span than control flies. Images PMID:2508089

  14. Distribution of elongation factor-1alpha in larval tissues of the fall armyworm, Spodoptera frugiperda.

    PubMed

    Habibi, Javad; Goodman, Cynthia L; Stuart, Melissa K

    2006-01-01

    Elongation factor-1alpha (EF-1alpha) promotes the delivery of aminoacyl-tRNA to the acceptor site of the ribosome during protein synthesis. The enzyme has a number of additional functions, including regulation of apoptosis and interaction with the cytoskeleton. We determined the distribution of EF-1alpha in larval tissues of the fall armyworm, Spodoptera frugiperda , with a monoclonal antibody generated to EF-1alpha from Sf21 cells, a cell line developed from ovarian tissue of S. frugiperda. Enzyme-linked immunosorbent assay showed that EF-1alpha comprised 1.9-9.9% of the total protein within the tissues that were examined, which included fat body, Malpighian tubules, midgut, muscle, salivary glands, trachea, and ventral nerve cord. To a certain extent, EF-1alpha concentrations reflected the expected metabolic activity level of each of the represented tissues. Closer examination by immunofluorescence microscopy revealed that EF-1alpha concentrations varied among different cell types within a given tissue, i.e. midgut columnar epithelial cells yielded strong signals, while goblet cells failed to react with the EF-1alpha-specific antibody.

  15. MIP-1alpha as a critical macrophage chemoattractant in murine wound repair.

    PubMed Central

    DiPietro, L A; Burdick, M; Low, Q E; Kunkel, S L; Strieter, R M

    1998-01-01

    At sites of injury, macrophages secrete growth factors and proteins that promote tissue repair. While this central role of the macrophage has been well studied, the specific stimuli that recruit macrophages into sites of injury are not well understood. This study examines the role of macrophage inflammatory protein 1alpha (MIP-1alpha), a C-C chemokine with monocyte chemoattractant capability, in excisional wound repair. Both MIP-1alpha mRNA and protein were detectable in murine wounds from 12 h through 5 d after injury. MIP-1alpha protein levels peaked 3 d after injury, coinciding with maximum macrophage infiltration. The contribution of MIP-1alpha to monocyte recruitment into wounds was assessed by treating mice with neutralizing anti-MIP-1alpha antiserum before injury. Wounds of mice treated with anti-MIP-1alpha antiserum had significantly fewer macrophages than control (41% decrease, P < 0. 01). This decrease in wound macrophages was paralleled by decreased angiogenic activity and collagen synthesis. When tested in the corneal micropocket assay, wound homogenates from mice treated with anti-MIP-1alpha contained significantly less angiogenic activity than control wound homogenates (27% positive for angiogenic activity versus 91% positive in the control group, P < 0.01). Collagen production was also significantly reduced in the wounds from anti-MIP-1alpha treated animals (29% decrease, P < 0.05). The results demonstrate that MIP-1alpha plays a critical role in macrophage recruitment into wounds, and suggest that appropriate tissue repair is dependent upon this recruitment. PMID:9541500

  16. Sirt1 is involved in energy metabolism: the role of chronic ethanol feeding and resveratrol.

    PubMed

    Oliva, Joan; French, Barbara A; Li, Jun; Bardag-Gorce, Fawzia; Fu, Paul; French, Samuel W

    2008-12-01

    Sirt1, a deacetylase involved in regulating energy metabolism in response to calorie restriction, is up regulated after chronic ethanol feeding using the intragastric feeding model of alcohol liver disease. PGC1 alpha is also up regulated in response to ethanol. These changes are consistent with activation of the Sirt1/PGC1 alpha pathway of metabolism and aging, involved in alcohol liver disease including steatosis, necrosis and fibrosis of the liver. To test this hypothesis, male rats fed ethanol intragastrically for 1 month were compared with rats fed ethanol plus resveratrol or naringin. Liver histology showed macrovesicular steatosis caused by ethanol and this change was unchanged by resveratrol or naringin treatment. Necrosis occurred with ethanol alone but was accentuated by resveratrol treatment, as was fibrosis. The expression of Sirt1 and PGC1 alpha was increased by ethanol but not when naringin or resveratrol was fed with ethanol. Sirt3 was also up regulated by ethanol but not when resveratrol was fed with ethanol. These results support the concept that ethanol induces the Sirt1/PGC1 alpha pathway of gene regulation and both naringin and resveratrol prevent the activation of this pathway by ethanol. However, resveratrol did not reduce the liver pathology caused by chronic ethanol feeding.

  17. In vino veritas: a tale of two sirt1s?

    PubMed

    Koo, Seung-Hoi; Montminy, Marc

    2006-12-15

    Resveratrol increases life span in lower organisms by activating the NAD(+)-dependent histone deacetylase Sirt1. Studies by and now show that resveratrol promotes longevity and improves glucose homeostasis in mice by stimulating the Sirt1-mediated deacetylation of the transcriptional coactivator PGC-1alpha.

  18. A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1{alpha}-mediated signaling

    SciTech Connect

    Hsieh, Ming-Chu; Hu, Wan-Ping; Yu, Hsin-Su; Wu, Wen-Chuan; Chang, Long-Sen; Kao, Ying-Hsien; Wang, Jeh-Jeng

    2011-09-01

    Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1{alpha} (SDF-1{alpha}) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1{alpha}-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1{alpha}-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1{alpha}-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency. - Research Highlights: > A novel carboxylate-PBD hybrid as anti-melanoma drug. > IN4CPBD interrupts melanoma cell

  19. NF-{kappa}B suppresses HIF-1{alpha} response by competing for P300 binding

    SciTech Connect

    Mendonca, Daniela B.S.; Mendonca, Gustavo; Aragao, Francisco J.L.; Cooper, Lyndon F.

    2011-01-28

    Research highlights: {yields} p65 completely blocked HIF-1{alpha} activity at the HRE on different cell lines. {yields} p65 caused minor changes in HIF-1{alpha} and HIF-1{alpha} target genes mRNA expression. {yields} p65 reduced transcription of VEGF promoter. {yields} p65 competes with HIF-1{alpha} for p300. -- Abstract: Hypoxia has emerged as a key determinant of osteogenesis. HIF-1{alpha} is the transcription factor mediating hypoxia responses that include induction of VEGF and related bone induction. Inflammatory signals antagonize bone repair via the NF-{kappa}B pathway. The present investigation explored the functional relationship of hypoxia (HIF-1{alpha} function) and inflammatory signaling (NF-{kappa}B) in stem like and osteoprogenitor cell lines. The potential interaction between HIF-1{alpha} and NF-{kappa}B signaling was explored by co-transfection studies in hFOB with p65, HIF-1{alpha} and 9x-HRE-luc or HIF-1{alpha} target genes reporter plasmids. Nuclear cross-talk was directly tested using the mammalian Gal4/VP16 two-hybrid, and confirmed by co-immunoprecipitation/western blotting assays. The results show that inflammatory stimulation (TNF-{alpha} treatment) causes a marked inhibition of HIF-1{alpha} function at the HRE in all cell lines studied. Also, co-transfection with p65 expression vector leads to reduced hVEGFp transcription after DFO-induced hypoxia. However, TNF-{alpha} treatment had little effect on HIF-1{alpha} mRNA levels. The functional interaction of Gal4-HIF-1{alpha} and VP16-p300 fusion proteins is effectively blocked by expression of p65 in a dose dependent manner. It was concluded that NF-{kappa}B-mediated inflammatory signaling is able to block HIF-1{alpha} transactivation at HRE-encoding genes by direct competition for p300 binding at the promoter. Inflammation may influence the stem cell niche and tissue regeneration by influencing cellular responses to hypoxia.

  20. Two genes encode related cytoplasmic elongation factors 1 alpha (EF-1 alpha) in Drosophila melanogaster with continuous and stage specific expression.

    PubMed Central

    Hovemann, B; Richter, S; Walldorf, U; Cziepluch, C

    1988-01-01

    We have characterized two previously cloned genes, F1 and F2 (1) that code for elongation factor EF - 1 alpha of Drosophila melanogaster. Genomic Southern blot hybridization revealed that they are the only gene copies present. We isolated cDNA clones of both transcripts from embryonal and pupal stage of development that cover the entire transcription unit. The 5' ends of both genes have been determined by primer extension and for F1 also by RNA sequencing. These start sites have been shown to be used consistently during development. Comparison of cDNA and genomic sequences revealed that EF - 1 alpha,F1 consists of two and EF - 1 alpha,F2 of five exons. The two described elongation factor genes exhibit several regions of strong sequence conservation when compared to five recently cloned eucaryotic elongation factors. Images PMID:3131735

  1. Immunohistochemical expression of HIF-1alpha in response to early myocardial ischemia.

    PubMed

    Blanco Pampín, José; García Rivero, Sonia Aranzazu; Otero Cepeda, Xosé Luis; Vázquez Boquete, Angel; Forteza Vila, Jerónimo; Hinojal Fonseca, Rafael

    2006-01-01

    This study aims to evaluate the effects of ischemia on the myocardial fibers and the expression of the transcriptional factor for angiogenesis hypoxia-inducible factor-1 alpha (HIF-1alpha) in human heart specimens. We have prospectively analyzed the HIF-1alpha expression in human ischemic hearts with the ABC-inmunohistochemistry technique and amplification by biotinylated tyramide. The relationship between the expression of HIF-1alpha and the temporal evolution of ischemia has also been evaluated. As pathomorphological diagnosis of early myocardial ischemia has many problems in human autopsy material with less than 4 to 6 h after clinical onset, we suggest that HIF-1alpha is helpful in the early acute myocardial infarction diagnosis, so it stains necrotic areas within the first 2 h. The amplification procedure provides a higher intensity of the final staining without losing specificity. It is concluded that in normal cardiac fibers, basal expression of HIF-1alpha is not appreciable, but it steadily increases after ischemia. With regard to the practical applicability in forensic field, our observations suggest that positive immunohistochemical expression of HIF-1alpha on heart samples may be used as a reliable indicator of myocardial damage in cases without cardiac lesion evidence, using conventional microscopy. This method is especially useful and may provide definitive proof of myocardial ischemia in unexpected deaths without previous symptoms, or in forensic cases with a short period of clinical manifestations. In addition, it may have been involved in possible future cardiovascular therapies.

  2. Effects of continuously administered murine interleukin-1 alpha: tolerance development and granuloma formation.

    PubMed Central

    Otterness, I G; Golden, H W; Brissette, W H; Seymour, P A; Daumy, G O

    1989-01-01

    Continuous infusion of murine recombinant interleukin-1 alpha (rIL-1 alpha) into rats by using intraperitoneally implanted osmotic pumps led to marked decreases in body weight, liver enzymes (serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, and sorbitol dehydrogenase), appetite, and mobility and increases in drinking, blood urea nitrogen, and total peripheral blood leukocytes within 3 days. Granuloma formation was found in the local area of rIL-1 alpha release. As early as day 3, a focal infiltrate of polymorphonuclear leukocytes, mononuclear leukocytes, and plasma cells filled the area; by day 6, extensive fibrosis was found. A loss of rIL-1 alpha-induced changes, with the exception of granuloma formation, occurred by day 10. A marked decrease in the response to rIL-1 alpha was also observed when animals were challenged by implantation of new pumps containing rIL-1 alpha, with monitoring of body weight, or by subcutaneous injection of rIL-1 alpha, with monitoring of serum colony-stimulating factor production. We propose that, even in the continuous presence of interleukin-1, replacement of the acute responses to interleukin-1 by restoration of more normal physiology may be advantageous upon acquisition of specific immunity. Images PMID:2788137

  3. Polymorphism of IL-1alpha(-889) Gene and Its Association with Aggressive Periodontitis.

    PubMed

    Kiani, Zahra; Tavakkol-Afshari, Jalil; Hojjat, Hamed; Arab, Hamid Reza; Radvar, Mehrdad; Sadeghizadeh, Mehrdad; Ebadian, Ahmad Reza

    2009-06-01

    Adult periodontitis is a complex multifactorial disease whose etiology is not well defined. The pro-inflammatory and bone resorption properties of Interleukine-1alpha (IL-1alpha) strongly suggest a role for this cytokine in the pathogenesis of periodontal disease. Eighty Iranian adult patients with periodontitis and 80 Iranian controls were investigated in this study.In this study we report that the frequency of IL-1alpha genotypes including allele 2 of the IL-1alpha(-889) restriction fragment length bi-allele polymorphism were significantly increased in patients with advanced aggressive adult periodontitis compared to those with early and moderate disease. Furthermore, allele 2 was associated with increased production of IL-1alpha by activated peripheral blood polymorphonuclear cells of patients with advanced disease, although this increase failed to reach statistical significance. Finally, the data obtained revealed significant linkage disequilibrium between allele 2 of the IL-1alpha (-889) polymorphism and allele 2 of the bi-allelic IL-1beta (+3953) polymorphism in both patients and orally normal controls. These findings provide new insight into the possible rate of IL-1alpha and beta genes polymorphism in the susceptibility to adult periodontitis.

  4. Interleukin-1 alpha has antiallodynic and antihyperalgesic activities in a rat neuropathic pain model.

    PubMed

    Mika, Joanna; Korostynski, Michal; Kaminska, Dorota; Wawrzczak-Bargiela, Agnieszka; Osikowicz, Maria; Makuch, Wioletta; Przewlocki, Ryszard; Przewlocka, Barbara

    2008-09-15

    Nerve injury and the consequent release of interleukins (ILs) are processes implicated in pain transmission. To study the potential role of IL-1 in the pathogenesis of allodynia and hyperalgesia, IL-1alpha and comparative IL-1beta, IL-6, and IL-10 mRNA levels were quantified using competitive RT-PCR of the lumbar spinal cord and dorsal root ganglia (DRG; L5-L6) three and seven days after chronic constriction injury (CCI) in rats. Microglial and astroglial activation in the ipsilateral spinal cord and DRG were observed after injury. In naive and CCI-exposed rats, IL-1alpha mRNA and protein were not detected in the spinal cord. IL-1beta and IL-6 mRNAs were strongly ipsilaterally elevated on day seven after CCI. In the ipsilateral DRG, IL-1alpha, IL-6, and IL-10 mRNA levels were increased on days three and seven; IL-1beta was elevated only on day seven. Western blot analysis revealed both the presence of IL-1alpha proteins (45 and 31 kDa) in the DRG and the down-regulation of these proteins after CCI. Intrathecal administration of IL-1alpha (50-500 ng) in naive rats did not influence nociceptive transmission, but IL-1beta (50-500 ng) induced hyperalgesia. In rats exposed to CCI, an IL-1alpha or IL-1 receptor antagonist dose-dependently attenuated symptoms of neuropathic pain; however, no effect of IL-1beta was observed. In sum, the first days after CCI showed a high abundance of IL-1alpha in the DRG. Together with the antiallodynic and antihyperalgesic effects observed after IL-1alpha administration, this finding indicates an important role for IL-1alpha in the development of neuropathic pain symptoms.

  5. Synergistic anti-Parkinsonism activity of high doses of B vitamins in a chronic cellular model.

    PubMed

    Jia, Haiqun; Liu, Zhongbo; Li, Xin; Feng, Zhihui; Hao, Jiejie; Li, Xuesen; Shen, Weili; Zhang, Hongyu; Liu, Jiankang

    2010-04-01

    We propose that elevation of mitochondrial enzyme cofactors may prevent or ameliorate neurodegenerative diseases by improving mitochondrial function. In the present study, we investigated the effects of high doses of B vitamins, the precursors of mitochondrial enzyme cofactors, on mitochondrial dysfunction, oxidative stress, and Parkinsonism in a 4-week long rotenone treatment-induced cellular model of Parkinson's disease (PD). Pretreatment with B vitamins (also 4 weeks) prevented rotenone-induced: (1) mitochondrial dysfunction, including reduced mitochondrial membrane potential and activities of complex I; (2) oxidative stress, including increase in reactive oxygen species, oxidative DNA damage and protein oxidation, and (3) Parkinsonism parameters, including accumulation of alpha-synuclein and poly-ubiquitin. The optimum doses were found around 2.5- and 5-fold of that in normal MEM medium. The 4-week pretreatment was chosen based on time-dependent experiments that pretreatments longer than 2 weeks resulted in a decrease in oxidants, an increase in oxygen consumption, and up-regulation of complex I activity and PGC-1alpha expression. Individual B vitamins at the same doses did not show a similar effect suggesting that these B vitamins work synergistically. These results suggest that administration of high doses of B vitamins sufficient to elevate mitochondrial enzyme cofactors may be effective in preventing PD by reducing oxidative stress and improving mitochondrial function.

  6. Combination effect of recombinant human interleukin-1 alpha with antimicrobial agents.

    PubMed Central

    Nakamura, S; Minami, A; Fujimoto, K; Kojima, T

    1989-01-01

    Combination effects of recombinant human interleukin-1 alpha with ceftazidime, moxalactam, gentamicin, enoxacin, amphotericin B, miconazole, or an immunoglobulin preparation were evaluated in systemic infections with Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans in normal mice and systemic infection with P. aeruginosa in mice with leukopenia induced by preadministration of cyclophosphamide. Synergistic effects were generally observed at interleukin-1 alpha doses as low as 1 to 30 ng per mouse with most combinations. The results show the possibility that recombinant human interleukin-1 alpha could be of help for treating obstinate infections not successfully treated with antimicrobial agents alone. PMID:2589847

  7. Macrophage inflammatory protein-1 alpha. A novel chemotactic cytokine for macrophages in rheumatoid arthritis.

    PubMed Central

    Koch, A E; Kunkel, S L; Harlow, L A; Mazarakis, D D; Haines, G K; Burdick, M D; Pope, R M; Strieter, R M

    1994-01-01

    We have shown that human macrophages (m phi s) play an important role in the elaboration of chemotactic cytokines in rheumatoid arthritis (RA) (Koch, A. E., S. L. Kunkel, J. C. Burrows, H. L. Evanoff, G. K. Haines, R. M. Pope, and R. M. Strieter. 1991. J. Immunol. 147:2187; Koch, A. E., S. L. Kunkel, L. A. Harlow, B. Johnson, H. L. Evanoff, G. K. Haines, M. D. Burdick, R. M. Pope, and R. M. Strieter. 1992. J. Clin. Invest. 90:772; Koch, A. E., P. J. Polverini, S. L. Kunkel, L. A. Harlow, L. A. DiPietro, V. M. Elner, S. G. Elner, and R. M. Strieter. 1992. Science (Wash. DC). 258:1798). Recently, m phi inflammatory protein-1 (MIP-1 alpha), a cytokine with chemotactic activity for m phi s and neutrophils (PMNs), has been described. We have examined the production of MIP-1 alpha using sera, synovial fluid (SF), and synovial tissue (ST) from 63 arthritic patients. MIP-1 alpha was higher in RA SF (mean, 29 +/- 8 ng/ml [SE]) compared with other forms of arthritis (2.8 +/- 1.7), or osteoarthritis (0.7 +/- 0.4; P < 0.05). RA SF MIP-1 alpha was greater than that found in either RA or normal peripheral blood (PB) (P < 0.05). Anti-MIP-1 alpha neutralized 36 +/- 3% (mean +/- SE) of the chemotactic activity for m phi s, but not PMNs, found in RA SFs. RA SF and PB mononuclear cells produced antigenic MIP-1 alpha. Mononuclear cell MIP-1 alpha production was augmented with phytohemagglutinin or LPS. Isolated RA ST fibroblast production of antigenic MIP-1 alpha was augmented upon incubation of cells with LPS, and to a lesser extent with tumor necrosis factor-alpha. Isolated RA ST m phi s expressed constitutive MIP-1 alpha mRNA and antigenic MIP-1 alpha. Using ST immunohistochemistry, MIP-1 alpha+ cells from RA compared with normal were predominantly m phi s and lining cells (P < 0.05). These results suggest that MIP-1 alpha plays a role in the selective recruitment of m phi s in synovial inflammation associated with RA. Images PMID:8132778

  8. Roles for the heliodynamic hormones, all trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3, in control of the hematopoietic cell cycle.

    PubMed

    Blazsek, I; Comisso, M; Farabos, C; Misset, J L

    1991-01-01

    It is now well established that the production of primary hematopoietic cells is controlled at different levels of the biological organization. Bone marrow (BM) stromal cells, the extracellular matrix (ECM), polypeptide hematopoietic growth factors (HGF) as well as endogenous cell-division cycle (CDC) related factors play a dominant role in this control. Recent information suggest that the 2 lipophilic hormones, transRA and 1 alpha,25D3, depending on and/or perhaps mediating solar energy, play a role in the maintenance of BM homeostasis. Here we show that both transRA and 1 alpha,25D3: a) modulate the growth and/or stimulate the adipocytic differentiation of fibroblastic stromal cells (F-CFU); b) inhibit the synthesis and extracellular processing but stimulate the solubilization of matrix collagen; c) modulate the clonal growth of myeloid progenitor cells (GM-CFU) in synergy with HGFs; and d) inhibit the production of lactic acid in standard, normal long-term BM cultures (LTBMC). Comparative analysis of normal, preleukemic and leukemic BM cells in LTBMC indicated a positive correlation between the induction of terminal differentiation and reduced lactate production elicited by transRA or 1 alpha,25D3. These results raise a hypothesis according to which the terminal differentiation induced by the helicodynamic hormones is dependent on the mitochondrial aerobic ATP-generating system whose impairment may be a critical step during the process of leukemic transformation.

  9. Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

    PubMed Central

    Habben, J E; Moro, G L; Hunter, B G; Hamaker, B R; Larkins, B A

    1995-01-01

    Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals. Images Fig. 1 Fig. 2 PMID:7567989

  10. IL-1 alpha (-889) promoter polymorphism is a risk factor for osteomyelitis.

    PubMed

    Asensi, Víctor; Alvarez, Victoria; Valle, Eulalia; Meana, Alvaro; Fierer, Joshua; Coto, Eliecer; Carton, José Antonio; Maradona, José Antonio; Paz, José; Dieguez, Maria Angeles; de la Fuente, Belén; Moreno, Alfonso; Rubio, Silvino; Tuya, Maria José; Sarasúa, Julián; Llames, Sara; Arribas, José Manuel

    2003-06-01

    As osteomyelitis (OM) induces the synthesis of inflammatory cytokines and IL-1 mediates bone resorption by osteoclasts we determined if there is an association between certain common polymorphisms of the genes encoding proinflammatory cytokines (IL-1 alpha and beta, IL-6, TNF-alpha) and OM in adults. The IL-1 alpha (-889) TT genotype was significantly more frequent among 52 OM patients than in 109 healthy controls (13/52, [25.0%] vs. 9/109, [8.3%], P = 0.0081, chi(2) = 7.01, OR = 3.7, 95% CI, 1.35-10.34). Patients who were homozygous for the T allele were younger than the rest of the OM patients (mean age 35.7 +/- 11.5 vs. 58.1 +/- 18.6 years, P = 0.001). IL-1 beta TT (+3953) polymorphism was also more frequent in OM patients (P = 0.014, chi(2) = 5.12, OR = 5.1, 95% CI, 1.21-52.14), but IL-1 beta is in linkage disequilibrium with the IL-1 alpha *T (P < 0.001). Route of infection, chronicity of the infection, type of microorganism isolated, and frequency of relapses were similar in patients with and without the IL-1 alpha TT genotype. There were no associations between OM and polymorphisms of other cytokines genes. IL-1 alpha serum levels were significantly increased in all the OM patients independently of their IL-1 genotype compared to the controls (P = 0.021). Although IL-1 alpha serum levels were not significantly higher in patients with the IL-1 alpha (-889) polymorphism, this does not exclude a difference in production of IL-1 alpha by osteoclasts or other inflammatory cells at the site of infection.

  11. Hypoxia-inducible factor-1alpha signaling in aquaporin upregulation after traumatic brain injury.

    PubMed

    Ding, Jamie Y; Kreipke, Christian W; Speirs, Susan L; Schafer, Patrick; Schafer, Steven; Rafols, José A

    2009-03-27

    Previous studies have demonstrated that traumatic brain injury (TBI) causes brain edema via aquaporins (AQPs), the water-transporting proteins. In the present study, we determined the role of hypoxia inducible factor-1alpha (HIF-1alpha), which is a transcription factor in response to physiological hypoxia, in regulating expression of AQP4 and AQP9. Adult male Sprague-Dawley rats (400-425g) received a closed head injury using the Marmarou weight drop model with a 450g weight and survived for 1, 4, 24 and 48h. Some animals were administered 30min after injury with 2-methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol which is known to post-transcriptionally down-regulate HIF-1alpha expression, and sacrificed 4h after injury. Real-time PCR and Western blot were used, respectively, to detect gene and protein expressions of manganese superoxide dismutase (MnSOD, showing hypoxic stress), HIF-1alpha, AQP4, and AQP9. ANOVA analysis demonstrated a significant (p<0.05) increase in gene expression of MnSOD, HIF-1alpha, AQP4, and AQP9, starting at 1h after injury through 48h. Western blot analysis further indicated a significant (p<0.05) increase in protein expression of these molecules at the same time points. Pharmacological inhibition of HIF-1alpha by 2ME2 reduced the up-regulated levels of AQP4 and AQP9 after TBI. The present study suggests that hypoxic conditions determined by MnSOD expression after closed head injury contribute to HIF-1alpha expression. HIF-1alpha, in turn, up-regulates expression of AQP4 and AQP9. These results characterize the pathophysiological mechanisms, and suggest possible therapeutic targets for TBI patients.

  12. Hypoxic regulation of VEGF, HIF-1(alpha) in coronary collaterals development.

    PubMed

    Sung, Ki Chul; Kim, Kyung Soo; Lee, Sang

    2005-12-01

    The interindividual variability for the development of collaterals in coronary artery disease is dependent on the hypoxic induction level of VEGF. To determine whether the hypoxic induction of VEGF is controlled by the transcription of HIF-1 (alpha), the VEGF and HIF-1 (alpha) m-RNA levels were correlated to hypoxia in monocytes harvested from patients with coronary artery disease. The collateral scoring system used was modified from the TIMI system. The mononuclear cell layer of the patients' blood was cultured in hypoxia (1% O2, 5% CO2, 94%N2) and normoxia (5% CO2, 95% room air) for 17 hours. The VEGF and HIF-1 (alpha) mRNA levels were measured using a RT-PCR technique. We calculated the fold inductionsof VEGF, HIF-1 (alpha) mRNA with hypoxia by dividing the hypoxic and the normoxic values. We found significantly higher hypoxic inductions of VEGF m-RNA in patients with collaterals compared to patients with no collaterals. However, there was no differencein the hypoxic inductions of HIF-1 (alpha) between the two groups (VEGF m-RNA mean fold inductions 3.71 +/- 3.30 versus 1.65 +/- 0.62, p=0.012, HIF-1(alpha) mRNA 1.42 +/- 0.58 versus 1.20 +/- 0.39, p=0.165). We concluded that the interindividual variability in the hypoxic inductions of VEGF m-RNA in monocytes in patients is not controlled by the transcriptional levels of HIF-1 (alpha) with hypoxia. These findings suggest that a mechanism such as the post-transcriptional modification of HIF-1(alpha) is involved in the hypoxic inductions of VEGF.

  13. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha.

    PubMed

    Elson, D A; Thurston, G; Huang, L E; Ginzinger, D G; McDonald, D M; Johnson, R S; Arbeit, J M

    2001-10-01

    Hypoxia-inducible factor-1alpha (HIF-1alpha) transactivates genes required for energy metabolism and tissue perfusion and is necessary for embryonic development and tumor explant growth. HIF-1alpha is overexpressed during carcinogenesis, myocardial infarction, and wound healing; however, the biological consequences of HIF-1alpha overexpression are unknown. Here, transgenic mice expressing constitutively active HIF-1alpha in epidermis displayed a 66% increase in dermal capillaries, a 13-fold elevation of total vascular endothelial growth factor (VEGF) expression, and a six- to ninefold induction of each VEGF isoform. Despite marked induction of hypervascularity, HIF-1alpha did not induce edema, inflammation, or vascular leakage, phenotypes developing in transgenic mice overexpressing VEGF cDNA in skin. Remarkably, blood vessel leakage resistance induced by HIF-1alpha overexpression was not caused by up-regulation of angiopoietin-1 or angiopoietin-2. Hypervascularity induced by HIF-1alpha could improve therapy of tissue ischemia.

  14. Whole-body irradiation transiently diminishes the adrenocorticotropin response to recombinant human interleukin-1{alpha}

    SciTech Connect

    Perlstein, R.S.; Mehta, N.R.; Neta, R.; Whitnall, M.H.; Mougey, E.H.

    1995-03-01

    Recombinant human interleukin-1{alpha} (rhIL-1{alpha}) has significant potential as a radioprotector and/or treatment for radiation-induced hematopoietic injury. Both IL-1 and whole-body ionizing irradiation acutely stimulate the hypothalamic-pituitary-adrenal axis. We therefore assessed the interaction of whole-body irradiation and rhIL-1{alpha} in altering the functioning of the axis in mice. Specifically, we determined the adrenocorticotropin (ACTH) and corticosterone responses to rhIL-1{alpha} administered just before and hours to days after whole-body or sham irradiation. Our results indicate that whole-body irradiation does not potentiate the rhIL-1{alpha}-induced increase in ACTH levels at the doses used. In fact, the rhIL-1{alpha}-induced increase in plasma ACTH is transiently impaired when the cytokine is administered 5 h after, but not 1 h before, exposure to whole-body irradiation. The ACTH response may be inhibited by elevated corticosterone levels after whole-body irradiation, or by other radiation-induced effects on the pituitary gland and hypothalamus. 36 refs., 3 figs.

  15. HNF1alpha is involved in tissue-specific regulation of CFTR gene expression.

    PubMed Central

    Mouchel, Nathalie; Henstra, Sytse A; McCarthy, Victoria A; Williams, Sarah H; Phylactides, Marios; Harris, Ann

    2004-01-01

    The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1alpha (hepatocyte nuclear factor 1alpha) transcription factor. HNF1alpha, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1alpha augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1 alpha transcription decreased the CFTR mRNA levels. Hnf1 alpha knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene. PMID:14656222

  16. [Effect of HIF-1alpha on sex hormone levels and germ cell apoptosis of mice].

    PubMed

    Chen, Xiang-Mei; Xiong, Yan-Lei; Gong, Hui; Xu, Cheng-Li

    2013-07-01

    To study the effect of hypoxia on hypothalamus-adenohypophysis-testis axis hormone levels, germ cell apoptosis and hypoxia-inducible factor-1alpha (HIF-1alpha) expression in testis of adolescent mice, and explore HIF-1alpha regulation on the reproductive function of male mice. Eighty SPF grade adolescent C57BL/6 mice were randomly divided into normoxia group, hypoxia 3, 7, 14 and 28 d groups. The level of serum testosterone (T), free testosterone (FT), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) was analyzed by ELISA. Detected the sperm count, motility rate and abnormal sperm rate of epididymal sperm suspension. The apoptosis cells in testis were determined using TUNEL method. The expression of HIF-1alpha was analyzed using Western blot. Compared with corresponding normoxia group, serum T, FT, FSH and LH concentrations in hypoxia 3 d group were significantly higher (P < 0.05); T and LH concentrations in hypoxia 14 d group were significantly lower (P < 0.05). Sperm count and motility rate in hypoxia 7 and 14 d groups significantly declined (P < 0.05); abnormal sperm rate in all hypoxia groups significantly increased (P < 0.05). The apoptosis index (AI) of germ cells in hypoxia 7, 14 and 28 d groups significantly increased (P < 0.05), and the levels of HIF-1alpha protein expression were significantly higher (P < 0.05). HIF-1alpha protein highly expressed in mice testis could induce germ cell apoptosis increased in chronic hypoxia environment.

  17. Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1 alpha.

    PubMed

    Qian, David Z; Kachhap, Sushant K; Collis, Spencer J; Verheul, Henk M W; Carducci, Michael A; Atadja, Peter; Pili, Roberto

    2006-09-01

    Hypoxia-inducible factor 1 alpha (HIF-1 alpha) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1 alpha protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1 alpha inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1 alpha protein via a VHL-independent mechanism and reduction of HIF-1 alpha transcriptional activity. HIF-1 alpha inhibition by LAQ824 was associated with HIF-1 alpha acetylation and polyubiquitination. HIF-1 alpha immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1 alpha. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1 alpha at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1 alpha only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1 alpha protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1 alpha protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1 alpha stability and provide a rationale for targeting HIF-1 alpha with HDAC inhibitors against class II isozymes.

  18. Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas.

    PubMed

    Lu, Huimin; Li, Yan; Shu, Minfeng; Tang, Jianjun; Huang, Yijun; Zhou, Yuxi; Liang, Yingjie; Yan, Guangmei

    2009-12-01

    Aberrant differentiation is a characteristic feature of neoplastic transformation, while hypoxia in solid tumors is believed to be linked to aggressive behavior and poor prognosis. However, the possible relationship between hypoxia and differentiation in malignancies remains poorly defined. Here we show that rat C6 and primary human malignant glioma cells can be induced to differentiate into astrocytes by the well-known adenylate cyclase activator forskolin. However, hypoxia-inducible factor-1alpha expression stimulated by the hypoxia mimetics cobalt chloride or deferoxamine blocks this differentiation and this effectiveness is reversible upon withdrawal of the hypoxia mimetics. Importantly, knockdown of hypoxia inducible factor-1alpha by RNA interference restores the differentiation capabilities of the cells, even in the presence of cobalt chloride, whereas stabilization of hypoxia-inducible factor-1alpha through retarded ubiquitination by von Hippel-Lindau tumor suppressor gene silence abrogates the induced differentiation. Moreover, targeting of HIF-1 using chetomin, a disrupter of HIF-1 binding to its transcriptional co-activator CREB-binding protein (CBP)/p300, abolishes the differentiation-inhibitory effect of hypoxia-inducible factor-1alpha. Administration of chetomin in combination with forskolin significantly suppresses malignant glioma growth in an in vivo xenograft model. Analysis of 95 human glioma tissues revealed an increase of hypoxia-inducible factor-1alpha protein expression with progressing tumor grade. Taken together, these findings suggest a key signal transduction pathway involving hypoxia-inducible factor-1alpha that contributes to a differentiation defect in malignant gliomas and sheds new light on the differentiation therapy of solid tumors by targeting hypoxia-inducible factor-1alpha.

  19. Hypoxia-inducible factor-1alpha suppresses the expression of macrophage scavenger receptor 1.

    PubMed

    Shirato, Ken; Kizaki, Takako; Sakurai, Takuya; Ogasawara, Jun-Etsu; Ishibashi, Yoshinaga; Iijima, Takehiko; Okada, Chikako; Noguchi, Izumi; Imaizumi, Kazuhiko; Taniguchi, Naoyuki; Ohno, Hideki

    2009-11-01

    Macrophages are distributed in all peripheral tissues and play a critical role in the first line of the innate immune defenses against bacterial infection by phagocytosis of bacterial pathogens through the macrophage scavenger receptor 1 (MSR1). Within tissues, the partial pressure of oxygen (pO2) decreases depending on the distance of cells from the closest O2-supplying blood vessel. However, it is not clear how the expression of MSR1 in macrophages is regulated by low pO2. On the other hand, hypoxia-inducible factor (HIF)-1alpha is well known to control hypoxic responses through regulation of hypoxia-inducible genes. Therefore, we investigated the effects of hypoxia and HIF-1alpha on MSR1 expression and function in the macrophage cell line RAW264. Exposure to 1% O2 or treatment with the hypoxia-mimetic agent cobalt chloride (CoCl2) significantly suppressed the expression of MSR1 mRNA, accompanied by a markedly increase in levels of nuclear HIF-1alpha protein. The overexpression of HIF-1alpha in RAW264 cells suppressed the expression of MSR1 mRNA and protein, transcriptional activity of the MSR1 gene, and phagocytic capacity against the Gram-positive bacteria Listeria monocytogenes. The suppression of MSR1 mRNA by hypoxia or CoCl2 was inhibited by YC-1, an inhibitor of HIF-1alpha, or by the depletion of HIF-1alpha expression by small interference RNA. These results indicate that hypoxia transcriptionally suppresses MSR1 expression through HIF-1alpha.

  20. Immunohistochemical detection of hypoxia-inducible factor-1alpha in human renal allograft biopsies.

    PubMed

    Rosenberger, Christian; Pratschke, Johann; Rudolph, Birgit; Heyman, Samuel N; Schindler, Ralf; Babel, Nina; Eckardt, Kai-Uwe; Frei, Ulrich; Rosen, Seymour; Reinke, Petra

    2007-01-01

    Although it generally is accepted that renal hypoxia may occur in various situations after renal transplantation, direct evidence for such hypoxia is lacking, and possible implications on graft pathophysiology remain obscure. Hypoxia-inducible factors (HIF) are regulated at the protein level by oxygen-dependent enzymes and, hence, allow for tissue hypoxia detection. With the use of high-amplification HIF-1alpha immunohistochemistry in renal biopsies, hypoxia is shown at specific time points after transplantation with clinicohistologic correlations. Immediately after engraftment, in primarily functioning grafts, abundant HIF-1alpha is present and correlates with cold ischemic time >15 h and/or graft age >50 yr (P < 0.04). In contrast, a low HIF-1alpha score correlates with primary nonfunction, likely reflecting loss of oxygen consumption for tubular transport. Protocol biopsies at 2 wk show widespread HIF-1alpha induction, irrespective of histology. Beyond 3 mo, both protocol biopsies and indicated biopsies are virtually void of HIF-1alpha, with the only exception being clinical/subclinical rejection. HIF-derived transcriptional adaptation to hypoxia may counterbalance, at least partly, the negative impact of cold preservation and warm reflow injury. Transient hypoxia at 2 wk may be induced by hyperfiltration, hypertrophy, calcineurin inhibitor-induced toxicity, or a combination of these. Lack of detectable HIF-1alpha at 3 mo and beyond suggests that at this time point, graft oxygen homeostasis occurs. The strong correlation between hypoxia and clinical/subclinical rejection in long-term grafts suggests that hypoxia is involved in such graft dysfunction, and HIF-1alpha immunohistochemistry could enhance the specific diagnosis of acute rejection.

  1. [Effects of the chemokine MIP-1alpha on anemia and inflammation in rheumatoid arthritis].

    PubMed

    Kullich, Werner; Niksic, Franz; Burmucic, Katharina; Pöllmann, Georg; Klein, Gert

    2002-10-01

    Macrophage inflammatory protein-1alpha (MIP-1alpha) is an interesting chemokine because in addition to its variety proinflammatory activities including chemotaxis and immunomodulation, it is a potent inhibitor of hematopoetic stem cell proliferation. Inhibition of erythroid progenitor cells due to MIP-1alpha or other cytokines can play a role in the pathogenesis of anemia which is one of the most common extra-articular features of active rheumatoid arthritis (RA). In 84 patients with RA, serological and immunological parameters were assessed to detect inflammatory mechanisms and anemia in relation to the serum concentrations of MIP-1alpha. All patients fulfilled the ACR criteria for the diagnosis of a definite or classic RA. We used a quantitative enzyme immuno assay for the detection of MIP-1alpha as well as for the measurement of the acute phase protein serum amyloid A (SAA), the erythropoiesis inducer erythropoietin (EPO) and the transferrin receptor (TfR). The immune activation marker neopterin was measured radioimmunologically. Half of the patients with RA were anemic with hemoglobin values below 12 g/dl. MIP-1alpha was found to be elevated significantly in serum of patients with active rheumatoid arthritis and in patients with anemia. Most of the anemic patients with markedly elevated acute phase reactions had an anemia with chronic diseases and not a functional iron deficiency alone. TfR correlated with EPO. The results show that enhanced expression of MIP-1alpha is indicative of systemic inflammation in RA. Moreover, besides the regulation of inflammatory processes, this chemokine may influence the pathogenesis of anemia in RA patients.

  2. Grape seed extract inhibits VEGF expression via reducing HIF-1alpha protein expression.

    PubMed

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-04-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1alpha. The inhibitory effect of GSE on HIF-1alpha expression was mainly through inhibiting HIF-1alpha protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1alpha protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1alpha and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1alpha, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1alpha protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE.

  3. P70S6K 1 regulation of angiogenesis through VEGF and HIF-1{alpha} expression

    SciTech Connect

    Bian, Chuan-Xiu; Shi, Zhumei; Meng, Qiao; Jiang, Yue; Liu, Ling-Zhi; Jiang, Bing-Hua

    2010-07-30

    Research highlights: {yields} P70S6K1 regulates VEGF expression; {yields} P70S6K1 induces transcriptional activation through HIF-1{alpha} binding site; {yields} P70S6K1 regulates HIF-1{alpha}, but not HIF-1{beta} protein expression; {yields} P70S6K1 mediates tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression. -- Abstract: The 70 kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1{alpha} binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1{alpha}, but not HIF-1{beta} protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1{alpha} expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

  4. Mitochondrial Diseases

    MedlinePlus

    ... disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are ... cells and cause damage. The symptoms of mitochondrial disease can vary. It depends on how many mitochondria ...

  5. A homology-derived structural model of the murine macrophage inflammatory protein, MIP-1 alpha.

    PubMed

    McKie, J H; Douglas, K T

    1994-01-01

    Macrophage inflammatory protein 1 alpha (MIP-1 alpha), a monocyte cytokine, has roles postulated for it in neutrophil chemoattraction, the inflammatory response and the control of haemopoietic stem cell proliferation. The three-dimensional structure of MIP-1 alpha has been modelled structurally, based on its sequence similarity to interleukin-8 and related proteins. The predicted dimeric form of MIP-1 alpha contains two symmetry-related antiparallel alpha-helices lying at an angle across a beta-sheet. The interhelical region and the beta-sheet flooring it are discussed as the potential receptor-binding site in terms of the distribution of negatively charged amino-acid side-chains, which contrasts remarkably with the corresponding positively-charged locations for IL-8. The general topographical features of this (alpha + beta) structural family of cytokines and related proteins (including HLA-A2, PF-4) are discussed. The members of this cytokine family fall into two structural groups as the antiparallel helices (N to C directed) mounted across the beta-sheet platform can be located in a clockwise (e.g. HLA-A2) or anticlockwise (e.g. MIP-1 alpha) sense with respect to the beta-floor).

  6. Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis.

    PubMed Central

    Moyer, C. F.; Sajuthi, D.; Tulli, H.; Williams, J. K.

    1991-01-01

    Interleukin-1 (IL-1) has been implicated as a regulatory protein in the development and clinical sequelae of atherosclerosis. To determine which cells in the atherosclerotic plaque synthesize IL-1 in situ, the authors evaluated histologic sections of iliac arteries from cynomolgus monkeys using probes for IL-1 alpha and beta. A polyclonal antibody to IL-1 alpha and beta was used to determine if proteins were concomitantly produced. The predominant cells expressing IL-1 alpha and beta mRNA were foam cells in the intima. Adherent leukocytes and vascular smooth muscle cells (VSMCs) expressed mRNA for IL-1 alpha. Microvascular endothelium expressed mRNA for both IL-1 alpha and beta. IL-1 proteins were located frequently in cells expressing IL-1 mRNA. These results indicate that endothelium and VSMCs, in conjunction with macrophages, serve as localized sources of IL-1 protein synthesis. These findings suggest that vascular cells may contribute directly to the pathogenesis of atherosclerotic vascular disease by actively secreting potent biologic mediators that modify vascular and immune cell function. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:2012178

  7. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL.

    PubMed

    Hon, Wai-Ching; Wilson, Michael I; Harlos, Karl; Claridge, Timothy D W; Schofield, Christopher J; Pugh, Christopher W; Maxwell, Patrick H; Ratcliffe, Peter J; Stuart, David I; Jones, E Yvonne

    2002-06-27

    Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1 alpha is the oxygen-regulated subunit of HIF-1, an alpha beta heterodimeric complex. HIF-1 alpha is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1 alpha by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1 alpha. The crystal structure of a hydroxylated HIF-1 alpha peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

  8. Down-regulation of cardiac lineage protein (CLP-1) expression in CLP-1 +/- mice affords.

    PubMed

    Mascareno, Eduardo; Manukyan, Irena; Das, Dipak K; Siddiqui, M A Q

    2009-08-01

    In order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

  9. HIF-1{alpha} is necessary to support gluconeogenesis during liver regeneration

    SciTech Connect

    Tajima, Toshihide; Goda, Nobuhito; Fujiki, Natsuko; Hishiki, Takako; Nishiyama, Yasumasa; Senoo-Matsuda, Nanami; Shimazu, Motohide; Soga, Tomoyoshi; Yoshimura, Yasunori; Johnson, Randall S.; Suematsu, Makoto

    2009-10-02

    Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1{alpha} (HIF-1{alpha}) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72 h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1{alpha} in the regulation of gluconeogenesis under liver regeneration.

  10. Diet, energy metabolism and mitochondrial biogenesis.

    PubMed

    Civitarese, Anthony E; Smith, Steven R; Ravussin, Eric

    2007-11-01

    This review highlights some recent findings regarding nutritional and endocrine regulators of mitochondrial mass and function and their association with insulin resistance. Insulin resistance is central to many chronic metabolic diseases, including obesity, type 2 diabetes, dyslipidemia, and hypertension. Insulin resistance in skeletal muscle is associated with lower mitochondrial mass and reduced oxidative phosphorylation. Part of the mitochondrial dysfunction can be triggered by adverse nutrition. Increased fatty acid exposure, resulting from high fats diets or overfeeding, is linked to both decreased mitochondrial number and markers of oxidative phosphorylation. Caloric restriction and the adiponectin signaling pathway, however, can stimulate mitochondrial biogenesis by elevating the transcriptional machinery that regulates mitochondrial mass, improving mitochondrial efficiency, activating the peroxisome proliferator-activated receptor coactivator 1alpha mediated reactive oxygen species scavenging mechanism, and lowering reactive oxygen species production. States of insulin resistance are characterized by defects in lipid and carbohydrate metabolism. Abnormalities in oxidative capacity, however, can be partially normalized by caloric restriction by modulating mitochondrial mass in an insulin sensitizing manner.

  11. Quantitative evaluation of 1-alpha-hydroxycholecalciferol as a cholecalciferol substitute for broilers.

    PubMed

    Edwards, H M; Shirley, R B; Escoe, W B; Pesti, G M

    2002-05-01

    Two experiments were conducted using a corn-soybean meal diet that meets or exceeds the NRC (1984) requirements for all nutrients except cholecalciferol (D3) to determine the effectiveness of 1-alpha-hydroxycholecalciferol (1alpha-OHD3) as a substitute for D3 in the diet of young broilers. Ross x Ross mixed-sex, 1-d-old chicks were reared in Petersime battery brooders not exposed to ultraviolet light with feed and water supplied ad libitum for 16 d. In Experiment 1, D3 was fed at 0, 2.5, 5, 10, 20, and 40 microg/kg and one source of 1alpha-OHD3-(Hoffmann-LaRoche, Inc.; HLR) was fed at 0.625, 1.25, 2.5, 5, and 10 microg/kg of diet. In Experiment 2, the D3 was fed at 0, 2.5, 5, and 10 microg and two sources of 1alpha-OHD3-[HLR and Majestic Research Inc. (MRI)] were fed at 0, 0.625, 1.25, and 5 microg/kg of diet. Slope ratio analysis of data from the measurement of 16-d body weight, plasma Ca, rickets, and bone ash indicated bioavailability of the 1alpha-OHD3 as compared to D3 from 1.88 to 21.2. Percentage bone ash gave the most precise values in both experiments. Considering all the data from both experiments, the 1alpha-OHD3 appears to be approximately eight times as effective as D3 for satisfying the requirements of several criteria in two experiments with broiler chickens.

  12. Expression of HIF-1 alpha in irradiated tissue is altered by topical negative-pressure therapy.

    PubMed

    Grimm, Andreas; Dimmler, Arno; Stange, Sebastian; Labanaris, Apostolos; Sauer, Rolf; Grabenbauer, Gerhard; Horch, Raymund E

    2007-03-01

    Despite the enormous therapeutic potential of modern radiotherapy, common side effects such as radiation-induced wound healing disorders remain a well-known clinical phenomenon. Topical negative pressure therapy (TNP) is a novel tool to alleviate intraoperative, percutaneous irradiation or brachytherapy. Since TNP has been shown to positively influence the perfusion of chronic, poorly vascularized wounds, the authors applied this therapeutic method to irradiated wounds and investigated the effect on tissue oxygenation in irradiated tissue in five patients. With informed patients' consent, samples prior to and 4 and 8 days after continuous TNP with -125 mmHg were obtained during routine wound debridements. Granulation tissue was stained with hematoxylin-eosin, and additionally with CD31, HIF-1 alpha (hypoxia-inducible factor-1 alpha), and D2-40 to detect blood vessels, measure indirect signs of hypoxia, and lymph vessel distribution within the pre- and post-TNP samples. In this first series of experiments, a positive influence of TNP onto tissue oxygenation in radiation-induced wounds could be demonstrated. TNP led to a significant decrease of 53% HIF-1 alpha-positive cell nuclei. At the same time, a slight reduction of CD31-stained capillaries was seen in comparison to samples before TNP. Immunostaining with D2-40 revealed an increased number of lymphatic vessels with distended lumina and an alteration of the parallel orientation within the post-TNP samples. This study is, to the authors' knowledge, the first report on a novel previously not described histological marker to demonstrate the effects of TNP on HIF-1 alpha expression as an indirect marker of tissue oxygenation in irradiated wounds, as demonstrated by a reduction of HIF-1 alpha concentration after TNP. Since this observation may be of significant value to develop possible new strategies to treat radiation-induced tissue injury, further investigations of HIF-1 alpha regulation under TNP are warranted.

  13. Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1alpha.

    PubMed

    Bando, Mika; Hiroshima, Yuka; Kataoka, Masatoshi; Herzberg, Mark C; Ross, Karen F; Shinohara, Yasuo; Yamamoto, Takenori; Nagata, Toshihiko; Kido, Jun-ichi

    2010-01-01

    Calprotectin is an antimicrobial complex composed of the S100A8 and S100A9 protein family subunits. Contributing to innate immunity, calprotectin expression is increased by interleukin-1alpha (IL-1alpha), which modulates keratinocyte differentiation. Keratinocyte growth factor (KGF) is produced by mesenchymal cells and has a mitogenic activity for epithelial cells. In this study, we investigated the effect of KGF on calprotectin expression in keratinocytes and modulation by IL-1alpha. Human keratinocytes were cultured with KGF in the presence or absence of a KGF receptor (KGFR) inhibitor or mitogen-activated protein kinase (MAPK) inhibitors. Calprotectin (S100A8/S100A9) expression was determined by northern blotting and enzyme-linked immunosorbent assay, respectively, whereas MAPK phosphorylation was analyzed by western blot analysis. KGF significantly decreased the expression of S100A8/S100A9-specific mRNAs and calprotectin protein. In the presence of KGF, KGFR inhibitor or extracellular-regulated kinase inhibitor restored KGF-downregulated expression of S100A8/S100A9. KGF increased IL-1alpha expression in keratinocytes, whereas IL-1alpha increased KGF expression in fibroblasts. Cocultured fibroblast and keratinocytes showed lower S100A8/S100A9 mRNA expression than keratinocytes alone in the presence or absence of IL-1alpha or KGF. These results suggest that fibroblast-derived KGF reduces or restricts calprotectin expression in keratinocytes, which supports our hypothesis that calprotectin expression in keratinocytes is modulated by factors associated with epithelial-mesenchymal interactions.

  14. Parkinson's syndrome and Parkinson's disease in mitochondrial disorders.

    PubMed

    Finsterer, Josef

    2011-04-01

    In the majority of cases, mitochondrial disorders are multisystem conditions that most frequently affect the skeletal muscle, followed by the central nervous system. One of the clinical manifestations of central nervous system involvement is Parkinson's syndrome (PS). Evidence for an association of mitochondrial defects with PS comes from mitochondrial disorder patients who have developed Parkinson's syndrome and from Parkinson's syndrome patients who have developed a mitochondrial disorder. In addition, there are a number of patients with Parkinson's syndrome or Parkinson's disease (PD) who later develop subclinical immunohistological or biochemical indications of mitochondrial defects or accumulates mitochondrial DNA mutations within various cerebral regions. There are also Parkinson's syndrome patients who present with elevated cerebrospinal-fluid lactate by magnetic resonance spectroscopy. Furthermore, it has been shown that mutations in genes causing PD, such as PINK1, parkin, DJ1, alpha-synuclein, and LRRK2, also cause mitochondrial dysfunction, which is one of the reasons why they are called mitochondrial nigropathies. Parkinson's syndrome in patients with a mitochondrial disorder may also result from oxidative stress or exogenous toxins. Treatment of mitochondrial Parkinson's syndrome is not at variance with the treatment of Parkinson's syndrome due to other causes, but because of the multisystem nature of mitochondrial disorders, mitochondrial Parkinson's syndrome requires additional therapeutic support.

  15. Menthol Enhances an Antiproliferative Activity of 1alpha,25-Dihydroxyvitamin D(3) in LNCaP Cells.

    PubMed

    Park, Eun-Jung; Kim, Su-Hwa; Kim, Byung-Joo; Kim, Sung-Young; So, Insuk; Jeon, Ju-Hong

    2009-03-01

    1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the most active form of vitamin D(3), and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1alpha,25(OH)(2)D(3) for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1alpha,25(OH)(2)D(3) in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1alpha,25(OH)(2)D(3)-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1alpha,25(OH)(2)D(3) does not induce acute Ca(2+) response, whereas menthol evokes an increase in [Ca(2+)](i), which suggests that cross-talks of menthol-induced Ca(2+) signaling with 1alpha,25(OH)(2)D(3)-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1alpha,25(OH)(2)D(3) and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1alpha,25(OH)(2)D(3)-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1alpha,25(OH)(2)D(3).

  16. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  17. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  18. Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease.

    PubMed

    McConoughey, Stephen J; Basso, Manuela; Niatsetskaya, Zoya V; Sleiman, Sama F; Smirnova, Natalia A; Langley, Brett C; Mahishi, Lata; Cooper, Arthur J L; Antonyak, Marc A; Cerione, Rick A; Li, Bo; Starkov, Anatoly; Chaturvedi, Rajnish Kumar; Beal, M Flint; Coppola, Giovanni; Geschwind, Daniel H; Ryu, Hoon; Xia, Li; Iismaa, Siiri E; Pallos, Judit; Pasternack, Ralf; Hils, Martin; Fan, Jing; Raymond, Lynn A; Marsh, J Lawrence; Thompson, Leslie M; Ratan, Rajiv R

    2010-09-01

    Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

  19. Mitochondrial Cardiomyopathies.

    PubMed

    El-Hattab, Ayman W; Scaglia, Fernando

    2016-01-01

    Mitochondria are found in all nucleated human cells and perform various essential functions, including the generation of cellular energy. Mitochondria are under dual genome control. Only a small fraction of their proteins are encoded by mitochondrial DNA (mtDNA), whereas more than 99% of them are encoded by nuclear DNA (nDNA). Mutations in mtDNA or mitochondria-related nDNA genes result in mitochondrial dysfunction leading to insufficient energy production required to meet the needs for various organs, particularly those with high energy requirements, including the central nervous system, skeletal and cardiac muscles, kidneys, liver, and endocrine system. Because cardiac muscles are one of the high energy demanding tissues, cardiac involvement occurs in mitochondrial diseases with cardiomyopathies being one of the most frequent cardiac manifestations found in these disorders. Cardiomyopathy is estimated to occur in 20-40% of children with mitochondrial diseases. Mitochondrial cardiomyopathies can vary in severity from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. Hypertrophic cardiomyopathy is the most common type; however, mitochondrial cardiomyopathies might also present as dilated, restrictive, left ventricular non-compaction, and histiocytoid cardiomyopathies. Cardiomyopathies are frequent manifestations of mitochondrial diseases associated with defects in electron transport chain complexes subunits and their assembly factors, mitochondrial transfer RNAs, ribosomal RNAs, ribosomal proteins, translation factors, mtDNA maintenance, and coenzyme Q10 synthesis. Other mitochondrial diseases with cardiomyopathies include Barth syndrome, Sengers syndrome, TMEM70-related mitochondrial complex V deficiency, and Friedreich ataxia.

  20. Mitochondrial Cardiomyopathies

    PubMed Central

    El-Hattab, Ayman W.; Scaglia, Fernando

    2016-01-01

    Mitochondria are found in all nucleated human cells and perform various essential functions, including the generation of cellular energy. Mitochondria are under dual genome control. Only a small fraction of their proteins are encoded by mitochondrial DNA (mtDNA), whereas more than 99% of them are encoded by nuclear DNA (nDNA). Mutations in mtDNA or mitochondria-related nDNA genes result in mitochondrial dysfunction leading to insufficient energy production required to meet the needs for various organs, particularly those with high energy requirements, including the central nervous system, skeletal and cardiac muscles, kidneys, liver, and endocrine system. Because cardiac muscles are one of the high energy demanding tissues, cardiac involvement occurs in mitochondrial diseases with cardiomyopathies being one of the most frequent cardiac manifestations found in these disorders. Cardiomyopathy is estimated to occur in 20–40% of children with mitochondrial diseases. Mitochondrial cardiomyopathies can vary in severity from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. Hypertrophic cardiomyopathy is the most common type; however, mitochondrial cardiomyopathies might also present as dilated, restrictive, left ventricular non-compaction, and histiocytoid cardiomyopathies. Cardiomyopathies are frequent manifestations of mitochondrial diseases associated with defects in electron transport chain complexes subunits and their assembly factors, mitochondrial transfer RNAs, ribosomal RNAs, ribosomal proteins, translation factors, mtDNA maintenance, and coenzyme Q10 synthesis. Other mitochondrial diseases with cardiomyopathies include Barth syndrome, Sengers syndrome, TMEM70-related mitochondrial complex V deficiency, and Friedreich ataxia. PMID:27504452

  1. Mitochondrial vasculopathy

    PubMed Central

    Finsterer, Josef; Zarrouk-Mahjoub, Sinda

    2016-01-01

    Mitochondrial disorders (MIDs) are usually multisystem disorders (mitochondrial multiorgan disorder syndrome) either on from onset or starting at a point during the disease course. Most frequently affected tissues are those with a high oxygen demand such as the central nervous system, the muscle, endocrine glands, or the myocardium. Recently, it has been shown that rarely also the arteries may be affected (mitochondrial arteriopathy). This review focuses on the type, diagnosis, and treatment of mitochondrial vasculopathy in MID patients. A literature search using appropriate search terms was carried out. Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy. Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy, migraine-like headache, stroke-like episodes, or peripheral retinopathy. Mitochondrial macroangiopathy manifests as atherosclerosis, ectasia of arteries, aneurysm formation, dissection, or spontaneous rupture of arteries. The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes. Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes. Mitochondrial vasculopathy exists and manifests as micro- or macroangiopathy. Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications. PMID:27231520

  2. Comparison of blocking effects of monoclonal antibodies anti-MO1-alpha and anti-LFA1-alpha on human neutrophil functions.

    PubMed Central

    Pham Huu, T; Chollet-Martin, S; Perianin, A; Marquetty, C; Sourbier, P; Babin-Chevaye, C; Olive, D; Gougerot-Pocidalo, M A; Debre, P; Hakim, J

    1987-01-01

    In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MO1) recognizing an epitope of the MO1-alpha chain and the other (25.31) an epitope of the LFA1-alpha chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean +/- 1 SD) on plastic nuclon plates; this figure decreased to 33 +/- 5% and 23 +/- 6% of control adherence when the neutrophils had been pretreated with anti-LFA1-alpha (anti-alpha L) and anti-MO1-alpha (anti-alpha M), respectively. On another support (plastic culture chambers), 84 +/- 6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-alpha L or anti-alpha M was 10% and 43% of the control figure, respectively. These results show that adherence of neutrophils is dependent upon the plastic used. Moreover, inhibition of adhesion by the two MAbs was also dependent upon the support used for the assay, suggesting that MO1 and LFA1 may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-alpha L inhibited (congruent to 50%) locomotion more than did anti-alpha M (congruent to 25%), without altering chemoattractant-induced shape changes. These results suggest that the two MAbs inhibit chemokinesis but not chemotaxis. Many other adherence-associated functions, such as ingestion of opsonized Klebsiella pneumoniae, and cytotoxicity towards K/562 cells were decreased more by anti-alpha L than by anti-alpha M. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-alpha M than by anti-alpha L. Degranulation induced by zymosan or opsonized zymosan was altered by anti-alpha M only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-alpha M than by anti-alpha L, while

  3. Inhibition of HIF-1{alpha} activity by BP-1 ameliorates adjuvant induced arthritis in rats

    SciTech Connect

    Shankar, J.; Thippegowda, P.B.; Kanum, S.A.

    2009-09-18

    Rheumatoid arthritis (RA) is a chronic inflammatory, angiogenic disease. Inflamed synovitis is a hallmark of RA which is hypoxic in nature. Vascular endothelial growth factor (VEGF), one of the key regulators of angiogenesis, is overexpressed in the pathogenesis of RA. VEGF expression is regulated by hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), a master regulator of homeostasis which plays a pivotal role in hypoxia-induced angiogenesis. In this study we show that synthetic benzophenone analogue, 2-benzoyl-phenoxy acetamide (BP-1) can act as a novel anti-arthritic agent in an experimental adjuvant induced arthritis (AIA) rat model by targeting VEGF and HIF-1{alpha}. BP-1 administered hypoxic endothelial cells and arthritic animals clearly showed down regulation of VEGF expression. Further, BP-1 inhibits nuclear translocation of HIF-1{alpha}, which in turn suppresses transcription of the VEGF gene. These results suggest a further possible clinical application of the BP-1 derivative as an anti-arthritic agent in association with conventional chemotherapeutic agents.

  4. UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

    PubMed

    Alexandru, Gabriela; Graumann, Johannes; Smith, Geoffrey T; Kolawa, Natalie J; Fang, Ruihua; Deshaies, Raymond J

    2008-09-05

    p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.

  5. HIV-1-infected macrophages induce astrogliosis by SDF-1{alpha} and matrix metalloproteinases

    SciTech Connect

    Okamoto, Mika; Wang, Xin; Baba, Masanori . E-mail: baba@m.kufm.kagoshima-u.ac.jp

    2005-11-04

    Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1{alpha} or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1{alpha} production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1{alpha} was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1{alpha} and MMP production, which implies a mechanism of astrogliosis in HAD.

  6. Differential localization of protein phosphatase-1alpha, beta and gamma1 isoforms in primate prefrontal cortex.

    PubMed

    Bordelon, Jill R; Smith, Yoland; Nairn, Angus C; Colbran, Roger J; Greengard, Paul; Muly, E Chris

    2005-12-01

    Prefrontal cortical functioning depends on D1 family receptors and their complex signal transduction cascade, including protein phosphatase-1 (PP1). Three PP1 isoforms are prominent in the brain: PP1alpha, PP1beta and PP1gamma1. PP1 localization by a variety of scaffolding proteins is critical for dopamine-mediated modulation of glutamatergic neurotransmission. We have quantified the subcellular distribution of each isoform in primate prefrontal cortex using immunoelectron microscopy. All three are found in spines, dendrites, axon terminals, axons and glia. However, PP1alpha and PP1gamma1 labeling is enriched in spines, whereas PP1beta label is enriched in dendrites. Using post-embedding immunogold labeling, we further examined the distribution of PP1alpha and PP1gamma1 within spines. PP1gamma1 is highly and specifically concentrated in the postsynaptic density (PSD) of these spines, while PP1alpha is enriched in the PSD but also found subjacent to the PSD in moderate amounts. Thus, PP1 isoforms are heterogeneously distributed in the cortical neuropil and within spines. These results suggest that each PP1 isoform has access to a different set of substrates and, furthermore, they demonstrate that the composition of signal transduction proteins varies in different parts of the neuron and even in different regions of a dendritic spine in the primate PFC.

  7. Biologic activity of interleukin 1 (IL-1) alpha in patients with refractory malignancies.

    PubMed

    Rosenthal, M A; Dennis, D; Liebes, L; Furmanski, P; Caron, D; Garrison, L; Wiprovnick, J; Peace, D; Oratz, R; Speyer, J; Chachoua, A

    1998-09-01

    Interleukin 1 alpha (IL-1 alpha) is a cytokine with pleiotropic effects, including cytotoxic-cytostatic activity against some tumor cell lines. We have conducted a phase I study of recombinant human IL-1 alpha (rhIL-1 alpha) in 17 patients with refractory malignancies to examine its toxicity and biologic activity. rhIL-1 alpha was given as a 2-h IV infusion daily for 5 days at five dose levels (0.08, 0.2, 0.8, 2.0, and 5.0 micrograms/m2). Seventeen patients with malignancies were treated, with no objective tumor responses noted. Common toxicities included: fever (100%), rigors and/or chills (96%), myalgia (54%), and headache (48%). Three patients developed grade III hypotension. The maximum tolerated dose was 2.0 micrograms/m2. rhIL-1 alpha induced a significant increase in absolute neutrophil count over baseline (p < 0.05), a delayed but significant increase in platelet count over baseline (p < 0.05), and there was a marked increase in the number of progenitors [colony-forming units (CFU)-G, CFU-M, CFU-GM, CFU-GEMM and burst-forming units (BFU-E)] observed in the peripheral blood. Nine of 12 evaluable patients showed an increase in bone marrow cellularity or myeloid:erthyroid ratio. Immunophenotyping did not demonstrate an increase in peripheral blood or bone marrow CD34+ cells. Interferon-gamma-mediated monocyte cytotoxicity (MCCTX) was significantly enhanced from baseline (p < 0.001), although an increase in direct MCCTX did not reach statistical significance. In summary, rhIL-1 alpha administration is well tolerated at a dose of 2.0 micrograms/m2 with fever, rigors, myalgia, and headache being the most frequent toxicities. Although there were no objective tumor responses, we have demonstrated significant biologic activity with increased neutrophil and platelet counts, increased peripheral blood progenitor cells, and enhanced interferon-gamma-mediated MCCTX.

  8. EFL GTPase in cryptomonads and the distribution of EFL and EF-1alpha in chromalveolates.

    PubMed

    Gile, Gillian H; Patron, Nicola J; Keeling, Patrick J

    2006-10-01

    EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1alpha, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1alpha. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1alpha (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1alpha PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1alpha in neither. On balance, we suggest the previously reported EF-1alpha from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group

  9. Phase II study of recombinant interleukin 1alpha and etoposide in patients with relapsed osteosarcoma.

    PubMed

    Worth, L L; Jaffe, N; Benjamin, R S; Papadopoulos, N E; Patel, S; Raymond, A K; Jia, S F; Rodriguez, C; Gano, J; Gianan, M A; Kleinerman, E S

    1997-10-01

    A Phase II trial using interleukin 1alpha (IL-1alpha) and etoposide for patients with relapsed osteosarcoma (OS) was undertaken to assess the feasibility and tolerability of combination therapy with biotherapy and chemotherapy. Nine patients with histologically proven relapsed OS were treated with IL-1alpha immediately followed by etoposide daily for 5 days every 3 weeks. Surgical resection of lung metastasis or peripheral tumor was performed after two or three cycles. We observed three partial responses; disease was stable in another case. One case could not be evaluated. The side effects associated with combination therapy were as predicted from known side effects of the individual agents; however, more profound neutropenia was observed. Four patients exhibited clinical signs of capillary leak syndrome, i.e., hypotension, edema, and weight gain. The etiology of the capillary leak was unclear, because serum IL-1alpha, IL-2, tumor necrosis factor, and nitric oxide levels could not be used to predict which patients would develop capillary leak. Histological analysis of tumor specimens obtained after two or more courses of therapy showed changes consistent with a response to a biological response modifier: peripheral fibrosis surrounded the metastasis with infiltration of chronic and acute inflammatory cells. Because the response of relapsed OS to any type of salvage regimen has been poor, we interpret the clinical response of this therapy as good. However, the significant side effects associated with this therapy must also be taken into consideration before deciding to use this combination therapy. It is unfortunate that the study was stopped early due to halted production of IL-1alpha. If this agent is again manufactured for clinical use, we conclude that additional evaluation in patients with relapsed OS is warranted.

  10. Increase in gene dosage is a mechanism of HIF-1alpha constitutive expression in head and neck squamous cell carcinomas.

    PubMed

    Secades, Pablo; Rodrigo, Juan Pablo; Hermsen, Mario; Alvarez, Cesar; Suarez, Carlos; Chiara, María-Dolores

    2009-05-01

    The HIF-1alpha protein plays a key role in the cellular response to hypoxia via transcriptional regulation of genes involved in erythropoiesis, angiogenesis, and metabolism. Overexpression of HIF-1alpha is commonly found in solid tumors in significant association with increased patient mortality and resistance to therapy. The predominant mode of HIF-1alpha regulation by hypoxia occurs at the level of protein stability. In addition to hypoxia, HIF-1alpha protein stability and synthesis is regulated by nonhypoxic signals such as inactivation of tumor suppressors and activation of oncogenes. Here, we show that an increase in gene dosage may contribute to HIF-1alpha mRNA and protein overexpression in a nonhypoxic environment in head and neck squamous cell carcinomas (HNSCC). Increased HIF-1alpha gene dosage was found in one out of five HNSCC-derived cell lines and three out of 27 HNSCC primary tumors. Significantly, increased gene dosage in those samples was associated with high HIF-1alpha mRNA and protein levels. Normoxic overexpression of HIF-1alpha protein in HNSCC-derived cell lines was also paralleled by higher expression levels of HIF-1alpha target genes. Array CGH analysis confirmed the copy number increase of HIF-1alpha gene and revealed that the gene is contained within a region of amplification at 14q23-q24.2 both in the cell line and primary tumors. In addition, FISH analysis revealed the presence of 11-13 copies on a tetraploid background in SCC2 cells. These data suggest that increased HIF-1alpha gene dosage is a mechanism of HIF-1alpha protein overexpression in HNSCC that possibly prepares the cells for a higher activity in an intratumoral hypoxic environment.

  11. Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    SciTech Connect

    Porcile, Carola; Bajetto, Adriana . E-mail: bajetto@cba.unige.it; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio . E-mail: florio@cba.unige.it; Schettini, Gennaro

    2005-08-15

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.

  12. Oxygen-dependent expression of hypoxia-inducible factor-1alpha in renal medullary cells of rats.

    PubMed

    Zou, A P; Yang, Z Z; Li, P L; Cowley AW, J R

    2001-08-28

    Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates the oxygen-dependent expression of a number of genes. This transcription factor may contribute to the abundant expression of many genes in renal medullary cells that function normally under hypoxic conditions. The present study was designed to determine the characteristics of HIF-1alpha cDNA cloned from the rat kidney and the expression profile of HIF-1alpha in different kidney regions and to explore the mechanism activating or regulating HIF-1alpha expression in renal medullary cells. A 3,718-bp HIF-1alpha cDNA from the rat kidney was first cloned and sequenced using RT-PCR and TA cloning technique. It was found that 823 amino acids deduced from this renal HIF-1alpha cDNA had 99%, 96%, and 90% identity with rat, mouse, or human HIF-1alpha deposited in GenBank, respectively. The 3'-untranslated region of HIF-1alpha mRNA from the rat kidney contained seven AUUUA instability elements, five of which were found to be conserved among rat, mouse, and human HIF-1alpha. Northern blot analyses demonstrated a corticomedullary gradient of HIF-1alpha mRNA expression in the kidney, with the greatest abundance in the renal inner medulla. Western blot analyses also detected a higher HIF-1alpha protein level in the nuclear extracts from the renal medulla than the renal cortex. A classic loop diuretic, furosemide (10 mg/kg ip), markedly increased renal medullary Po(2) levels from 22.5 to 52.2 mmHg, which was accompanied by a significant reduction of HIF-1alpha transcripts in renal medullary tissue. In in vitro experiments, low Po(2), but not elevated osmolarity, was found to significantly increase HIF-1alpha mRNA in renal medullary interstitial cells and inner medullary collecting duct cells. These results indicate that HIF-1alpha is more abundantly expressed in the renal medulla compared with the renal cortex. Increased abundance of HIF-1alpha mRNA in the renal medulla may represent an adaptive

  13. Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

    PubMed Central

    Graham, G J; Wilkinson, P C; Nibbs, R J; Lowe, S; Kolset, S O; Parker, A; Freshney, M G; Tsang, M L; Pragnell, I B

    1996-01-01

    We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes. Images PMID:8978677

  14. Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

    PubMed

    Graham, G J; Wilkinson, P C; Nibbs, R J; Lowe, S; Kolset, S O; Parker, A; Freshney, M G; Tsang, M L; Pragnell, I B

    1996-12-02

    We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.

  15. D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

    SciTech Connect

    Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il; Baek, Won-Ki

    2009-04-24

    D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

  16. Management of the aging risk factor for Parkinson's disease.

    PubMed

    Phillipson, Oliver T

    2014-04-01

    The aging risk factor for Parkinson's disease is described in terms of specific disease markers including mitochondrial and gene dysfunctions relevant to energy metabolism. This review details evidence for the ability of nutritional agents to manage these aging risk factors. The combination of alpha lipoic acid, acetyl-l-carnitine, coenzyme Q10, and melatonin supports energy metabolism via carbohydrate and fatty acid utilization, assists electron transport and adenosine triphosphate synthesis, counters oxidative and nitrosative stress, and raises defenses against protein misfolding, inflammatory stimuli, iron, and other endogenous or xenobiotic toxins. These effects are supported by gene expression via the antioxidant response element (ARE; Keap/Nrf2 pathway), and by peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1 alpha), a transcription coactivator, which regulates gene expression for energy metabolism and mitochondrial biogenesis, and maintains the structural integrity of mitochondria. The effectiveness and synergies of the combination against disease risks are discussed in relation to gene action, dopamine cell loss, and the accumulation and spread of pathology via misfolded alpha-synuclein. In addition there are potential synergies to support a neurorestorative role via glial derived neurotrophic factor expression. Copyright © 2014 The Author. Published by Elsevier Inc. All rights reserved.

  17. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1{alpha} survival pathways

    SciTech Connect

    Oommen, Deepu; Prise, Kevin M.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer KNK437, a benzylidene lactam compound, is a novel radiosensitizer. Black-Right-Pointing-Pointer KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1{alpha} under hypoxia. Black-Right-Pointing-Pointer KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1{alpha} (HIF-1{alpha}). HIF-1{alpha} is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1{alpha}. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1{alpha} in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1{alpha} levels in KNK437-treated cells. This suggested that the absence of HIF-1{alpha} in hypoxic cells was not due to the enhanced protein degradation. HIF-1{alpha} is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1{alpha} mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1{alpha} levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  18. Expression of HIF-1alpha and VEGF in skeletal muscle of plateau animals in response to hypoxic stress.

    PubMed

    Xie, H-C; He, J-P; Zhu, J-F; Li, J-G

    2014-01-01

    Hypoxia-inducible factor-1alpha (HIF-1alpha) transcriptionally regulates expression of several target genes in protecting tissues against hypoxia. With hypoxic stress, vascular endothelial growth factor (VEGF) is a signal protein produced by cells and further contributes to improvement of vascular functions and restoring the oxygen supply to tissues. In this current study, we first hypothesized that the protein levels of HIF-1alpha and VEGF are reduced in skeletal muscles of plateau animals [China Qinghai-Tibetan plateau pikas (ochotona curzoniae)] in response to hypoxia as compared with control animals [normal lowland Sprague-Dawley (SD) rats]. We further hypothesized that HIF-1alpha plays a role in regulating expression of VEGF in skeletal muscle. Note that HIF-1alpha and VEGF were determined by using two-site immunoenzymatic assay (ELISA) methods. Our results demonstrated that hypoxic stress induced by exposure of lower O(2) (6 h) significantly increased the levels of HIF-1alpha and VEGF in the oxidative and glycolytic muscles of SD rats and pikas (P<0.05 vs. normoxic conditions). Notably, the increases in HIF-1alpha and VEGF were significantly less in pikas (P<0.05, vs. SD controls) than in SD rats. In addition, a linear relationship was observed between amplified HIF-1alpha and VEGF in oxidative muscle (r=0.76 and P<0.01) and glycolytic muscle (r=0.72 and P<0.01) and inhibiting HIF-1alpha significantly decreased expression of VEGF induced by hypoxic stress in skeletal muscles (P<0.05). Overall, our findings suggest that (1) responsiveness of HIF-1alpha and VEGF in skeletal muscles to hypoxic stress is blunted in plateau animals, and (2) HIF-1alpha has a regulatory effect on VEGF under hypoxic environment.

  19. Hypoxia-inducible factor 1-alpha release after intracoronary versus intramyocardial stem cell therapy in myocardial infarction.

    PubMed

    Gyöngyösi, Mariann; Hemetsberger, Rayyan; Posa, Aniko; Charwat, Silvia; Pavo, Noemi; Petnehazy, Ors; Petrasi, Zsolt; Pavo, Imre J; Hemetsberger, Hani; Benedek, Imre; Benedek, Teodora; Benedek, Istvan; Kovacs, Istvan; Kaun, Christoph; Maurer, Gerald

    2010-04-01

    We have investigated the effect of stem cell delivery on the release of hypoxia-inducible factor 1 alpha (HIF-1alpha) in peripheral circulation and myocardium in experimental myocardial ischemia. Closed-chest, reperfused myocardial infarction (MI) was created in domestic pigs. Porcine mesenchymal stem cells (MSCs) were cultured and delivered (9.8 +/- 1.2 x 10(6)) either percutaneously NOGA-guided transendocardially (Group IM) or intracoronary (Group IC) 22 +/- 4 days post-MI. Pigs without MSC delivery served as sham control (Group S). Plasma HIF-1alpha was measured at baseline, immediately post- and at follow-up (FUP; 2 h or 24 h) post-MSC delivery by ELISA kit. Myocardial HIF-1alpha expression of infarcted, normal myocardium, or border zone was determined by Western blot. Plasma level of HIF-1alpha increased immediately post-MI (from 278 +/- 127 to 631 +/- 375 pg/ml, p < 0.05). Cardiac delivery of MSCs elevated the plasma levels of HIF-1alpha significantly (p < 0.05) in groups IC and IM immediately post-MSC delivery, and returned to baseline level at FUP, without difference between the groups IC and IM. The myocardial tissue HIF-1alpha expression in the infarcted area was higher in Group IM than in Group IC or S (1,963 +/- 586 vs. 1,307 +/- 392 vs. 271 +/- 110 activity per square millimeter, respectively, p < 0.05), while the border zone contained similarly lower level of HIF-1alpha, but still significantly higher as compared with Group S. Trend towards increase in myocardial expression of HIF-1alpha was measured in Group IM at 24 h, in contrast to Group IC. In conclusion, both stem cell delivery modes increase the systemic and myocardial level of HIF-1alpha. Intramyocardial delivery of MSC seems to trigger the release of angiogenic HIF-1alpha more effectively than does intracoronary delivery.

  20. Novel 1alpha,25-dihydroxyvitamin D3 analogues with the side chain at C12.

    PubMed

    González-Avión, Xosé C; Mouriño, Antonio; Rochel, Natacha; Moras, Dino

    2006-03-09

    The plethora of actions of 1alpha,25(OH)2D3 in various systems suggested wide clinical applications of vitamin D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis, cancers, and autoimmune diseases. More than 3000 vitamin D analogues have been synthesized in order to reduce the calcemic side effects while maintaining the transactivation potency of the natural ligand. In light of the crystal structures of the vitamin D nuclear receptor (VDR), novel analogues of the hormone 1alpha,25(OH)2D3 with side chains attached to C-12 were synthesized via the convergent Wittig-Horner approach. Among the compounds studied, the analogue 2b showed the highest binding affinity for VDR and was the most potent at inducing VDR transcriptional activity in a transient transfection assay (20% of the transactivation activity of the natural ligand).

  1. The Structure of Neurexin 1[alpha] Reveals Features Promoting a Role as Synaptic Organizer

    SciTech Connect

    Chen, Fang; Venugopal, Vandavasi; Murray, Beverly; Rudenko, Gabby

    2014-10-02

    {alpha}-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The {alpha}-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how {alpha}-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1{alpha} extracellular domain (n1{alpha}) to 2.65 {angstrom}. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 {angstrom} structure of n1{alpha} carrying splice insert SS3 in LNS4 reveals that SS3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables {alpha}-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.

  2. Role of hypoxia-inducible factor 1{alpha} in modulating cobalt-induced lung inflammation.

    PubMed

    Saini, Yogesh; Kim, Kyung Y; Lewandowski, Ryan; Bramble, Lori A; Harkema, Jack R; Lapres, John J

    2010-02-01

    Hypoxia plays an important role in development, cellular homeostasis, and pathological conditions, such as cancer and stroke. There is also growing evidence that hypoxia is an important modulator of the inflammatory process. Hypoxia-inducible factors (HIFs) are a family of proteins that regulate the cellular response to oxygen deficit, and loss of HIFs impairs inflammatory cell function. There is little known, however, about the role of epithelial-derived HIF signaling in modulating inflammation. Cobalt is capable of eliciting an allergic response and promoting HIF signaling. To characterize the inflammatory function of epithelial-derived HIF in response to inhaled cobalt, a conditional lung-specific HIF1alpha, the most ubiquitously expressed HIF, deletion mouse, was created. Control mice showed classic signs of metal-induced injury following cobalt exposure, including fibrosis and neutrophil infiltration. In contrast, HIF1alpha-deficient mice displayed a Th2 response that resembled asthma, including increased eosinophilic infiltration, mucus cell metaplasia, and chitinase-like protein expression. The results suggest that epithelial-derived HIF signaling has a critical role in establishing a tissue's inflammatory response, and compromised HIF1alpha signaling biases the tissue towards a Th2-mediated reaction.

  3. Anti-IL-1 alpha autoantibodies in patients with rheumatic diseases and in healthy subjects.

    PubMed Central

    Suzuki, H; Ayabe, T; Kamimura, J; Kashiwagi, H

    1991-01-01

    We have developed a quantitative assay for IgG autoantibodies against IL-1 alpha using protein A-Sepharose CL-4B. We examined the autoantibodies in sera from 107 healthy subjects, 151 patients with rheumatoid arthritis (RA), 64 patients with systemic lupus erythematosus (SLE) and 16 patients with systemic sclerosis. The frequency of positive sera for the autoantibodies in patients with RA was 16.6%, which was about three times more frequent (P less than 0.01) than that in healthy subjects (5.6%) or that in patients with SLE (4.7%). Only one serum of 16 patients with systemic sclerosis was positive for the autoantibodies. Neutralizing activity of the autoantibodies was demonstrated by murine thymocyte proliferation assay. The concentrations of IgG at 50% inhibition of IL-1 alpha (15 pM) induced thymocyte proliferation ranged between 0.1 and 0.5 mg/ml. A time-course study showed fluctuations of the titres of the autoantibodies in parallel with the disease activity of RA. These results suggest that the anti-IL-1 alpha autoantibodies present in the sera and possibly some other body fluids may be involved in the regulation of IL-1 activity in vivo. Images Fig. 2 PMID:1893621

  4. Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

    PubMed

    Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

    2005-08-01

    Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

  5. Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio).

    PubMed

    Burzio, Veronica; Antonelli, Marcelo; Allende, Catherine C; Allende, Jorge E

    2002-01-01

    Protein kinase CK1 (previously known as casein kinase I) conforms to a subgroup of the great protein kinase family found in eukaryotic organisms. The CK1 subgroup of vertebrates contains seven members known as alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon. The CK1alpha gene can generate four variants (CK1alpha, CK1alphaS, CK1alphaL, and CK1alphaLS) through alternate splicing, characterized by the presence or absence of two additional coding sequences. Exon "L" encodes a 28-amino acid stretch that is inserted after lysine 152, in the center of the catalytic domain. The "S" insert encodes 12 amino acid residues and is located close to the carboxyl terminus of the protein. This work reports some biochemical and cellular properties of the four CK1alpha variants found to be expressed in zebrafish (Danio rerio). The results obtained indicate that the presence of the "L" insert affects several biochemical properties of CK1alpha: (a) it increases the apparent Km for ATP twofold, from approximately 30 to approximately 60 microM; (b) it decreases the sensitivity to the CKI-7 inhibitor, raising the I50 values from 113 to approximately 230 microM; (c) it greatly decreases the heat stability of the enzyme at 40 degrees C. In addition, the insertion of the "L" fragment exerts very important effects on some cellular properties of the enzyme. CK1alphaL concentrates in the cell nucleus, excluding nucleoli, while the CK1alpha variant is predominantly cytoplasmic, although some presence is observed in the nucleus. This finding supports the thesis that the basic-rich region found in the "L" insert acts as a nuclear localization signal. The "L" insert-containing variant was also found to be more rapidly degraded (half-life of 100 min) than the CK1alpha variant (half-life of 400 min) in transfected Cos-7 cells.

  6. Extended ischemia prevents HIF1alpha degradation at reoxygenation by impairing prolyl-hydroxylation: role of Krebs cycle metabolites.

    PubMed

    Serra-Pérez, Anna; Planas, Anna M; Núñez-O'Mara, Analía; Berra, Edurne; García-Villoria, Judit; Ribes, Antònia; Santalucía, Tomàs

    2010-06-11

    Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that activates the cellular response to hypoxia. The HIF1alpha subunit is constantly synthesized and degraded under normoxia, but degradation is rapidly inhibited when oxygen levels drop. Oxygen-dependent hydroxylation by prolyl-4-hydroxylases (PHD) mediates HIF1alpha proteasome degradation. Brain ischemia limits the availability not only of oxygen but also of glucose. We hypothesized that this circumstance could have a modulating effect on HIF. We assessed the separate involvement of oxygen and glucose in HIF1alpha regulation in differentiated neuroblastoma cells subjected to ischemia. We report higher transcriptional activity and HIF1alpha expression under oxygen deprivation in the presence of glucose (OD), than in its absence (oxygen and glucose deprivation, OGD). Unexpectedly, HIF1alpha was not degraded at reoxygenation after an episode of OGD. This was not due to impairment of proteasome function, but was associated with lower HIF1alpha hydroxylation. Krebs cycle metabolites fumarate and succinate are known inhibitors of PHD, while alpha-ketoglutarate is a co-substrate of the reaction. Lack of HIF1alpha degradation in the presence of oxygen was accompanied by a very low alpha-ketoglutarate/fumarate ratio. Furthermore, treatment with a fumarate analogue prevented HIF1alpha degradation under normoxia. In all, our data suggest that postischemic metabolic alterations in Krebs cycle metabolites impair HIF1alpha degradation in the presence of oxygen by decreasing its hydroxylation, and highlight the involvement of metabolic pathways in HIF1alpha regulation besides the well known effects of oxygen.

  7. Genetic signatures of differentiation induced by 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells.

    PubMed

    Pálmer, Héctor G; Sánchez-Carbayo, Marta; Ordóñez-Morán, Paloma; Larriba, María Jesús; Cordón-Cardó, Carlos; Muñoz, Alberto

    2003-11-15

    Epidemiological and preclinical data indicate that vitamin D and its most active metabolite 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] have anticancer activity. Accordingly, clinical trials are under way using several nonhypercalcemic 1alpha,25(OH)(2)D(3) analogues against various neoplasms including colon cancer. 1alpha,25(OH)(2)D(3) induces proliferation arrest and epithelial differentiation of human SW480-ADH colon cancer cells. We examined the gene expression profiles associated with 1alpha,25(OH)(2)D(3) exposure using oligonucleotide microarrays. 1alpha,25(OH)(2)D(3) changed the expression levels of numerous previously unreported genes, including many involved in transcription, cell adhesion, DNA synthesis, apoptosis, redox status, and intracellular signaling. Most genes were up-regulated, and only a small fraction were down-regulated. Fourteen of 17 candidate genes studied were validated as 1alpha,25(OH)(2)D(3) target genes by Northern and Western blotting or immunocytochemistry. They included c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, keratin-13, G(0)S2, and the putative tumor suppressors NES-1 and protease M. There was little overlap between genes regulated after short (4 h) or long (48 h) exposure. Gene regulatory effects of 1alpha,25(OH)(2)D(3) in SW480-ADH cells differed from those in LS-174T cells, which lack E-cadherin and do not differentiate in response to 1alpha,25(OH)(2)D(3). Data from this study reveal that 1alpha,25(OH)(2)D(3) causes a profound change in gene expression profiles and provide a mechanistic basis to the ongoing clinical studies using nonhypercalcemic vitamin D(3) derivatives for colon cancer prevention and treatment.

  8. Assay of 25-hydroxy vitamin D3-1 alpha-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry

    SciTech Connect

    Holmberg, I.; Saarem, K.; Pedersen, J.I.; Bjoerkhem, I.

    1986-12-01

    An assay of 1 alpha-hydroxylation of 25-hydroxy vitamin D3 in pig kidney mitochondria, based on selected ion monitoring, has been developed. Trideuterium-labeled 1,25-dihydroxy vitamin D3 was synthesized and used as internal standard. This standard was added immediately after incubation of 25-hydroxy vitamin D3 with the mitochondrial fraction. The incubation extracts were purified by high-performance liquid chromatography. After formation of the trimethylsilyl derivative, the product was quantitated by mass fragmentography using the ion at m/z 452 and m/z 455. With the use of this assay it was found that formation of 1,25-dihydroxy vitamin D3 was linear with the amount of mitochondrial protein and time of incubation. Substrate saturation was obtained at about 20 microM of 25-hydroxy vitamin D3. The maximal rate of conversion obtained under the conditions employed was about 0.1 pmol/mg protein X minute.

  9. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  10. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  11. Mitochondrial genetics

    PubMed Central

    Chinnery, Patrick Francis; Hudson, Gavin

    2013-01-01

    Introduction In the last 10 years the field of mitochondrial genetics has widened, shifting the focus from rare sporadic, metabolic disease to the effects of mitochondrial DNA (mtDNA) variation in a growing spectrum of human disease. The aim of this review is to guide the reader through some key concepts regarding mitochondria before introducing both classic and emerging mitochondrial disorders. Sources of data In this article, a review of the current mitochondrial genetics literature was conducted using PubMed (http://www.ncbi.nlm.nih.gov/pubmed/). In addition, this review makes use of a growing number of publically available databases including MITOMAP, a human mitochondrial genome database (www.mitomap.org), the Human DNA polymerase Gamma Mutation Database (http://tools.niehs.nih.gov/polg/) and PhyloTree.org (www.phylotree.org), a repository of global mtDNA variation. Areas of agreement The disruption in cellular energy, resulting from defects in mtDNA or defects in the nuclear-encoded genes responsible for mitochondrial maintenance, manifests in a growing number of human diseases. Areas of controversy The exact mechanisms which govern the inheritance of mtDNA are hotly debated. Growing points Although still in the early stages, the development of in vitro genetic manipulation could see an end to the inheritance of the most severe mtDNA disease. PMID:23704099

  12. [Mitochondrial myopathies].

    PubMed

    Finsterer, J

    2009-11-01

    The organ most frequently affected in mitochondrial disorders is the skeletal muscle (mitochondrial myopathy). Mitochondrial myopathies may be part of syndromic as well as non-syndromic mitochondrial disorders. Involvement of the skeletal muscle may remain subclinical, may manifest as isolated elevation of the creatine-kinase, or as weakness and wasting of one or several muscle groups. The course of mitochondrial myopathies is usually slowly progressive and only rarely rapidly progressive leading to restriction of mobility and requirement of a wheel chair or even muscular respiratory insufficiency. Frequently reported symptoms of mitochondrial myopathies are permanent tiredness, easy fatigability, muscle aching at rest or already after moderate exercise, muscle cramps, muscle stiffness, fasciculations and muscle weakness. The diagnosis is based on the history, clinical neurologic examination, blood chemical investigations, lactate stress test, electromyography, magnetic resonance imaging, magnetic resonance spectroscopy, muscle biopsy, biochemical investigations of the skeletal muscles, and genetic investigations. Only symptomatic therapy is available and includes physiotherapy and orthopedic supportive devices, diet, symptomatic drug therapy (analgetics, cramp-releasing drugs, antioxidants, lactate-lowering drugs, alternative energy sources, co-factors), avoidance of mitochondrion-toxic drugs, surgery (correction of ptosis or orthopedic problems), and invasive or non-invasive mechanical ventilation. General anesthesia needs to be performed in the same way as in patients with susceptibility for malignant hyperthermia. Georg Thieme Verlag KG Stuttgart, New York.

  13. HNF-1alpha participates in glucose regulation of sucrase-isomaltase gene expression in epithelial intestinal cells.

    PubMed

    Gu, Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke

    2007-02-16

    Sucrase-isomaltase (SI) gene expression is negatively regulated by glucose, but its molecular mechanism is not completely clear. The purpose of this study is to investigate whether HNF-1alpha and HNF-1beta contribute to glucose regulation of SI gene expression. To explore this question, we examined the association of gene expressions between SI and HNF-1alpha and HNF-1beta in Caco-2 cells cultured in medium containing 2.0 and 16.7 mM glucose. We found that gene expression of HNF-1alpha but not HNF-1beta exhibits a positive correlation with that of SI regulated by glucose. Moreover, to elucidate whether glucose regulation of SI gene expression is changed when HNF-1alpha and HNF-1beta are inhibited, we produced three stable cell lines, in which dominant-negative mutant HNF-1alphaT539fsdelC, mutant HNF-1betaR177X, and empty vector (as a control), respectively, were stably expressed. We found that the glucose regulation of SI gene expression was significantly attenuated in HNF-1alphaT539fsdelC cells, but it was well maintained in empty vector and HNF-1betaR177X cells. These results suggest that HNF-1alpha participates in glucose regulation of SI gene expression.

  14. Human eosinophils can express the cytokines tumor necrosis factor-alpha and macrophage inflammatory protein-1 alpha.

    PubMed Central

    Costa, J J; Matossian, K; Resnick, M B; Beil, W J; Wong, D T; Gordon, J R; Dvorak, A M; Weller, P F; Galli, S J

    1993-01-01

    By in situ hybridization, 44-100% of the blood eosinophils from five patients with hypereosinophilia and four normal subjects exhibited intense hybridization signals for TNF-alpha mRNA. TNF-alpha protein was detectable by immunohistochemistry in blood eosinophils of hypereosinophilic subjects, and purified blood eosinophils from three atopic donors exhibited cycloheximide-inhibitable spontaneous release of TNF-alpha in vitro. Many blood eosinophils (39-91%) from hypereosinophilic donors exhibited intense labeling for macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA, whereas eosinophils of normal donors demonstrated only weak or undetectable hybridization signals for MIP-1 alpha mRNA. Most tissue eosinophils infiltrating nasal polyps were strongly positive for both TNF-alpha and MIP-1 alpha mRNA. By Northern blot analysis, highly enriched blood eosinophils from a patient with the idiopathic hypereosinophilic syndrome exhibited differential expression of TNF-alpha and MIP-1 alpha mRNA. These findings indicate that human eosinophils represent a potential source of TNF-alpha and MIP-1 alpha, that levels of expression of mRNA for both cytokines are high in the blood eosinophils of hypereosinophilic donors and in eosinophils infiltrating nasal polyps, that the eosinophils of normal subjects express higher levels of TNF-alpha than MIP-1 alpha mRNA, and that eosinophils purified from the blood of atopic donors can release TNF-alpha in vitro. Images PMID:8514874

  15. Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1 [alpha]. Search for a function.

    PubMed

    Joshi, R L; Ravel, J M; Haenni, A L

    1986-06-01

    The 3'-terminal tRNA-like structure in turnip yellow mosaic virus (TYMV) RNA can be adenylated by tRNA nucleotidyltransferase and subsequently aminoacylated by valyl-tRNA synthetase. Here we present evidence that TYMV Val-RNA can form a stable complex with eukaryotic wheat germ elongation factor EF-1alpha and GTP: the Val-RNA is protected by EF-1alpha.. GTP against digestion by RNase A. By affinity chromatography of TYMV Val-RNA fragments on immobilized EF-1alpha . GTP, it has been established that the valylated aminoacyl RNA domain, which in TYMV RNA is formed by the 3' half of the tRNA-like region, is sufficient for complex formation with EF-1alpha . GTP. The aminoacyl RNA domain is equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop. In line with these results, the aminoacyl RNA domain in TYMV Val-RNA complexed to EF-1 alpha . GTP is resistant to digestion by RNase A. It is also shown that the TYMV RNA replicase (RNA-dependent RNA polymerase) isolated from TYMV-infected Chinese cabbage leaves does not contain tRNA nucleotidyltransferase, valyl-tRNA synthetase or EF-1alpha. This suggests that interaction of TYMV RNA with EF-1alpha is not mandatory for replicase activity.

  16. Interaction of SDF-1alpha and CXCR4 plays an important role in pulmonary cellular infiltration in differentiation syndrome.

    PubMed

    Zhou, Jin; Hu, Longhu; Cui, Zhe; Jiang, Xian; Wang, Guifang; Krissansen, Geoffrey W; Sun, Xueying

    2010-03-01

    This study aims to investigate the role of stromal cell-derived factor 1alpha (SDF-1alpha) and its receptor CXCR4 in cellular infiltration of the lung in differentiation syndrome (DS). The acute promyelocytic leukemia (APL) NB4 cells and freshly prepared APL cells from the patients were differentiated by all-trans retinoic acid (ATRA). The expression of SDF-1alpha in human lung tissues was examined by RT-PCR and Western blot analysis. The cells were subjected to adhesion, migration or invasion assays, and co-cultured with human lung tissues in a microgravity rotary cell culture system to examine cellular infiltration in situ. ATRA-differentiated cells expressed high levels of CXCR4, and adhered more strongly to matrigel. Their ability to migrate and invade was enhanced by SDF-1alpha and lung homogenate, and diminished by pre-treatment with an anti-CXCR4 blocking antibody. SDF-1alpha was expressed in the lung tissues of all seven human donors. ATRA-differentiated NB4 cells infiltrated into lung tissues, and this was reduced by pre-treatment with an anti-CXCR4 blocking antibody. The interaction of SDF-1alpha and CXCR4 plays an important role in pulmonary cellular infiltration during DS, suggesting that targeting SDF-1alpha and CXCR4 may provide the basis for potential treatments in the management of DS.

  17. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1{alpha} involved in cell fusion, we measured the ability of nectin-1{alpha}/nectin-2{alpha} chimeras, nectin-1{alpha}/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1{alpha} to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1{alpha} cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1{alpha} and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1{alpha} interaction in fusion.

  18. Involvement of PPAR gamma co-activator-1, nuclear respiratory factors 1 and 2, and PPAR alpha in the adaptive response to endurance exercise.

    PubMed

    Baar, Keith

    2004-05-01

    Endurance exercise training induces an increase in the respiratory capacity of muscle, resulting in an increased capacity to generate ATP as well as improved efficiency of muscle contraction. Such adaptations are largely the result of a coordinated genetic response that increases mitochondrial proteins, fatty acid oxidation enzymes and the exercise- and insulin-stimulated glucose transporter GLUT4, and shifts the contractile and regulatory proteins to their more efficient isoforms. In recent years a number of the transcriptional regulators involved in this genetic response have been identified and these factors can be classified into two different groups. The first group comprises transcription factors such as nuclear respiratory factors (NRF) 1 and 2 and PPAR alpha that bind DNA in a sequence-specific manner. The second group, referred to as transcriptional co-activators, alter transcription without directly binding to DNA. The PPAR gamma co-activator (PGC) family of proteins have been identified as the central family of transcriptional co-activators for induction of mitochondrial biogenesis. PGC-1 alpha is activated by exercise, and is sufficient to produce the endurance phenotype through direct interactions with NRF-1 and PPAR alpha, and potentially NRF-2. Furthering the understanding of the activation of PGC proteins following exercise has implications beyond improving athletic performance, including the possibility of providing targets for the treatment of frailty in the elderly, obesity and diseases such as mitochondrial myopathies and diabetes.

  19. Ontogenic loss of brown adipose tissue sensitivity to beta-adrenergic stimulation in the ovine.

    PubMed

    Lomax, Michael A; Sadiq, Fouzia; Karamanlidis, Georgios; Karamitri, Angeliki; Trayhurn, Paul; Hazlerigg, David G

    2007-01-01

    In ruminants and other large animals, expression of uncoupling protein-1 (UCP1) in brown adipose tissue (BAT) is confined to the perinatal period when it plays a key role in nonshivering thermogenesis. This study determined whether loss of expression of the BAT phenotype was due to reduced response to a beta-agonist, isoprenaline, and expression of the peroxisome proliferator-activated receptor (PPAR) family [PPARalpha, PPARgamma, PPAR coactivator 1alpha (PGC-1alpha)], which regulates UCP1 gene expression. Perirenal adipose tissue (PAT) was sampled from ovine fetuses, newborn lambs, and lambs on d 1, 5, 7, and 21 of life. UCP1 mRNA and protein in PAT increased from d 123 of fetal life to reach a maximum at birth followed by a rapid decrease over the first 5 d of life. Expression of the coactivator, PGC-1alpha and PPAR alpha, peaked between fetal day 123 and birth, and then declined to undetectable levels in the first days of life. In vivo administration of isoprenaline was able to induce expression of UCP1, PGC-1alpha, and PPARalpha in BAT up to 5 d of age but thereafter was ineffective. In vitro addition of beta-receptor, PPARalpha, and PPARgamma agonists were unable to overcome the suppression of UCP1, PPARalpha, and PPARgamma expression observed in differentiated adipocytes prepared from 30-d-old compared with 1-d-old lambs. These data are consistent with a model in which postnatal loss of UCP1 expression and beta-adrenergic induction of the brown adipocyte phenotype is due to loss of expression of PGC-1alpha and PPARalpha.

  20. Fasting induced up-regulation of activating transcription factor 5 in mouse liver.

    PubMed

    Shimizu, Yusuke I; Morita, Momoko; Ohmi, Asako; Aoyagi, Shun; Ebihara, Hitomi; Tonaki, Daijuro; Horino, Yoko; Iijima, Mika; Hirose, Hidenori; Takahashi, Shigeru; Takahashi, Yuji

    2009-06-19

    Food deprivation (fasting) is commonly encountered in the lives of animals and humans. In mammals, adaptive responses predominantly include the induction of hepatic gluconeogenesis, but the regulatory mechanisms remain unclear. Atf5 (activating transcription factor 5) is a transcription factor of the ATF/cAMP response element-binding protein family and is expressed abundantly in human liver. Atf5 has been associated with stress responses, cell differentiation, proliferation, and survival. However, its role in the liver response to in vivo food deprivation has not yet been investigated. Adult mice were food-deprived for 48 h and the expression of two Atf5 mRNA subtypes (Atf5-R1 and Atf-R2) and gluconeogenic factors was investigated. Using in vitro cell culture, Pgc-1alpha (peroxisome proliferator-activated receptor-gamma coactivator-1alpha) promoter activities after ectopic expression of Atf5 and Cebpg (CCAAT/enhancer-binding protein gamma) proteins were measured. The Atf5-R1 transcript was found to be abundant in liver and other energy metabolism-related organs; Atf5-R2 was prominent in the testis. Fasting resulted in elevation of the expression of both Atf5-R1 and R2 in the liver. Interestingly, up-regulation of Atf5 was accompanied by increased expression of Cebpg and Pgc-1alpha. In human hepatoma cells (HepG2), but not in human cervical carcinoma cells (HeLa), forced expression of Atf5 and Cebpg cooperatively stimulated Pgc-1alpha promoter activity, suggesting that hepatic Pgc-1alpha could be induced by Atf5 and Cebpg in cooperation with other hepatic factors. Hepatic Atf5 might be potentially involved in the induction of gluconeogenetic factors during in vivo fasting stress.

  1. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha.

    PubMed

    Rius, Jordi; Guma, Monica; Schachtrup, Christian; Akassoglou, Katerina; Zinkernagel, Annelies S; Nizet, Victor; Johnson, Randall S; Haddad, Gabriel G; Karin, Michael

    2008-06-05

    The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2) (ref. 1) and controlled by hypoxia-inducible transcription factor-1 (HIF-1), whose alpha subunit is rapidly degraded under normoxia but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain are inhibited. Thus, the amount of HIF-1alpha, which controls genes involved in energy metabolism and angiogenesis, is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-kappaB plays a central role. NF-kappaB activation is controlled by IkappaB kinases (IKK), mainly IKK-beta, needed for phosphorylation-induced degradation of IkappaB inhibitors in response to infection and inflammation. IKK-beta is modestly activated in hypoxic cell cultures when PHDs that attenuate its activation are inhibited. However, defining the relationship between NF-kappaB and HIF-1alpha has proven elusive. Using in vitro systems, it was reported that HIF-1alpha activates NF-kappaB, that NF-kappaB controls HIF-1alpha transcription and that HIF-1alpha activation may be concurrent with inhibition of NF-kappaB. Here we show, with the use of mice lacking IKK-beta in different cell types, that NF-kappaB is a critical transcriptional activator of HIF-1alpha and that basal NF-kappaB activity is required for HIF-1alpha protein accumulation under hypoxia in cultured cells and in the liver and brain of hypoxic animals. IKK-beta deficiency results in defective induction of HIF-1alpha target genes including vascular endothelial growth factor. IKK-beta is also essential for HIF-1alpha accumulation in macrophages experiencing a bacterial infection. Hence, IKK-beta is an important physiological contributor to the hypoxic response, linking it to innate immunity and inflammation.

  2. Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells.

    PubMed

    Kang, Gi Soo; Li, Qin; Chen, Haobin; Costa, Max

    2006-11-07

    Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 microM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 microM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.

  3. The ML1/alpha =2 Instability Strip of ZZ Ceti Stars

    NASA Astrophysics Data System (ADS)

    Kepler, S. O.; da Costa, A. F. M.; Giovannini, O.; Koester, D.

    We are obtaining high signal-to-noise optical spectra and time-series photometry of all known DA white dwarfs around the ZZ Ceti instability strip to derive their atmospheric parameters and masses. In this work we report the fundamental parameters for 13 DA stars using ML1/alpha =2 model atmospheres and analyze the ZZ Ceti instability strip with a sample of 92 DA white dwarfs. This sample has an average mass of 0.59 +/- 0.02 Msun with a dispersion of 0.15 Msun. Our study shows that the limits of the ZZ Ceti instability strip depend on the effective temperature and mass of the star.

  4. Nuclear receptor signaling and cardiac energetics.

    PubMed

    Huss, Janice M; Kelly, Daniel P

    2004-09-17

    The heart has a tremendous capacity for ATP generation, allowing it to function as an efficient pump throughout the life of the organism. The adult myocardium uses either fatty acid or glucose oxidation as its main energy source. Under normal conditions, the adult heart derives most of its energy through oxidation of fatty acids in mitochondria. However, the myocardium has a remarkable ability to switch between carbohydrate and fat fuel sources so that ATP production is maintained at a constant rate in diverse physiological and dietary conditions. This fuel selection flexibility is important for normal cardiac function. Although cardiac energy conversion capacity and metabolic flux is modulated at many levels, an important mechanism of regulation occurs at the level of gene expression. The expression of genes involved in multiple energy transduction pathways is dynamically regulated in response to developmental, physiological, and pathophysiological cues. This review is focused on gene transcription pathways involved in short- and long-term regulation of myocardial energy metabolism. Much of our knowledge about cardiac metabolic regulation comes from studies focused on mitochondrial fatty acid oxidation. The genes involved in this key energy metabolic pathway are transcriptionally regulated by members of the nuclear receptor superfamily, specifically the fatty acid-activated peroxisome proliferator-activated receptors (PPARs) and the nuclear receptor coactivator, PPARgamma coactivator-1alpha (PGC-1alpha). The dynamic regulation of the cardiac PPAR/PGC-1 complex in accordance with physiological and pathophysiological states will be described.

  5. Reptilian uncoupling protein: functionality and expression in sub-zero temperatures.

    PubMed

    Rey, Benjamin; Sibille, Brigitte; Romestaing, Caroline; Belouze, Maud; Letexier, Dominique; Servais, Stéphane; Barré, Hervé; Duchamp, Claude; Voituron, Yann

    2008-05-01

    Here we report the partial nucleotide sequence of a reptilian uncoupling protein (repUCP) gene from the European common lizard (Lacerta vivipara). Overlapping sequence analysis reveals that the protein shows 55%, 72% and 77% sequence homology with rat UCP1, UCP2 and UCP3, respectively, and 73% with bird and fish UCPs. RepUCP gene expression was ubiquitously detected in 4 degrees C cold-acclimated lizard tissues and upregulated in muscle tissues by a 20 h exposure to sub-zero temperatures in a supercooling state or after thawing. In parallel, we show an increase in the co-activators, peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and peroxisome proliferator-activated receptors (PPAR), mRNA expression, suggesting that the mechanisms regulating UCP expression may be conserved between mammals (endotherms) and reptiles (ectotherms). Furthermore, mitochondria extracted from lizard skeletal muscle showed a guanosine diphosphate (GDP)-sensitive non phosphorylating respiration. This last result indicates an inhibition of extra proton leakage mediated by an uncoupling protein, providing arguments that repUCP is functional in lizard tissues. This result is associated with a remarkable GDP-dependent increase in mitochondrial endogenous H(2)O(2) production. All together, these data support a physiological role of the repUCP in superoxide limitation by lizard mitochondria in situations of stressful oxidative reperfusion following a re-warming period in winter.

  6. Regulatory roles for L-arginine in reducing white adipose tissue

    PubMed Central

    Tan, Bi’e; Li, Xinguo; Yin, Yulong; Wu, Zhenlong; Liu, Chuang; Tekwe, Carmen D.; Wu, Guoyao

    2012-01-01

    As the nitrogenous precursor of nitric oxide, L-arginine regulates multiple metabolic pathways involved in the metabolism of fatty acids, glucose, amino acids, and proteins through cell signaling and gene expression. Specifically, arginine stimulates lipolysis and the expression of key genes responsible for activation of fatty acid oxidation to CO2 and water. The underlying mechanisms involve increases in the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), mitochondrial biogenesis, and the growth of brown adipose tissue growth. Furthermore, arginine regulates adipocyte-muscle crosstalk and energy partitioning via the secretion of cytokines and hormones. In addition, arginine enhances AMP-activated protein kinase (AMPK) expression and activity, thereby modulating lipid metabolism and energy balance toward the loss of triacylglycerols. Growing evidence shows that dietary supplementation with arginine effectively reduces white adipose tissue in Zucker diabetic fatty rats, diet-induced obese rats, growing-finishing pigs, and obese patients with type II diabetes. Thus, arginine can be used to prevent and treat adiposity and the associated metabolic syndrome. PMID:22652774

  7. Regulatory roles for L-arginine in reducing white adipose tissue.

    PubMed

    Tan, Bi'e; Li, Xinguo; Yin, Yulong; Wu, Zhenlong; Liu, Chuang; Tekwe, Carmen D; Wu, Guoyao

    2012-06-01

    As the nitrogenous precursor of nitric oxide, L-arginine regulates multiple metabolic pathways involved in the metabolism of fatty acids, glucose, amino acids, and proteins through cell signaling and gene expression. Specifically, arginine stimulates lipolysis and the expression of key genes responsible for activation of fatty acid oxidation to CO2 and water. The underlying mechanisms involve increases in the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), mitochondrial biogenesis, and the growth of brown adipose tissue growth. Furthermore, arginine regulates adipocyte-muscle crosstalk and energy partitioning via the secretion of cytokines and hormones. In addition, arginine enhances AMP-activated protein kinase (AMPK) expression and activity, thereby modulating lipid metabolism and energy balance toward the loss of triacylglycerols. Growing evidence shows that dietary supplementation with arginine effectively reduces white adipose tissue in Zucker diabetic fatty rats, diet-induced obese rats, growing-finishing pigs, and obese patients with type II diabetes. Thus, arginine can be used to prevent and treat adiposity and the associated metabolic syndrome.

  8. Skeletal muscle NAMPT is induced by exercise in humans.

    PubMed

    Costford, Sheila R; Bajpeyi, Sudip; Pasarica, Magdalena; Albarado, Diana C; Thomas, Shantele C; Xie, Hui; Church, Timothy S; Jubrias, Sharon A; Conley, Kevin E; Smith, Steven R

    2010-01-01

    In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects (P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content (R(2) = 0.28, P < 0.01), MRS-measured maximal ATP synthesis (R(2) = 0.37, P = 0.002), and Vo(2max) (R(2) = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training (P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an approximately 2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an approximately 3.4-fold increase in NAMPT mRNA (P < 0.05) as well as modest increases in NAMPT protein (P < 0.05) and mitochondrial content (P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.

  9. Hypoxia-inducible factor-1alpha suppresses squamous carcinogenic progression and epithelial-mesenchymal transition.

    PubMed

    Scortegagna, Marzia; Martin, Rebecca J; Kladney, Raleigh D; Neumann, Robert G; Arbeit, Jeffrey M

    2009-03-15

    Hypoxia-inducible factor-1 (HIF-1) is a known cancer progression factor, promoting growth, spread, and metastasis. However, in selected contexts, HIF-1 is a tumor suppressor coordinating hypoxic cell cycle suppression and apoptosis. Prior studies focused on HIF-1 function in established malignancy; however, little is known about its role during the entire process of carcinogenesis from neoplasia induction to malignancy. Here, we tested HIF-1 gain of function during multistage murine skin chemical carcinogenesis in K14-HIF-1alpha(Pro402A564G) (K14-HIF-1alphaDPM) transgenic mice. Transgenic papillomas appeared earlier and were more numerous (6 +/- 3 transgenic versus 2 +/- 1.5 nontransgenic papillomas per mouse), yet they were more differentiated, their proliferation was lower, and their malignant conversion was profoundly inhibited (7% in transgenic versus 40% in nontransgenic mice). Moreover, transgenic cancers maintained squamous differentiation whereas epithelial-mesenchymal transformation was frequent in nontransgenic malignancies. Transgenic basal keratinocytes up-regulated the HIF-1 target N-myc downstream regulated gene-1, a known tumor suppressor gene in human malignancy, and its expression was maintained in transgenic papillomas and cancer. We also discovered a novel HIF-1 target gene, selenium binding protein-1 (Selenbp1), a gene of unknown function whose expression is lost in human cancer. Thus, HIF-1 can function as a tumor suppressor through transactivation of genes that are themselves targets for negative selection in human cancers.

  10. Selective solid-phase extraction of urinary 2,3-dinor-6-ketoprostaglandin F1 alpha for determination with radioimmunoassay.

    PubMed

    Riutta, A; Nurmi, E; Weber, C; Hansson, G; Vapaatalo, H; Mucha, I

    1994-08-01

    This paper describes a method for selective two-step solid-phase extraction of urinary 2,3-dinor-6-ketoprostaglandin F1 alpha for reliable determination with radioimmunoassay. In the immunoreactivity profile of non-selectively extracted urine after HPLC separation, over 90% of the total 2,3-dinor-6-ketoprostaglandin F1 alpha immunoreactivity consisted of interfering material coeluting with 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha. Among the alkyl silica sorbents studied (methyl, butyl, octyl, and octadecyl), an efficient separation of 2,3-dinor-6-ketoprostaglandin F1 alpha from 6-ketoprostaglandin F1 alpha and the lowest immunoreactive concentration of analyte were achieved in extraction on the methyl silica sorbent by elution of 2,3-dinor-6-ketoprostaglandin F1 alpha with chloroform: hexane (85:15, v/v) from the cartridge. The proportion of specific immunoreactivity could be further increased by two-step extraction of sample on methyl silica cartridges, first at pH 3 and then at pH 10 using diethyl ether:hexane (85:15, v/v) and chloroform as eluent, respectively. After this, a high correlation was found with concentrations of samples determined by radioimmunoassay using three different antisera. A significant correlation of values was also observed between samples measured by radioimmunoassay and those measured by GC-MS. The values of 12-h excretion of 2,3-dinor-6-ketoprostaglandin F1 alpha in eight volunteers (268 +/- 204 ng/g creatinine, mean +/- SD) as well as the inhibitory effect of acetylsalicylic acid (74 +/- 12%) are in accordance with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring subsequent TLC or HPLC purification.

  11. Over-expression of CCL3 MIP-1alpha in a blastoid mantle cell lymphoma with hypercalcemia.

    PubMed

    Hattori, Norimichi; Nakamaki, Tsuyoshi; Ariizumi, Hirotsugu; Homma, Mayumi; Yamochi-Onizuka, Toshiko; Ota, Hidekazu; Tomoyasu, Shigeru

    2010-05-01

    We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1alpha in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-alpha, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1alpha significantly stimulated (3)H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1alpha. In the present case, MIP-1alpha, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1alpha is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1alpha is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

  12. Synapse loss regulated by matrix metalloproteinases in traumatic brain injury is associated with hypoxia inducible factor-1alpha expression.

    PubMed

    Ding, Jamie Y; Kreipke, Christian W; Schafer, Patrick; Schafer, Steven; Speirs, Susan L; Rafols, José A

    2009-05-01

    The present study assessed the role of matrix metalloproteinase-2 (MMP-2) and -9 in synapse loss after traumatic brain injury (TBI) and the role of hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor up-regulated during hypoxia, in the regulation of MMP-2 and -9 expression post-TBI. Adult male Sprague-Dawley rats (n=6 per group, 400 g-425 g) were injured using Marmarou's closed-head acceleration impact model and allowed to survive for 1, 4, 24 and 48 h. In another set of experiments, 30 min after TBI, animals were treated with Minocycline (inhibitor of MMPs), or 2-Methoxyestradiol (2ME2, inhibitor of HIF-1alpha) and sacrificed at 4 h after injury. Relative amounts of synaptophysin, a presynaptic vesicular protein, HIF-1alpha, as well as MMP-2 and -9 were assessed by real-time PCR and Western blotting. Activity levels of MMP-2 and -9 were determined by zymography. Synaptophysin expression was significantly (p<0.05) decreased at 1 h through 48 h after TBI. A significant increase in gene and protein expressions of HIF-1alpha, MMP-2 and -9, as well as enzyme activity of MMP-2 and -9 at the same time points was also detected. Inhibition of either MMPs or HIF-1alpha significantly reversed the TBI-induced decrease in synaptophysin. Inhibition of HIF-1alpha reduced expression of MMP-2 and -9. This study showed an early detection of a correlation between synaptic loss and MMP expression after TBI. The data also supports a role for HIF-1alpha in the MMP regulatory cascade in synapse loss after TBI, suggesting potential targets for reducing loss of synaptic terminals.

  13. Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha

    SciTech Connect

    McFarlane, Steven; Nicholl, Mary Jane; Sutherland, Jane S.; Preston, Chris M.

    2011-05-25

    The cellular protein hypoxia-inducible factor 1 alpha (HIF-1{alpha}) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1{alpha} was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1{alpha}-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1{alpha} to occur. HIF-1{alpha} controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology.

  14. Dehydroepiandrosterone effects on the mRNA levels of peroxisome proliferator-activated receptors and their coactivators in human hepatoma HepG2 cells.

    PubMed

    Poczatková, H; Bogdanová, K; Uherková, L; Cervenková, K; Riegrová, D; Rypka, M; Veselý, J

    2007-12-01

    To investigate the effect of dehydroepiandrosterone (DHEA) on intracellular mRNA levels of peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs), we conducted a quantitative real-time RT-PCR study using HepG2 cells. Treatment with 100 micromol/l DHEA for 2-20 h caused a time-dependent elevation of mRNA levels in the cells. Upon 20 h, PPAR-alpha, -gamma1, and -gamma2 mRNAs and PGC-1alpha and -1beta mRNAs increased to 157, 161, 155, 656, and 475% of control levels, respectively (p < 0.05 each). Treatment with actinomycin D for 2.5-8 h revealed a significant stabilization effect of DHEA on PPAR-gamma1 and PGC-1alpha mRNAs at both 2.5 and 8 h incubation periods and a mild but significant stabilization effect on PGC-1beta mRNA at the 8 h incubation period suggesting that DHEA can modulate turnover of these mRNA transcripts. Basal mRNA levels of PPAR-alpha and PGC-1alpha were significantly suppressed upon 20 h treatment with cycloheximide, while those of PPAR-gamma1, -gamma2, and PGC-1beta were elevated. Cycloheximide also significantly reduced DHEA-induced accumulation of PPAR-alpha, -gamma1, -gamma2, and PGC-1alpha mRNAs, demonstrating the dependence of the DHEA action on de novo protein synthesis. The findings demonstrate that a supraphysiological concentration of DHEA can substantially influence gene expression of the PPAR signalling machinery at both transcriptional and posttranscriptional levels.

  15. Inhibition of Androgen-Independent Growth of Prostate Cancer by siRNA-Mediated Androgen Receptor Gene Silencing

    DTIC Science & Technology

    2006-02-01

    member FOXO1 (105, 125). The FKHR family proteins play an important role in many cellular processes including cell proliferation through regulation... FOXO1 ) through a proteolytic mechanism in prostate cancer cells. J Biol Chem. 2004; 279(14):13866-77. 126. Medema RH, Kops GJ, Bos JL, Burgering BM...BM. Insulin-regulated hepatic gluconeogenesis through FOXO1 -PGC-1alpha interaction. Nature. 2003; 423: 550-5. 137. Nakamura N, Ramaswamy S

  16. PGC-1beta is downregulated by training in human skeletal muscle: no effect of training twice every second day vs. once daily on expression of the PGC-1 family.

    PubMed

    Mortensen, Ole Hartvig; Plomgaard, Peter; Fischer, Christian P; Hansen, Anne K; Pilegaard, Henriette; Pedersen, Bente Klarlund

    2007-11-01

    We hypothesized that the peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) family of transcriptional coactivators (PGC-1alpha, PGC-1beta, and PRC) is differentially regulated by training once daily vs. training twice daily every second day and that this difference might be observed in the acute response to endurance exercise. Furthermore, we hypothesized that expression levels of the PGC-1 family differ with muscular fiber-type composition. Thus, before and after 10 wk of knee extensor endurance training, training one leg once daily and the other leg twice daily every second day, keeping the total amount of training for the legs equal, skeletal muscle mRNA expression levels of PGC-1alpha, PGC-1beta, and PRC were determined in young healthy men (n = 7) in response to 3 h of acute exercise. No significant difference was found between the two legs, suggesting that regulation of the PGC-1 family is independent of training protocol. Training decreased PGC-1beta in both legs, whereas PGC-1alpha was increased, but not significantly, in the leg training once daily. PRC did not change with training. Both PGC-1alpha and PRC were increased by acute exercise both before and after endurance training, whereas PGC-1beta did not change. The mRNA levels of the PGC-1 family were examined in different types of human skeletal muscle (triceps, soleus, and vastus lateralis; n = 7). Only the expression level of PGC-1beta differed and correlated inversely with percentage of type I fibers. In conclusion, there was no difference between training protocols on the acute exercise and training response of the PGC-1 family. However, training caused a decrease in PGC-1beta mRNA levels.

  17. mRNA levels of peroxisome proliferator-activated receptors and their coactivators are affected by glucose deprivation and oleate in human hepatoma hepG2 cells.

    PubMed

    Bogdanova, Katerina; Uherkova, Lenka; Poczatkova, Hana; Rypka, Miroslav; Vesely, Jaroslav

    2007-12-01

    Very modest changes in mRNA stability can affect critical points in cellular energy pathways. The aim of this study was to investigate the impact of energy abundant substrates on peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs) mRNA's steady-state levels. Quantitative RT-PCR study was performed to assess the effect of zero or normal (5 mmol/l) glucose and/or oleic acid (0.3 mmol/l) on mRNA levels of (PPARs) (PGCs) in HepG2 cells. PGC-1alpha mRNA was significantly upregulated in glucose deprived cells (123 % of the control level; p < 0.05), while PGC-1beta mRNA was significantly enhanced in oleate-fed cells (134 % and 160 % of control levels for zero glucose plus oleate and normal glucose plus oleate, respectively; p < 0.05) during the 0.5 h incubation. Upon the 4 h incubation, PPAR-gamma1 and PGC-1alpha mRNAs were significantly elevated in cells lacking glucose (142 % and 163 % of control levels, respectively; p < 0.05). Oleate significantly suppressed PPAR-alpha and PGC-1beta mRNA levels in glucose-deprived cells (58 % and 49 % of control levels, respectively; p < 0.05). PPAR-gamma1 and -gamma2 mRNAs were significantly superinduced when the cells were treated with cycloheximide, whereas PPAR-alpha and PGC-1alpha and-1beta mRNAs were destabilized. Upon actinomycin D treatment, glucose shortage significantly stabilized PPAR-alpha mRNA, while PGC-1alpha mRNA was destabilized by oleate in glucose-deprived cells. Our findings provide evidence that transcriptional processes that are under the control of energetic substrates are interconnected with concurrent translational processes that can change stability of mRNAs.

  18. Molecular basis of maple syrup urine disease: novel mutations at the E1 alpha locus that impair E1(alpha 2 beta 2) assembly or decrease steady-state E1 alpha mRNA levels of branched-chain alpha-keto acid dehydrogenase complex.

    PubMed Central

    Chuang, J. L.; Fisher, C. R.; Cox, R. P.; Chuang, D. T.

    1994-01-01

    We report the occurrence of three novel mutations in the E1 alpha (BCKDHA) locus of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1 alpha gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1 alpha subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1 alpha mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1 alpha subunit impairs its proper assembly with the normal E1 beta. Unassembled as well as misassembled E1 alpha and E1 beta subunits are degraded in the cell. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:8037208

  19. Sequence analysis of the EF-1 alpha gene family of Mucor racemosus.

    PubMed Central

    Sundstrom, P; Lira, L M; Choi, D; Linz, J E; Sypherd, P S

    1987-01-01

    Our previous studies have shown that Mucor racemosus possesses three genes (TEF-1, -2 and -3) for EF-1 alpha, and that all three genes are transcribed. However, the level of transcription varies markedly between the three genes, with TEF-1 mRNA levels being approximately two fold higher than TEF-3 and 6 fold higher than TEF-2. We have now completed the DNA sequence of both strands of all three genes and have found that these genes are highly homologous. TEF-2 and TEF-3 are more similar to each other than they are to TEF-1. The TEF-2 and the TEF-3 coding regions differ from TEF-1 at 30 and 37 positions respectively out of 1374 nucleotides. Twenty-six of these nucleotide substitutions were common to both TEF-2 and TEF-3, and the majority of the substitutions were clustered in the 5' region of the coding sequences. While the majority of these changes were silent, TEF-2 and TEF-3 differed from TEF-1 by having a lysine instead of a glutamate at amino acid position 41. In addition, TEF-2 and -3, but not TEF-1, each have an intron located near the 5' end of the coding region, although its size and sequence is not conserved between the two genes. All three genes have a conserved intron near the 3' end of the coding region. The sequence data have been analyzed with respect to the structure and function of EF-1 alpha in protein biosynthesis. PMID:3697088

  20. RANTES, MDC and SDF-1alpha, prevent the HIVgp120-induced food and water intake decrease in rats.

    PubMed

    Guzmán, Khalil; Guevara-Martínez, Marcela; Montes-Rodríguez, Corinne J; Prospéro-García, Oscar

    2006-03-20

    Human immunodeficiency virus (HIV)-wasting syndrome might be facilitated by the HIVgp120 affecting the immunological system. We studied the effect (subchronic administration: 5 days) of HIVgp120, and a few immune-response mediators: regulated upon activation normal T-cell expressed and presumably secreted (RANTES), stromal derived factor-1alpha (SDF-1alpha), macrophage-derived chemokine (MDC), and their combination, on food and water intake in rats, motor control and pain perception. Eighty male adult Wistar rats received an intracerebroventricular (icv) administration of: vehicle 5 microl/day or 0.92 nmol daily of HIVgp120IIIB, RANTES, SDF-1alpha, or MDC, and the combination of RANTES+HIVgp120IIIB, SDF-1alpha+HIVgp120IIIB, or MDC+HIVgp120IIIB. Food and water intake was measured every day during administration, and 24 and 48 h after the last administration. Rats were also weighed the first and the last day of experiment in order to detect the impact of these treatments in the body weight. HIVgp120IIIB significantly decreased food and water intake. These rats gain less weight than the control (vehicle) and chemokines-treated subjects with exception of those treated with SDF-1alpha that also gain less weight. In addition, HIVgp120 deteriorated motor control. HIVgp120IIIB effects on food and water intake, and motor control were prevented by these chemokines. HIVgp120+RANTES, HIVgp120+SDF-1alpha, and SDF-1alpha alone induced hyperalgesia. Results suggest an interaction between HIVgp120 and the chemokine system to generate the HIV-wasting syndrome, the motor abnormalities and changes in pain perception.

  1. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts.

    PubMed

    Vengellur, A; LaPres, J J

    2004-12-01

    Cobalt has been widely used in the treatment of anemia and as a hypoxia mimic in cell culture and it is known to activate hypoxic signaling by stabilizing the hypoxia inducible transcription factor 1alpha (HIF1alpha). However, cobalt exposure can lead to tissue and cellular toxicity. These studies were conducted to determine the role of HIF1alpha in mediating cobalt-induced toxicity. Mouse embryonic fibroblasts (MEFs) that were null for the HIF1alpha protein were used to show that HIF1alpha protein plays a major role in mediating cobalt-induced cytotoxicity. Previous work from our lab and others has shown that two BH3 domain containing cell death genes, BNip3 and NIX, are targets of hypoxia signaling. These experiments document that BNip3 and NIX expression is HIF1alpha-dependent, and cobalt induces their expression in a time and dose dependent manner. In addition, their expression is correlated with an increase in BNIP3 and NIX protein. Characteristically, the elevated level of BNIP3 was correlated with an increased presence of chromatin condensation, one marker for cell injury. Interestingly, this increased chromosomal condensation was not coupled to caspase-3 activation as usually seen in a typical apoptotic response. These results show that HIF1alpha is playing a major role in mediating cobalt-induced toxicity in mouse embryonic fibroblasts and may offer a possible mechanism for the underlying pathology of injuries seen in workers exposed to environmental contaminants that can influence the hypoxia signaling system, such as cobalt.

  2. Modulation of in vitro porcine natural killer cell activity by recombinant interleukin-1 alpha, interleukin-2 and interleukin-4.

    PubMed Central

    Knoblock, K F; Canning, P C

    1992-01-01

    In order to understand better how cytokines modulate porcine lymphocyte-mediated natural cytotoxicity and to develop a rapid and reliable colorimetric assay to study that activity in young pigs, we studied inherent and cytokine induced in vitro natural killer (NK) activity. The cytokines we studied were human recombinant interleukin-1 alpha (IL-1 alpha), IL-2, IL-4 and interferon-gamma (IFN-gamma). Natural killer activity by peripheral blood mononuclear cells (PBMC), reported as per cent specific lysis (%SL), was determined by the colorimetric measurement of lactate dehydrogenase released from tumour cell targets, YAC-1 and K562. Inherent NK activity was low and remained relatively unchanged by alterations of assay length or effector cell concentration. Low NK activity was also observed in response to IL-4 and IFN-gamma. IL-2 and, to a lesser extent, IL-1 alpha induced significant NK activity with trends towards increasing %SL with increasing cytokine dose. Optimal IL-1 alpha- and IL-2-induced NK activity could be observed at 18 hr, with significant activity stimulated by IL-2 as early as 4 hr. IL-2-induced NK activity was sensitive to effector cell concentration; %SL decreased as the effector to target ratio decreased. IL-1 alpha- and IL-2-induced NK activities were decreased in the presence of IL-4. These results indicate porcine PBMC are sensitive to in vitro modulation by human recombinant IL-1 alpha, IL-2 and IL-4. The ability of IL-1 alpha and IL-2 to induce swine NK activity and the ability of IL-4 to inhibit that activity are similar to the actions of those cytokines in human NK systems. PMID:1634252

  3. The distribution of Elongation Factor-1 Alpha (EF-1alpha), Elongation Factor-Like (EFL), and a non-canonical genetic code in the ulvophyceae: discrete genetic characters support a consistent phylogenetic framework.

    PubMed

    Gile, Gillian H; Novis, Philip M; Cragg, David S; Zuccarello, Giuseppe C; Keeling, Patrick J

    2009-01-01

    The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1alpha (EF-1alpha) while most Chlorophyta instead possess the related GTPase Elongation Factor-Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non-canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF-1alpha, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF-1alpha of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non-canonical genetic code previously described in A. acetabulum was observed in EF-1alpha sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis, and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.

  4. Thermosensitive chitosan-based hydrogels releasing stromal cell derived factor-1 alpha recruit MSC for corneal epithelium regeneration.

    PubMed

    Tang, Qiaomei; Luo, Chenqi; Lu, Bing; Fu, Qiuli; Yin, Houfa; Qin, Zhenwei; Lyu, Danni; Zhang, Lifang; Fang, Zhi; Zhu, Yanan; Yao, Ke

    2017-10-01

    Corneal epithelium integrity depends on continuous self-renewing of epithelium and connections between adjacent cells or between the cells and the basement membrane. Self-renewing epithelium cells mainly arise from the continuous proliferation and differentiation of the basal layer and limbal stem cells. The aim of the present study was to generate a bioactive, thermosensitive chitosan-gelatin hydrogel (CHI hydrogel) by incorporating exogenous recombinant human stromal cell-derived factor-1 alpha (SDF-1 alpha) for corneal epithelium regeneration. The exogenous SDF-1 alpha could enhance the stem cells proliferation, chemotaxis and migration, and the expression levels of related genes were significantly elevated in LESCs and mesenchymal stem cells (MSCs) in vitro. Moreover, the MSCs promoted the proliferation and maintained the corneal fate of the LESCs. The rat alkali injury model was used for in vivo study. The injured eyes were covered with CHI hydrogel alone or rhSDF-1 alpha-loaded CHI hydrogel. All rats were followed for 13days. Histological examination showed that the SDF-1 alpha/CHI hydrogel complex group had a nearly normal thickness; moreover, it was also found that this group could upregulate the expression of some genes and had more ΔNp63-positive cells. The SDF-1 alpha/CHI hydrogel complex group had a more tightly arranged epithelium compared with the control group using transmission electron microscopy (TEM). The mechanism for this may have involved the activation of stem cell homing and the secretion of growth factors via the SDF-1/CXCR4 chemokine axis. Therefore, SDF-1 alpha/CHI hydrogel complexes could provide a new idea for the clinical application. The clarity of cornea is important for normal vision. The loss or dysfunction of LESCs leads to the impairment of corneal epithelium. The complete regeneration of corneal epithelium has not been achieved. Our study demonstrated that the incorporation of rhSDF-1 alpha with CHI hydrogel accelerated corneal

  5. PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion.

    PubMed

    Pilegaard, Henriette; Birk, Jesper B; Sacchetti, Massimo; Mourtzakis, Marina; Hardie, D Graham; Stewart, Greg; Neufer, P Darrell; Saltin, Bengt; van Hall, Gerrit; Wojtaszewski, Jorgen F P

    2006-11-01

    To investigate pyruvate dehydrogenase (PDH)-E1alpha subunit phosphorylation and whether free fatty acids (FFAs) regulate PDH activity, seven subjects completed two trials: saline (control) and intralipid/heparin (intralipid). Each infusion trial consisted of a 4-h rest followed by a 3-h two-legged knee extensor exercise at moderate intensity. During the 4-h resting period, activity of PDH in the active form (PDHa) did not change in either trial, yet phosphorylation of PDH-E1alpha site 1 (PDH-P1) and site 2 (PDH-P2) was elevated in the intralipid compared with the control trial. PDHa activity increased during exercise similarly in the two trials. After 3 h of exercise, PDHa activity remained elevated in the intralipid trial but returned to resting levels in the control trial. Accordingly, in both trials PDH-P1 and PDH-P2 decreased during exercise, and the decrease was more marked during intralipid infusion. Phosphorylation had returned to resting levels at 3 h of exercise only in the control trial. Thus, an inverse association between PDH-E1alpha phosphorylation and PDHa activity exists. Short-term elevation in plasma FFA at rest increases PDH-E1alpha phosphorylation, but exercise overrules this effect of FFA on PDH-E1alpha phosphorylation leading to even greater dephosphorylation during exercise with intralipid infusion than with saline.

  6. Detection of pyruvate dehydrogenase E1 alpha-subunit deficiencies in females by immunohistochemical demonstration of mosaicism in cultured fibroblasts.

    PubMed

    Lib, Margarita Y; Brown, Ruth M; Brown, Garry K; Marusich, Michael F; Capaldi, Roderick A

    2002-07-01

    Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1 alpha-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1 alpha deficiency. We also show that anti-E1 alpha and anti-E2 MAbs, when used in immunocytochemical analysis, can detect mosaicism in cell cultures from female patients in which as few as 2-5% of cells express the deficiency. This immunocytochemical approach, which is fast, reliable, and quantitative, will be particularly useful in identifying females with PDH E1 alpha-subunit deficiency as a precursor to mutation analysis.

  7. Continuous infusion of macrophage inflammatory protein MIP-1alpha enhances leucocyte recovery and haemopoietic progenitor cell mobilization after cyclophosphamide.

    PubMed Central

    Marshall, E.; Woolford, L. B.; Lord, B. I.

    1997-01-01

    Macrophage inflammatory protein 1alpha (MIP-1alpha) inhibits haemopoietic stem cell proliferation. This property has been exploited in a murine chemotherapy model and has been shown to ameliorate cytotoxic-induced myelosuppression after S-phase-specific cytotoxic therapy. We have now shown that BB-10010, a stable mutant of MIP-1alpha, (a) is more effective when administered as a continuous infusion than when bolus injected and (b), when administered via a 7-day infusion during and after cyclophosphamide treatment, results in an earlier recovery of leucocyte numbers. This effect was accompanied by progenitor cell mobilization into the peripheral blood and included primitive cells with marrow-repopulating ability (MRA). Maximal mobilization and recovery of leucocytes occurred when MIP-1alpha was combined with granulocyte colony-stimulating factor (G-CSF) therapy. The findings suggest that MIP1-alpha used alone or in combination with G-CSF may allow delivery of a greater chemotherapy dose intensity as a consequence of both accelerated leucocyte recovery and maintenance of high-quality mobilized progenitor cells for harvesting and peripheral blood stem cell transplantation. PMID:9192972

  8. Covarion shifts cause a long-branch attraction artifact that unites microsporidia and archaebacteria in EF-1alpha phylogenies.

    PubMed

    Inagaki, Yuji; Susko, Edward; Fast, Naomi M; Roger, Andrew J

    2004-07-01

    Microsporidia branch at the base of eukaryotic phylogenies inferred from translation elongation factor 1alpha (EF-1alpha) sequences. Because these parasitic eukaryotes are fungi (or close relatives of fungi), it is widely accepted that fast-evolving microsporidian sequences are artifactually "attracted" to the long branch leading to the archaebacterial (outgroup) sequences ("long-branch attraction," or "LBA"). However, no previous studies have explicitly determined the reason(s) why the artifactual allegiance of microsporidia and archaebacteria ("M + A") is recovered by all phylogenetic methods, including maximum likelihood, a method that is supposed to be resistant to classical LBA. Here we show that the M + A affinity can be attributed to those alignment sites associated with large differences in evolutionary site rates between the eukaryotic and archaebacterial subtrees. Therefore, failure to model the significant evolutionary rate distribution differences (covarion shifts) between the ingroup and outgroup sequences is apparently responsible for the artifactual basal position of microsporidia in phylogenetic analyses of EF-1alpha sequences. Currently, no evolutionary model that accounts for discrete changes in the site rate distribution on particular branches is available for either protein or nucleotide level phylogenetic analysis, so the same artifacts may affect many other "deep" phylogenies. Furthermore, given the relative similarity of the site rate patterns of microsporidian and archaebacterial EF-1alpha proteins ("parallel site rate variation"), we suggest that the microsporidian orthologs may have lost some eukaryotic EF-1alpha-specific nontranslational functions, exemplifying the extreme degree of reduction in this parasitic lineage.

  9. Pro-gliogenic effect of IL-1alpha in the differentiation of embryonic neural precursor cells in vitro.

    PubMed

    Ajmone-Cat, Maria Antonietta; Cacci, Emanuele; Ragazzoni, Ylenia; Minghetti, Luisa; Biagioni, Stefano

    2010-05-01

    Inflammation is regarded as a main obstacle to brain regeneration. Major detrimental effects are attributed to microglial/macrophagic products, such as TNF-alpha and interleukin (IL)-6. The role of cytokines of the IL-1 family, particularly of IL-1alpha, in the modulation of neural precursor cell (NPC) properties is less characterized. IL-1alpha is one of the most abundant cytokines released upon acute stimulation of microglia with lipopolysaccharide and is down-regulated upon chronic stimulation. As we recently demonstrated, acutely activated microglia reduces NPC survival, prevent neuronal differentiation and promote glial differentiation. Chronically activated microglia are instead permissive to NPC survival and neuronal differentiation, and less effective in promoting astrocytic differentiation. We thus investigated whether IL-1alpha could contribute to the effects of acutely activated microglia on NPC. We found that NPC express functional IL-1 receptors and that exposure to recombinant IL-1alpha strongly enhances NPC differentiation into astrocytes, without affecting cell viability and neuronal differentiation. In the same conditions, recombinant IL-1beta has pro-gliogenic effects at concentrations 10-fold higher than those found in activated microglial conditioned media. Interestingly, immunodepletion of IL-1alpha in activated microglial conditioned media fails to revert microglial pro-gliogenic action and slightly enhances neuronal differentiation, revealing that other microglial-derived factors contribute to the modulation of NPC properties.

  10. Persistent induction of HIF-1alpha and -2alpha in cardiomyocytes and stromal cells of ischemic myocardium.

    PubMed

    Jürgensen, Jan Steffen; Rosenberger, Christian; Wiesener, Michael S; Warnecke, Christina; Hörstrup, Jan H; Gräfe, Michael; Philipp, Sebastian; Griethe, Wanja; Maxwell, Patrick H; Frei, Ulrich; Bachmann, Sebastian; Willenbrock, Roland; Eckardt, Kai-Uwe

    2004-09-01

    Hypoxia-inducible factor (HIF)-1alpha and -2alpha are key regulators of the transcriptional response to hypoxia and pivotal in mediating the consequences of many disease states. In the present work, we define their temporo-spatial accumulation after myocardial infarction and systemic hypoxia. Rats were exposed to hypoxia or underwent coronary artery ligation. Immunohistochemistry was used for detection of HIF-1alpha and -2alpha proteins and target genes, and mRNA levels were determined by RNase protection. Marked nuclear accumulation of HIF-1alpha and -2alpha occurred after both systemic hypoxia and coronary ligation in cardiomyocytes as well as interstitial and endothelial cells (EC) without pronounced changes in HIF mRNA levels. While systemic hypoxia led to widespread induction of HIF, expression after coronary occlusion occurred primarily at the border of infarcted tissue. This expression persisted for 4 wk, included infiltrating macrophages, and colocalized with target gene expression. Subsets of cells simultaneously expressed both HIF-alpha subunits, but EC more frequently induced HIF-2alpha. A progressive increase of HIF-2alpha but not HIF-1alpha occurred in areas remote from the infarct, including the interventricular septum. Cardiomyocytes and cardiac stromal cells exhibit a marked potential for a prolonged transcriptional response to ischemia mediated by HIF. The induction of HIF-1alpha and -2alpha appears to be complementary rather than solely redundant.

  11. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  12. Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

    PubMed

    Koo, Hyun-Na; Hong, Seung-Heon; Song, Bong-Keun; Kim, Cheorl-Ho; Yoo, Young-Hyun; Kim, Hyung-Min

    2004-01-16

    Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

  13. The role of AMP-activated protein kinase in mitochondrial biogenesis.

    PubMed

    Reznick, Richard M; Shulman, Gerald I

    2006-07-01

    While it has been known for more than 75 years that physical activity is associated with increased mitochondrial content in muscle, the molecular mechanism for this adaptive process has only recently been elucidated. This brief review examines existing studies that have identified AMPK-activated protein kinase (AMPK) and several other key regulators of mitochondrial biogenesis, including peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta, calcium/calmodulin-dependent protein kinase IV, and nitric oxide. In addition, the potential role of mitochondrial dysfunction in the pathogenesis of insulin resistance associated with ageing and type 2 diabetes mellitus is also discussed.

  14. In cultured astrocytes, p53 and MDM2 do not alter hypoxia-inducible factor-1alpha function regardless of the presence of DNA damage.

    PubMed

    Rempe, David A; Lelli, Katherine M; Vangeison, Grace; Johnson, Randall S; Federoff, Howard J

    2007-06-01

    A principal molecular mechanism by which cells respond to hypoxia is by activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Several studies describe a binding of p53 to HIF-1alpha in a protein complex, leading to attenuated function, half-life, and abundance of HIF-1alpha. However, these reports almost exclusively utilized transformed cell lines, and many employed transfection of p53 or HIF-1alpha plasmid constructs and/or p53 and HIF-1alpha reporter constructs as surrogates for endogenous protein activity and target expression, respectively. Thus, it remains an open and important question as to whether p53 inhibits HIF-1alpha-mediated transactivation of endogenous HIF-1alpha targets in nontransformed cells. After determining in primary astrocyte cultures the HIF-1alpha targets that were most dependent on HIF-1alpha function, we examined the effect of the loss of p53 function either alone or in combination with MDM2 on expression of these targets. Although p53 null astrocyte cultures resulted in markedly increased HIF-1alpha-dependent target expression compared with controls, this altered expression was determined to be the result of increased cell density of p53 null cultures and the accompanying acidosis, not loss of p53 protein. Although activation of p53 by DNA damage induced p53 target expression in astrocytes, it did not alter hypoxia-induced HIF-1alpha target expression. Finally, a combined loss of MDM2 and p53 did not alter HIF-1alpha target expression compared with loss of p53 alone. These data strongly suggest that p53 and MDM2 do not influence the hypoxia-induced transactivation of HIF-1alpha targets, regardless of p53 activation, in primary astrocytes.

  15. Curcumin Attenuates Gentamicin-Induced Kidney Mitochondrial Alterations: Possible Role of a Mitochondrial Biogenesis Mechanism

    PubMed Central

    Negrette-Guzmán, Mario; García-Niño, Wylly Ramsés; Tapia, Edilia; Zazueta, Cecilia; Huerta-Yepez, Sara; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Aparicio-Trejo, Omar Emiliano; Madero, Magdalena; Pedraza-Chaverri, José

    2015-01-01

    It has been shown that curcumin (CUR), a polyphenol derived from Curcuma longa, exerts a protective effect against gentamicin- (GM-) induced nephrotoxicity in rats, associated with a preservation of the antioxidant status. Although mitochondrial dysfunction is a hallmark in the GM-induced renal injury, the role of CUR in mitochondrial protection has not been studied. In this work, LLC-PK1 cells were preincubated 24 h with CUR and then coincubated 48 h with CUR and 8 mM GM. Treatment with CUR attenuated GM-induced drop in cell viability and led to an increase in nuclear factor (erythroid-2)-related factor 2 (Nrf2) nuclear accumulation and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) cell expression attenuating GM-induced losses in these proteins. In vivo, Wistar rats were injected subcutaneously with GM (75 mg/Kg/12 h) during 7 days to develop kidney mitochondrial alterations. CUR (400 mg/Kg/day) was administered orally 5 days before and during the GM exposure. The GM-induced mitochondrial alterations in ultrastructure and bioenergetics as well as decrease in activities of respiratory complexes I and IV and induction of calcium-dependent permeability transition were mostly attenuated by CUR. Protection of CUR against GM-induced nephrotoxicity could be in part mediated by maintenance of mitochondrial functions and biogenesis with some participation of the nuclear factor Nrf2. PMID:26345660

  16. Curcumin Attenuates Gentamicin-Induced Kidney Mitochondrial Alterations: Possible Role of a Mitochondrial Biogenesis Mechanism.

    PubMed

    Negrette-Guzmán, Mario; García-Niño, Wylly Ramsés; Tapia, Edilia; Zazueta, Cecilia; Huerta-Yepez, Sara; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Aparicio-Trejo, Omar Emiliano; Madero, Magdalena; Pedraza-Chaverri, José

    2015-01-01

    It has been shown that curcumin (CUR), a polyphenol derived from Curcuma longa, exerts a protective effect against gentamicin- (GM-) induced nephrotoxicity in rats, associated with a preservation of the antioxidant status. Although mitochondrial dysfunction is a hallmark in the GM-induced renal injury, the role of CUR in mitochondrial protection has not been studied. In this work, LLC-PK1 cells were preincubated 24 h with CUR and then coincubated 48 h with CUR and 8 mM GM. Treatment with CUR attenuated GM-induced drop in cell viability and led to an increase in nuclear factor (erythroid-2)-related factor 2 (Nrf2) nuclear accumulation and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) cell expression attenuating GM-induced losses in these proteins. In vivo, Wistar rats were injected subcutaneously with GM (75 mg/Kg/12 h) during 7 days to develop kidney mitochondrial alterations. CUR (400 mg/Kg/day) was administered orally 5 days before and during the GM exposure. The GM-induced mitochondrial alterations in ultrastructure and bioenergetics as well as decrease in activities of respiratory complexes I and IV and induction of calcium-dependent permeability transition were mostly attenuated by CUR. Protection of CUR against GM-induced nephrotoxicity could be in part mediated by maintenance of mitochondrial functions and biogenesis with some participation of the nuclear factor Nrf2.

  17. Telomere dysfunction induces metabolic and mitochondrial compromise

    PubMed Central

    Sahin, Ergün; Colla, Simona; Liesa, Marc; Moslehi, Javid; Müller, Florian L.; Guo, Mira; Cooper, Marcus; Kotton, Darrell; Fabian, Attila J.; Walkey, Carl; Maser, Richard S.; Tonon, Giovanni; Foerster, Friedrich; Xiong, Robert; Wang, Y. Alan; Shukla, Sachet A.; Jaskelioff, Mariela; Martin, Eric S.; Heffernan, Timothy P.; Protopopov, Alexei; Ivanova, Elena; Mahoney, John E.; Kost-Alimova, Maria; Perry, Samuel R.; Bronson, Roderick; Liao, Ronglih; Mulligan, Richard; Shirihai, Orian S.; Chin, Lynda; DePinho, Ronald A.

    2013-01-01

    Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere–p53–PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction. PMID:21307849

  18. Exercise intensity-dependent regulation of peroxisome proliferator-activated receptor coactivator-1 mRNA abundance is associated with differential activation of upstream signalling kinases in human skeletal muscle.

    PubMed

    Egan, Brendan; Carson, Brian P; Garcia-Roves, Pablo M; Chibalin, Alexander V; Sarsfield, Fiona M; Barron, Niall; McCaffrey, Noel; Moyna, Niall M; Zierath, Juleen R; O'Gorman, Donal J

    2010-05-15

    Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)-dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC-1alpha expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC-1alpha transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 +/- 4.0 min; HI, 36.0 +/- 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC-1alpha mRNA abundance increased in an intensity-dependent manner +3 h after exercise (LO, 3.8-fold; HI, 10.2-fold, P < 0.05). AMP-activated protein kinase (AMPK) (2.8-fold, P < 0.05) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen-activated protein kinase (MAPK) phosphorylation increased after both trials (2.0-fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor-2 (ATF-2), increased only after HI (2.4-fold, P < 0.05). Cyclic-AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0-fold, P < 0.05). In conclusion, exercise intensity regulates PGC-1alpha mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of

  19. Prognostic Significance of Tumor Hypoxia Inducible Factor-1{alpha} Expression for Outcome After Radiotherapy in Oropharyngeal Cancer

    SciTech Connect

    Silva, Priyamal; Slevin, Nick J.; Sloan, Philip; Valentine, Helen; Cresswell, Jo; Ryder, David; Price, Patricia; Homer, Jarrod J.; West, Catharine

    2008-12-01

    Purpose: Head-and-neck squamous cell carcinoma (HNSCC) represents a heterogeneous group of patients in terms of subsite, treatment, and biology. Currently most management decisions are based on clinical parameters with little appreciation of patient differences in underlying tumor biology. We investigated the prognostic significance of clinicopathologic features and tumor hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) expression in a homogeneous series of patients who underwent radiotherapy. Methods and Materials: An audit identified 133 consecutive patients with histologically proven squamous cell carcinoma of the tonsil or tongue base. All patients received primary radiotherapy between 1996 and 2001. Tumor HIF-1{alpha} expression was examined in 79 patients. Results: Features associated with poor locoregional control were low Hb level (p = 0.05) and advancing T (p = 0.008), N (p = 0.03), and disease (p = 0.008) stage. HIF-1{alpha} expression was a more significant adverse prognostic factor in the tonsil (hazard ratio [HR], 23.1; 95% confidence interval [CI]. 3.04-176.7) than the tongue-base tumor (HR, 2.86; 95% CI, 1.14-7.19) group (p = 0.03, test for interaction). High tumor HIF-1{alpha} expression was associated with low blood Hb levels (p = 0.03). In a multivariate analysis HIF-1{alpha} expression retained prognostic significance for locoregional control (HR, 7.10; 95% CI, 3.07-16.43) and cancer-specific survival (HR, 9.19; 95% CI, 3.90-21.6). Conclusions: There are significant differences in radiation therapy outcome within a homogeneous subsite of the oropharynx related to molecular marker expression. The work highlights the importance of studying homogeneous groups of patients in HNSCC, and the complex interrelationships between tumor biology and clinicopathologic factors. The establishment of tumor-type specific markers would represent a major advance in this area.

  20. Functional response to SDF1 alpha through over-expression of CXCR4 on adult subventricular zone progenitor cells.

    PubMed

    Liu, Xian Shuang; Chopp, Michael; Santra, Manoranjan; Hozeska-Solgot, Ann; Zhang, Rui Lan; Wang, Lei; Teng, Hua; Lu, Mei; Zhang, Zheng Gang

    2008-08-21

    The chemokine receptor CXCR4 and its ligand, stromal cell derived factor-1 alpha (SDF1 alpha) regulate neuroblast migration towards the ischemic boundary after stroke. Using loss- and gain-function, we investigated the biological effect of CXCR4/SDF1 alpha on neural progenitor cells. Neural progenitor cells, from the subventricular zone (SVZ) of the adult rat, were transfected with rat CXCR4-pLEGFP-C1 and pSIREN-RetroQ-CXCR4-siRNA retroviral vectors. Migration assay analysis showed that inhibition of CXCR4 by siRNA significantly reduced cell migration compared to the empty vector, indicating that CXCR4 mediated neural progenitor cell motility. When neural progenitor cells were cultured in growth medium containing bFGF (20 ng/ml), over-expression of CXCR4 significantly reduced the cell proliferation as measured by the number of bromodeoxyuridine+ (BrdU+) cells (26.4%) compared with the number in the control group (54.0%). Addition of a high concentration of SDF1 alpha (500 ng/ml) into the progenitor cells with over-expression of CXCR4 reversed the cell proliferation back to the control levels (57.6%). Immunostaining analysis showed that neither over-expression nor inhibition of CXCR4 altered the population of neurons and astrocytes, when neural progenitor cells were cultured in differentiation medium. These in vitro results suggest that CXCR4/SDF1 alpha primarily regulates adult neural progenitor cell motility but not differentiation, while over-expression of CXCR4 in the absence of SDF1 alpha decreases neural progenitor cell proliferation.

  1. Contrasting effects of rh-MIP-1 alpha and TGF-beta 1 on chronic myeloid leukemia progenitors in vitro.

    PubMed

    Holyoake, T L; Freshney, M G; Sproul, A M; Richmond, L J; Alcorn, M J; Steward, W P; Fitzsimons, E; Dunlop, D J; Franklin, I M; Pragnell, I B

    1993-10-01

    In chronic myeloid leukemia (CML) an abnormality at the stem cell level results in unregulated expansion of myeloid progenitors. The mechanism underlying this uncontrolled proliferation remains unclear. An in vitro clonogenic assay which detects the human counterpart of the murine colony forming unit (CFU) CFU-A/CFU-S day 12 was described in a report of our recent findings. CML bone marrow samples were found to proliferate in the CFU-A assay, producing colonies morphologically indistinguishable from normal controls. The bcr/abl transcripts were sought in the RNA from individual colonies using the polymerase chain reaction (PCR). For the five CML samples tested to date, the majority of CFU-A colonies at diagnosis or in early chronic phase were found to be bcr/abl positive. For normal controls both macrophage inflammatory protein-1 alpha (MIP-1 alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibited the proliferation of CFU-A colonies when directly added to the assay. In contrast, CML progenitors responded normally to TGF-beta 1, but showed no response to MIP-1 alpha. In suicide assays, for five normal bone marrow samples, CFU-A progenitors induced into S-phase returned to a quiescent state after treatment with MIP-1 alpha. CML progenitors demonstrated inherently high cycle status which showed no definite response to MIP-1 alpha. However, TGF-beta 1 resulted in quiescence of CML progenitor cycling. In conclusion, the primitive progenitors from CML samples were inhibited normally by TGF-beta 1 but showed no response to MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Epstein-Barr virus latent membrane protein 1 induces synthesis of hypoxia-inducible factor 1 alpha.

    PubMed

    Wakisaka, Naohiro; Kondo, Satoru; Yoshizaki, Tomokazu; Murono, Shigeyuki; Furukawa, Mitsuru; Pagano, Joseph S

    2004-06-01

    Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1 alpha and HIF-1 beta that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1 alpha is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1 alpha protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1 alpha by LMP1. Catalase, an H(2)O(2) scavenger, strongly suppresses LMP1-induced production of H(2)O(2), which results in a decrease in the expression of HIF-1 alpha induced by LMP1. Inhibition of the NF-kappa B, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1 alpha expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1 alpha protein expression, which is controlled by p42/p44 MAPK activity and H(2)O(2). The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1 alpha along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.

  3. What Is Mitochondrial DNA?

    MedlinePlus

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  4. Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism

    PubMed Central

    Ugrankar, Rupali; Berglund, Eric; Akdemir, Fatih; Tran, Christopher; Kim, Min Soo; Noh, Jungsik; Schneider, Rebekka; Ebert, Benjamin; Graff, Jonathan M.

    2015-01-01

    Circulating carbohydrates are an essential energy source, perturbations in which are pathognomonic of various diseases, diabetes being the most prevalent. Yet many of the genes underlying diabetes and its characteristic hyperglycaemia remain elusive. Here we use physiological and genetic interrogations in D. melanogaster to uncover the ‘glucome', the complete set of genes involved in glucose regulation in flies. Partial genomic screens of ∼1,000 genes yield ∼160 hyperglycaemia ‘flyabetes' candidates that we classify using fat body- and muscle-specific knockdown and biochemical assays. The results highlight the minor glucose fraction as a physiological indicator of metabolism in Drosophila. The hits uncovered in our screen may have conserved functions in mammalian glucose homeostasis, as heterozygous and homozygous mutants of Ck1alpha in the murine adipose lineage, develop diabetes. Our findings demonstrate that glucose has a role in fly biology and that genetic screenings carried out in flies may increase our understanding of mammalian pathophysiology. PMID:25994086

  5. The relationships between hypoxia-dependent markers: HIF-1alpha, EPO and EPOR in colorectal cancer.

    PubMed

    Baltaziak, Marek; Wincewicz, Andrzej; Kanczuga-Koda, Luiza; Lotowska, Joanna M; Koda, Mariusz; Sulkowska, Urszula; Baltaziak, Marcin; Podbielski, Monika; Sobaniec-Lotowska, Maria E; Sulkowski, Stanislaw

    2013-01-01

    Hypoxia triggers production of several cytoprotective proteins. Hypoxia-inducible factor 1alpha (HIF-1α) is a powerful stimulator of transcription of many genes, including erythropoietin (EPO) in hypoxia-affected cells. Recent data have also implicated signaling by EPO receptor (EPOR) as a new factor influencing tumor progression. The aim of the study was to detect by immunohistochemistry the presence of HIF-1α, EPO and EPOR in colorectal cancer (CRC) in reference to clinicopathological variables. We found the presence of the studied proteins in specimens of all 125 CRC patients which is suggestive of the occurrence of hypoxia in colorectal cancer tissues. The expression of HIF-1α correlated significantly with the presence of EPO and EPOR in all samples (P < 0.001, r = 0.549 and P < 0.001, r = 0.536, respectively). Significant correlations (from P < 0.024 to P < 0.001) were found in the analyses of CRC subgroups such as histopathological type tumor, tumor grade, tumor stage and patients with lymph nodes metastases. The same high significant correlations (P < 0.001) were observed in group of sex, age and tumor location. However, the values of the correlation coefficients (r) which usually ranged from 0.5 to 0.6 suggest the existence of independent or concurrent mechanism stimulating generation of these proteins in colorectal cancer.

  6. Stromal Cell-Derived Factor-1 Alpha is Cardioprotective After Myocardial Infarction

    PubMed Central

    Saxena, Ankur; Fish, Jason E.; White, Michael D.; Yu, Sangho; Smyth, James WP; Shaw, Robin M.; DiMaio, J. Michael; Srivastava, Deepak

    2009-01-01

    Background Heart disease is a leading cause of mortality throughout the world. Tissue damage from vascular occlusive events results in the replacement of contractile myocardium by nonfunctional scar tissue. The potential of new technologies to regenerate damaged myocardium is significant, although cell-based therapies must overcome several technical barriers. One possible cell-independent alternative is the direct administration of small proteins to damaged myocardium. Methods and Results Here we show that the secreted signaling protein stromal cell-derived factor-1 alpha (SDF-1α), which activates the cell-survival factor protein kinase B (PKB/Akt) via the G-protein-coupled receptor CXCR4, protected tissue after an acute ischemic event in mice and activated Akt within endothelial cells and myocytes of the heart. Significantly better cardiac function than in control mice was evident as early as 24 hours post-infarction as well as at 3, 14 and 28 days post-infarction. Prolonged survival of hypoxic myocardium was followed by an increase in levels of vascular endothelial growth factor (VEGF) protein and neo-angiogenesis. Consistent with improved cardiac function, mice exposed to SDF-1α demonstrated significantly decreased scar formation than control mice. Conclusions These findings suggest that SDF-1α may serve a tissue-protective and regenerative role for solid organs suffering a hypoxic insult. PMID:18427137

  7. Elongation factor 1-alpha is released into the culture medium during growth of Giardia intestinalis trophozoites.

    PubMed

    Skarin, Hanna; Ringqvist, Emma; Hellman, Ulf; Svärd, Staffan G

    2011-04-01

    The molecular pathogenesis of the intestinal parasite Giardia intestinalis is still not fully understood but excretory-secretory products have been suggested to be important during host-parasite interactions. Here we used SDS-PAGE gels and MALDI-TOF analysis to identify proteins released by Giardia trophozoites during in vitro growth. Serum proteins (mainly bovine serum albumin) in the growth medium, bind to the parasite surface and they are continuously released, which interfere with parasite secretome characterization. However, we identified two released Giardia proteins: elongation factor-1 alpha (EF-1α) and a 58 kDa protein, identified as arginine deiminase (ADI). This is the first description of EF-1α as a released/secreted Giardia protein, whereas ADI has been identified in an earlier secretome study. Two genes encoding EF-1α were detected in the Giardia WB genome 35 kbp apart with almost identical coding sequences but with different promoter and 3' regions. Promoter luciferase-fusions showed that both genes are transcribed in trophozoites. The EF-1α protein localizes to the nuclear region in trophozoites but it relocalizes to the cytoplasm during host-cell interaction. Recombinant EF-1α is recognized by serum from giardiasis patients. Our results suggest that released EF-1α protein can be important during Giardia infections.

  8. The cellular and behavioral consequences of interleukin-1 alpha penetration through the blood-brain barrier of neonatal rats: a critical period for efficacy.

    PubMed

    Tohmi, M; Tsuda, N; Zheng, Y; Mizuno, M; Sotoyama, H; Shibuya, M; Kawamura, M; Kakita, A; Takahashi, H; Nawa, H

    2007-11-30

    Proinflammatory cytokines circulating in the periphery of early postnatal animals exert marked influences on their subsequent cognitive and behavioral traits and are therefore implicated in developmental psychiatric diseases such as schizophrenia. Here we examined the relationship between the permeability of the blood-brain barrier to interleukin-1 alpha (IL-1 alpha) in neonatal and juvenile rats and their later behavioral performance. Following s.c. injection of IL-1 alpha into rat neonates, IL-1 alpha immunoreactivity was first detected in the choroid plexus, brain microvessels, and olfactory cortex, and later diffused to many brain regions such as neocortex and hippocampus. In agreement, IL-1 alpha administration to the periphery resulted in a marked increase in brain IL-1 alpha content of neonates. Repeatedly injecting IL-1 alpha to neonates triggered astrocyte proliferation and microglial activation, followed by behavioral abnormalities in startle response and putative prepulse inhibition at the adult stage. Analysis of covariance with a covariate of startle amplitude suggested that IL-1 alpha administration may influence prepulse inhibition. However, adult rats treated with IL-1 alpha as neonates exhibited normal learning ability as measured by contextual fear conditioning, two-way passive shock avoidance, and a radial maze task and had no apparent sign of structural abnormality in the brain. In comparison, when IL-1 alpha was administered to juveniles, the blood-brain barrier permeation was limited. The increases in brain IL-1 alpha content and immunoreactivity were less pronounced following IL-1 alpha administration and behavioral abnormalities were not manifested at the adult stage. During early development, therefore, circulating IL-1 alpha efficiently crosses the blood-brain barrier to induce inflammatory reactions in the brain and influences later behavioral traits.

  9. Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism.

    PubMed

    Wicks, Shawna E; Vandanmagsar, Bolormaa; Haynie, Kimberly R; Fuller, Scott E; Warfel, Jaycob D; Stephens, Jacqueline M; Wang, Miao; Han, Xianlin; Zhang, Jingying; Noland, Robert C; Mynatt, Randall L

    2015-06-23

    The correlations between intramyocellular lipid (IMCL), decreased fatty acid oxidation (FAO), and insulin resistance have led to the hypothesis that impaired FAO causes accumulation of lipotoxic intermediates that inhibit muscle insulin signaling. Using a skeletal muscle-specific carnitine palmitoyltransferase-1 KO model, we show that prolonged and severe mitochondrial FAO inhibition results in increased carbohydrate utilization, along with reduced physical activity; increased circulating nonesterified fatty acids; and increased IMCLs, diacylglycerols, and ceramides. Perhaps more importantly, inhibition of mitochondrial FAO also initiates a local, adaptive response in muscle that invokes mitochondrial biogenesis, compensatory peroxisomal fat oxidation, and amino acid catabolism. Loss of its major fuel source (lipid) induces an energy deprivation response in muscle coordinated by signaling through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) to maintain energy supply for locomotion and survival. At the whole-body level, these adaptations result in resistance to obesity.

  10. Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism

    PubMed Central

    Wicks, Shawna E.; Vandanmagsar, Bolormaa; Haynie, Kimberly R.; Fuller, Scott E.; Warfel, Jaycob D.; Stephens, Jacqueline M.; Wang, Miao; Han, Xianlin; Zhang, Jingying; Noland, Robert C.; Mynatt, Randall L.

    2015-01-01

    The correlations between intramyocellular lipid (IMCL), decreased fatty acid oxidation (FAO), and insulin resistance have led to the hypothesis that impaired FAO causes accumulation of lipotoxic intermediates that inhibit muscle insulin signaling. Using a skeletal muscle-specific carnitine palmitoyltransferase-1 KO model, we show that prolonged and severe mitochondrial FAO inhibition results in increased carbohydrate utilization, along with reduced physical activity; increased circulating nonesterified fatty acids; and increased IMCLs, diacylglycerols, and ceramides. Perhaps more importantly, inhibition of mitochondrial FAO also initiates a local, adaptive response in muscle that invokes mitochondrial biogenesis, compensatory peroxisomal fat oxidation, and amino acid catabolism. Loss of its major fuel source (lipid) induces an energy deprivation response in muscle coordinated by signaling through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) to maintain energy supply for locomotion and survival. At the whole-body level, these adaptations result in resistance to obesity. PMID:26056297

  11. “Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

    USDA-ARS?s Scientific Manuscript database

    The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated...

  12. Mitochondrial biosensors.

    PubMed

    De Michele, Roberto; Carimi, Francesco; Frommer, Wolf B

    2014-03-01

    Biosensors offer an innovative tool for measuring the dynamics of a wide range of metabolites in living organisms. Biosensors are genetically encoded, and thus can be specifically targeted to specific compartments of organelles by fusion to proteins or targeting sequences. Mitochondria are central to eukaryotic cell metabolism and present a complex structure with multiple compartments. Over the past decade, genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of mitochondrial physiology. To date, sensors for ATP, NADH, pH, hydrogen peroxide, superoxide anion, redox state, cAMP, calcium and zinc have been used in the matrix, intermembrane space and in the outer membrane region of mitochondria of animal and plant cells. This review summarizes the different types of sensors employed in mitochondria and their main limits and advantages, and it provides an outlook for the future application of biosensor technology in studying mitochondrial biology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Mitochondrial ataxias.

    PubMed

    Finsterer, Josef

    2009-09-01

    Mitochondrial disorders (MIDs) are an increasingly recognized condition. The second most frequently affected organ in MIDs is the central nervous system. One of the most prevalent clinical CNS manifestations of MIDs is ataxia. Ataxia may be even the dominant manifestation of a MID. This is why certain MIDs should be included in the classification of heredoataxias or at least considered as differentials of classical heredoataxias. MIDs due to mutations of the mitochondrial DNA, which develop ataxia include the MERRF, NARP, MILS, or KSS syndrome. More rarely, ataxia may be a feature of MELAS, LHON, PS, MIDD, or MSL. MIDs due to mutations of the nuclear DNA, which develop ataxia include LS, SANDO, SCAE, AHS, XSLA/A, IOSCA, MIRAS, MEMSA, or LBSL syndrome. More rarely ataxia can be found in AD-CPEO, AR-CPEO, MNGIE, DIDMOAD, CoQ-deficiency, ADOAD, DCMA, or PDC-deficiency. MIDs most frequently associated with ataxia are the non-syndromic MIDs. Syndromic and non-syndromic MIDs with ataxia should be delineated from classical heredoataxias to initiate appropriate symptomatic or supportive treatment.

  14. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.

    PubMed

    Li, Tiangang; Chiang, John Y L

    2005-01-01

    Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7 alpha-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4 alpha (HNF4 alpha, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4 alpha. PXR also interacted with peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha), which interacted with HNF4 alpha and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4 alpha and reduced PGC-1 alpha interaction with HNF4 alpha. Chromatin immunoprecipitation assay showed that PXR, HNF4 alpha, and PGC-1 alpha bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1 alpha from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4 alpha and blocks PGC-1 alpha activation with HNF4 alpha and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis.

  15. Sesquiterpenes and an intermediate 1alpha, 6beta, 11-eudesmanetriol in the biosynthesis of geosmin from Streptomyces sp.

    PubMed

    Yang, Ya-Bin; Yang, Zhi; Yang, Xue-Qiong; Zhang, Yong; Zhao, Li-Xing; Xu, Li-Hua; Ding, Zhong-Tao

    2012-03-01

    One new sesquiterpene was isolated from the fermentation broth of Streptomyces sp. and the structure was elucidated by spectral analysis as caryolane-1, 6beta-diol (1). An intermediate 1alpha, 6beta, 11-eudesmanetriol (2) in the biosynthesis of geosmin was also found in this strain which proved sequence for the reactions, especially bicyclization preceding dealkylation.

  16. Cloning, expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain.

    PubMed

    Masullo, Mariorosario; Cantiello, Piergiuseppe; Lamberti, Annalisa; Longo, Olimpia; Fiengo, Antonio; Arcari, Paolo

    2003-01-28

    The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.

  17. Activation of JAK2/STAT1-alpha-dependent signaling events during Mycobacterium tuberculosis-induced macrophage apoptosis.

    PubMed

    Rojas, Mauricio; Olivier, Martin; García, Luis F

    2002-01-01

    Induction of apoptosis by Mycobacterium tuberculosis in murine macrophage involves TNF-alpha and nitric oxide (NO) production and caspase cascade activation; however, the intracellular signaling pathways implicated remain to be established. Our results indicate that infection of the B10R murine macrophage line with M. tuberculosis induces apoptosis independent of mycobacterial phagocytosis and that M. tuberculosis induces protein tyrosine kinase (PTK) activity, JAK2/STAT1-alpha phosphorylation, and STAT1-alpha nuclear translocation. Inhibitors of PTK (AG-126), or JAK2 (AG-490) inhibited TNF-alpha and NO production, caspase 1 activation and apoptosis, suggesting that M. tuberculosis-induction of these events depends on JAK2/STAT1-alpha activation. In addition, we have obtained evidence that ManLAM capacity to inhibit M. tuberculosis-induced apoptosis involves the activation of the PTP SHP-1. The finding that M. tuberculosis infection activate JAK2/STAT1-alpha pathway suggests that M. tuberculosis might mimic macrophage-activating stimuli.

  18. Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene

    Treesearch

    Amy L. Ross-Davis; John W. Hanna; Mee-Sook Kim; Ned B. Klopfenstein

    2012-01-01

    The translation elongation factor-1 alpha gene was used to examine the phylogenetic relationships among 30 previously characterized isolates representing ten North American Armillaria species: A. solidipes (=A. ostoyae), A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, North American biological species X, A. cepistipes, and A. tabescens. The...

  19. Interleukin-1alpha and tumor necrosis factor-alpha expression during the early phases of orthodontic tooth movement in rats.

    PubMed

    Bletsa, Athanasia; Berggreen, Ellen; Brudvik, Pongsri

    2006-10-01

    Remodelling of the periodontium after application of mechanical forces constitutes the basis of clinical orthodontics and various immunoregulatory molecules are involved in this process. The aim of this study was to investigate the expression of the cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) in dental tissues during the early phases of orthodontic tooth movement. Eightteen male Wistar rats were used. All maxillary right first molars were moved orthodontically, with a force of 0.5 N, for 3 h, 1 d, and 3 d. The contralateral sides served as untreated controls. Parasagittal sections of the maxillary molars and the surrounding tissues were subjected to immunohistochemical staining for IL-1alpha or TNF-alpha, and were evaluated with light microscopy. IL-1alpha and TNF-alpha were expressed in the bone and periodontal ligament (PDL) along the roots of the orthodontically moved molars and in the gingiva. Increased expression of both cytokines was observed in the aforementioned areas after 1 and 3 d of tooth movement. The pulp tissue exhibited only minor changes in cytokine expression during tooth movement. The results suggest that mechanical stress results in almost immediate inflammatory reactions in various dental tissues.

  20. Mitochondrial fusion, fission, and mitochondrial toxicity.

    PubMed

    Meyer, Joel N; Leuthner, Tess C; Luz, Anthony L

    2017-08-05

    Mitochondrial dynamics are regulated by two sets of opposed processes: mitochondrial fusion and fission, and mitochondrial biogenesis and degradation (including mitophagy), as well as processes such as intracellular transport. These processes maintain mitochondrial homeostasis, regulate mitochondrial form, volume and function, and are increasingly understood to be critical components of the cellular stress response. Mitochondrial dynamics vary based on developmental stage and age, cell type, environmental factors, and genetic background. Indeed, many mitochondrial homeostasis genes are human disease genes. Emerging evidence indicates that deficiencies in these genes often sensitize to environmental exposures, yet can also be protective under certain circumstances. Inhibition of mitochondrial dynamics also affects elimination of irreparable mitochondrial DNA (mtDNA) damage and transmission of mtDNA mutations. We briefly review the basic biology of mitodynamic processes with a focus on mitochondrial fusion and fission, discuss what is known and unknown regarding how these processes respond to chemical and other stressors, and review the literature on interactions between mitochondrial toxicity and genetic variation in mitochondrial fusion and fission genes. Finally, we suggest areas for future research, including elucidating the full range of mitodynamic responses from low to high-level exposures, and from acute to chronic exposures; detailed examination of the physiological consequences of mitodynamic alterations in different cell types; mechanism-based testing of mitotoxicant interactions with interindividual variability in mitodynamics processes; and incorporating other environmental variables that affect mitochondria, such as diet and exercise. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Insufficient renal 1-alpha hydroxylase and bone homeostasis in aged rats with insulin resistance or type 2 diabetes mellitus.

    PubMed

    Chang-Quan, Huang; Bi-Rong, Dong; Ping, He; Zhen-Chan, Lu

    2008-01-01

    This study aimed to explore the relationship between insufficient renal 1-alpha hydroxylase (IRH) and bone homeostasis in type 2 diabetes mellitus (T2DM) or insulin resistance (IR) and to investigate whether IR plays a major role in the pathogenesis of both IRH and bone loss in T2DM. The experimental animal models of T2DM, IR, IR treated with vitamin D (VD), IR treated with 1-alpha hydroxyvitamin D (1alpha(OH) D, the product of renal 1-alpha hydroxylase), T2DM treated with VD, and T2DM treated with 1alpha(OH) D were established on 18-month-old male Wistar rats. For rats in each animal model and normal control rats, IR was detected by euglycemic insulin clamp technique (EICT) and glucose infusion rate (GIR, an index of IR) was calculated. Levels of serum 25-hydroxyvitamin D (25(OH)D) and serum active vitamin D (1,25(OH)(2)D) were determined by radioimmunoassay (RIA), and 1,25(OH)(2)D/25(OH)D ratio (1,25-25-R, an index of renal 1-alpha hydroxylase activity in vivo) was calculated; and bone mineral density (BMD) in femoral bone and lumbar vertebrae was measured by dual-energy X-ray absorption (DEXA). No significant difference was observed among the levels of 25(OH)D in all the rats. In IR rats, 1,25(OH)(2)D level, 1,25-25-R, and BMD level were significantly higher than those in T2DM rats and were lower than those in normal control rats. In the aged rats with T2DM or IR, administration of VD had no effect on 25(OH)D level, 1,25(OH)(2)D level, 1,25-25-R, and BMD level. Administration of 1alpha(OH) D had also no effect on 25(OH)D level but increased 1,25(OH)(2)D level, 1,25-25-R, and BMD level. For the aged rats with T2DM or IR, GIR positively correlated with both levels of 1,25(OH)(2)D and BMD, and 1,25-25-R positively and significantly correlated with levels of BMD. In T2DM or IR, IRH is a precipitating factor for bone loss. IR seems to play a major role in the pathogenesis of both IRH and bone loss in T2DM.

  2. SAG/ROC2/RBX2 is a HIF-1 target gene that promotes HIF-1 alpha ubiquitination and degradation.

    PubMed

    Tan, M; Gu, Q; He, H; Pamarthy, D; Semenza, G L; Sun, Y

    2008-02-28

    SAG (sensitive to apoptosis gene) or ROC2/RBX2 is the second family member of ROC1/RBX1, a component of SCF (Skp1, Cullin, F-box protein) and VCB (von Hippel-Lindau (VHL), Cullin and Elongin B/C) E3 ubiquitin ligases. SAG protected cells from hypoxia-induced apoptosis when overexpressed. We report here that SAG was subjected to hypoxia induction at the levels of mRNA and protein. Hypoxia induction of SAG was largely HIF-1alpha dependent. A consensus HIF-1-binding site, GCGTG was identified in the first intron of the SAG gene. In response to hypoxia, HIF-1 bound to this site and transactivated SAG expression. SAG transactivation required both the intact binding site in cis and HIF-1alpha in trans. On the other hand, like its family member, ROC1, SAG promoted VHL-mediated HIF-1alpha ubiquitination and degradation, which was significantly inhibited upon small interfering RNA silencing of SAG or ROC1. Furthermore, the endogenous HIF-1alpha at both basal and hypoxia-induced levels was significantly increased upon SAG silencing. Finally, SAG forms in vivo complex with Cul-5 and VHL under hypoxia condition. These results suggest an HIF-1-SAG feedback loop in response to hypoxia, as follows: hypoxia induces HIF-1 to transactivate SAG. Induced SAG then promotes HIF-1alpha ubiquitination and degradation. This feedback loop may serve as a cellular defensive mechanism to reduce potential cytotoxic effects of prolonged HIF-1 activation under hypoxia.

  3. SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

    SciTech Connect

    Campos, D; Peeters, W; Nickel, K; Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M

    2015-06-15

    Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

  4. Lipopolysaccharide induces the expression of interleukin-1alpha distinctly in different compartments of term and preterm human placentae.

    PubMed

    Huleihel, Mahmoud; Amash, Alaa; Sapir, Olga; Maor, Ester; Levy, Sharon; Katz, Miriam; Dukler, Doron; Myatt, Lesly; Holcberg, Gershon

    2004-01-01

    The aim of the study was to investigate the stimulatory effect of lipopolysaccharide (LPS) on IL-lalpha production in different compartments of term and preterm placental tissues. Homogenates from amnion, chorion, and from fetal (subchorionic placental tissues, maternal decidua, and mid-placental tissue before and after perfusion of isolated placental cotyledons of 5 term placentas and 4 placentas obtained after preterm birth (28-34 W of gestation) were examined. Isolated placental cotyledons were dually perfused LPS (100 ng/kg perfused placental tissue) was perfused into the maternal side during 10 hours. Homogenates of the samples were examined by ELISA for IL-1alpha levels, and paraffin sections of the samples were stained by immunohistochemical staining, to characterize the cellular origin of placental IL-1alpha. Paired t test and ANOVA determined statistical significance. In the homogenates, there was a tendency towards higher IL-lalpha levels in all preterm placental compartments as compared to the term compartments before perfusion. A significant increase was observed only in the chorion compartment (p = 0.035). LPS had significantly increased IL-la levels only in the decidua compartment of term placentas as compared to other placental compartments (p = 0.0004), and had decreased IL-1alpha levels in the mid-placenta (p = 0.034). In preterm placentas, addition of LPS did not affect the expression levels of IL-1alpha in either fetal or maternal compartments as determined by ELISA and immunohistochemical staining. IL-la levels in the chorion compartment of preterm placenta were significantly higher as compared to term placenta. LPS affects placental tissues of term and preterm placentas differently. Also, in the term placentas, LPS affected the different compartments differently. Thus, IL-1alpha may have a key role (as a autocrine/paracrine factor) in the regulation of normal and pathological pregnancy and parturition.

  5. High glucose concentrations attenuate hypoxia-inducible factor-1{alpha} expression and signaling in non-tumor cells

    SciTech Connect

    Dehne, Nathalie; Bruene, Bernhard

    2010-04-15

    Hypoxia-inducible factor (HIF) is the major transcription factor mediating adaption to hypoxia e.g. by enhancing glycolysis. In tumor cells, high glucose concentrations are known to increase HIF-1{alpha} expression even under normoxia, presumably by enhancing the concentration of tricarboxylic acid cycle intermediates, while reactions of non-tumor cells are not well defined. Therefore, we analyzed cellular responses to different glucose concentrations in respect to HIF activation comparing tumor to non-tumor cells. Using cells derived from non-tumor origin, we show that HIF-1{alpha} accumulation was higher under low compared to high glucose concentrations. Low glucose allowed mRNA expression of HIF-1 target genes like adrenomedullin. Transfection of C{sub 2}C{sub 12} cells with a HIF-1{alpha} oxygen-dependent degradation domaine-GFP fusion protein revealed that prolyl hydroxylase (PHD) activity is impaired at low glucose concentrations, thus stabilizing the fusion protein. Mechanistic considerations suggested that neither O{sub 2} redistribution nor an altered redox state explains impaired PHD activity in the absence of glucose. In order to affect PHD activity, glucose needs to be metabolized. Amino acids present in the medium also diminished HIF-1{alpha} expression, while the addition of fatty acids did not. This suggests that glucose or amino acid metabolism increases oxoglutarate concentrations, which enhances PHD activity in non-tumor cells. Tumor cells deprived of glutamine showed HIF-1{alpha} accumulation in the absence of glucose, proposing that enhanced glutaminolysis observed in many tumors enables these cells to compensate reduced oxoglutarate production in the absence of glucose.

  6. Wortmannin influences hypoxia-inducible factor-1 alpha expression and glycolysis in esophageal carcinoma cells.

    PubMed

    Zeng, Ling; Zhou, Hai-Yun; Tang, Na-Na; Zhang, Wei-Feng; He, Gui-Jun; Hao, Bo; Feng, Ya-Dong; Zhu, Hong

    2016-05-28

    To investigate the influence of phosphatidylinositol-3-kinase protein kinase B (PI3K/AKT)-HIF-1α signaling pathway on glycolysis in esophageal carcinoma cells under hypoxia. Esophageal carcinoma cell lines Eca109 and TE13 were cultured under hypoxia environment, and the protein, mRNA and activity levels of hypoxia inducible factor-1 alpha (HIF-1α), glucose transporter 1, hexokinase-II, phosphofructokinase 2 and lactate dehydrogenase-A were determined. Supernatant lactic acid concentrations were also detected. The PI3K/AKT signaling pathway was then inhibited with wortmannin, and the effects of hypoxia on the expression or activities of HIF-1α, associated glycolytic enzymes and lactic acid concentrations were observed. Esophageal carcinoma cells were then transfected with interference plasmid with HIF-1α-targeting siRNA to assess impact of the high expression of HIF-1α on glycolysis. HIF-1α is highly expressed in the esophageal carcinoma cell lines tested, and with decreasing levels of oxygen, the expression of HIF-1α and the associated glycolytic enzymes and the extracellular lactic acid concentration were enhanced in the esophageal carcinoma cell lines Eca109 and TE13. In both normoxia and hypoxic conditions, the level of glycolytic enzymes and the secretion of lactic acid were both reduced by wortmannin. The expression and activities of glycolytic enzymes and the lactic acid concentration in cells were reduced by inhibiting HIF-1α, especially the decreasing level of glycolysis was significant under hypoxic conditions. The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells.

  7. Stromal Cell-Derived Factor-1 Alpha Is Decreased in Women With Migraine With Aura.

    PubMed

    Liman, Thomas G; Neeb, Lars; Rosinski, Jana; Reuter, Uwe; Endres, Matthias

    2016-09-01

    Endothelial dysfunction may contribute to the pathophysiology of migraine with aura. Stromal cell-derived factor-1 alpha (SDF-1α) is involved in the maintenance of endothelial integrity via mobilization of vascular stem cells. We sought to determine whether SDF-1α levels are decreased in women with MA. In this post hoc analysis of a case-cohort study, levels of SDF-1α were determined by enzyme-linked immunosorbent assay. Endothelial function was assessed using peripheral arterial tonometry. Arterial stiffness was assessed by fingertip tonometry derived and heart-rate-adjusted augmentation index (AI). Twenty-eight women with MA and 27 age-matched healthy women were included in this study. Levels of SDF-1α were significantly lower in women with MA compared to age- and risk factor-matched healthy women (1763 ± 281 vs 2013 ± 263 pg/mL, P = 0.006). SDF-1α levels were positively correlated with AI in healthy women (r = 0.49, P = 0.009), but not in women with MA (r = 0.05, P = 0.78). SDF-1α levels were negatively correlated with CD144-positive endothelial microparticles (EMP; r = -0.31, P = .02), and activated CD62E-positive EMP (r = -0.35, P = .01). Levels of SDF-1α are decreased in women with MA and are associated with EMPs as a surrogate marker of endothelial dysfunction. This might contribute to the pathophysiology and vascular risk in MA, but evidence from larger prospective studies is warranted. © 2016 American Headache Society.

  8. Clinicopathologic findings in (anti-FcepsilonR1alpha) autoimmune-related chronic urticaria.

    PubMed

    Rojanapremsuk, Theera; Kasprowicz, Sarah; Schafer, Ewa; Story, Rachel; Clarke, Michael S; Walls, Timothy; Snyder, Vivian; Gleason, Briana C; Thomas, Antoinette B; Cibull, Thomas

    2015-05-01

    One cause of chronic urticaria is autoreactivity which is diagnosed by detecting autoantibodies against the IgE receptor alpha subunit (anti-Fc R1alpha). To compare the histopathologic features of chronic urticaria patients testing positive for anti-IgE receptor antibody (Ab) to those testing negative. Totally, 438 patients with a clinical presentation of chronic urticaria (2011-2013) had anti-IgE receptor Ab tested and 37 of those patients had skin biopsy. We evaluated microscopic features including: spongiosis, dermal edema, presence of mast cells, density of lymphocytic infiltration, predomination of eosinophils/neutrophils; intravascular neutrophils and presence of vasculitis. The aforementioned features were compared between negative and positive anti-IgE receptor Ab groups. Of 37 patients , 69% were women and 31% were men. 49% had positive anti-IgE receptor Ab and 51% had negative anti-IgE receptor Ab. In the positive anti-IgE receptor Ab group, 83% showed intravascular neutrophils. Eosinophil predominance was identified in 72% and neutrophil predominance was identified in 28%. In the negative anti-IgE receptor Ab group, 89% showed intravascular neutrophils. Eosinophil predominance was identified in 53% and neutrophil predominance was identified in 47%. There was no evidence of vasculitis in either group. There were no significant histopathologic differences between the anti-IgE receptor Ab positive and negative cases. Therefore, serum testing for anti-IgE receptor Ab is required to identify this subgroup of chronic urticaria patients. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle.

    PubMed

    Takahashi, Natsumi; Davy, Philip M C; Gardner, Lauren H; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C

    2016-01-01

    Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells.

  10. F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction

    PubMed Central

    1996-01-01

    The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin- binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F- actin, we have studied the effect of F-actin on the ability of EF- 1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa- tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF- 1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus. PMID:8922379

  11. Cloning of hypoxia-inducible factor 1alpha cDNA from a high hypoxia tolerant mammal-plateau pika (Ochotona curzoniae).

    PubMed

    Zhao, T B; Ning, H X; Zhu, S S; Sun, P; Xu, S X; Chang, Z J; Zhao, X Q

    2004-04-02

    Hypoxia-inducible factor 1 is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species living only at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Furthermore, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa.

  12. The involvement of hypoxia-inducible factor-1alpha in the susceptibility to gamma-rays and chemotherapeutic drugs of oral squamous cell carcinoma cells.

    PubMed

    Sasabe, Eri; Zhou, Xuan; Li, Dechao; Oku, Naohisa; Yamamoto, Tetsuya; Osaki, Tokio

    2007-01-15

    The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) is the key regulator that controls the hypoxic response of mammalian cells. The overexpression of HIF-1alpha has been demonstrated in many human tumors. However, the role of HIF-1alpha in the therapeutic efficacy of chemotherapy and radiotherapy in cancer cells is poorly understood. In this study, we investigated the influence of HIF-1alpha expression on the susceptibility of oral squamous cell carcinoma (OSCC) cells to chemotherapeutic drugs (cis-diamminedichloroplatinum and 5-fluorouracil) and gamma-rays. Treatment with chemotherapeutic drugs and gamma-rays enhanced the expression and nuclear translocation of HIF-1alpha, and the susceptibility of OSCC cells to the drugs and gamma-rays was negatively correlated with the expression level of HIF-1alpha protein. The overexpression of HIF-1alpha induced OSCC cells to become more resistant to the anticancer agents, and down-regulation of HIF-1alpha expression by small interfering RNA enhanced the susceptibility of OSCC cells to them. In the HIF-1alpha-knockdown OSCC cells, the expression of P-glycoprotein, heme oxygenase-1, manganese-superoxide dismutase and ceruloplasmin were downregulated and the intracellular levels of chemotherapeutic drugs and reactive oxygen species were sustained at higher levels after the treatment with the anticancer agents. These results suggest that enhanced HIF-1alpha expression is related to the resistance of tumor cells to chemo- and radio-therapy and that HIF-1alpha is an effective therapeutic target for cancer treatment.

  13. The efficacy of an IL-1alpha vaccine depends on IL-1RI availability and concomitant myeloid-derived suppressor cell reduction.

    PubMed

    Weiss, Tobias; Vitacolonna, Mario; Zöller, Margot

    2009-01-01

    We recently reported that tumor-derived interleukin (IL)-1beta strongly promotes tumor growth by inducing myeloid-derived suppressor cell (MDSC) and regulatory T-cell (T(reg)) expansion. To see whether redirection of an immune response can be achieved through immune response-supporting IL-1alpha application, IL-1RI competent (IL-1RI(comp)) and IL-1RI-deficient (IL-1RI(-/-)) mice received IL-1alpha cDNA-transformed attenuated Salmonella typhimurium (SL-IL-1alpha) and/or lysates from methycholanthrene-induced IL-1(comp) or IL-1(-/-) fibrosarcoma cells. Vaccination with SL-IL-1alpha and/or tumor lysate exerted only a minor effect on the survival of IL-1alpha/beta(-/-) and none on IL-1alpha(comp) tumor-bearing mice despite induction of a potent antitumor response, that was overridden by intratumoral and systemic expansion of MDSC. Application of all-trans-retinoic acid together with anti-CD25 efficiently coped with MDSC and T(reg) expansion. Vaccination concomitantly with application of all-trans-retinoic acid and anti-CD25 treatment significantly increased the survival time and rate of IL-1alpha/beta(comp), but even of IL-1alpha(-/-)beta(comp) IL-1RI(comp) tumor-bearing mice. Instead, in IL-1RI(-/-) mice, though MDSC expansion was weaker, SL-IL-1alpha application hardly displayed any therapeutic efficacy, which implies signal transduction through IL-1alpha binding to the IL-1RI as an essential component for immune response induction. Taken together, IL-1alpha can efficiently support tumor vaccination, as far as expansion of MDSC and T(reg) is controlled. However, care should be taken to interfere with MDSC expansion/activation not through a blockade of the IL-1RI, which is the preferential target of IL-1alpha.

  14. Mitochondrial DNA polymorphism in mitochondrial myopathy.

    PubMed

    Holt, I J; Harding, A E; Morgan-Hughes, J A

    1988-05-01

    In order to test the hypothesis that mitochondrial myopathy may be caused by mutation of the mitochondrial (mt) genome, restriction fragment length polymorphism in leucocyte mt DNA has been studied in 38 patients with mitochondrial myopathy, 44 of their unaffected matrilineal relatives, and 35 normal control subjects. Previously unreported mt DNA polymorphisms were identified in both patients and controls. No differences in restriction fragment patterns were observed between affected and unaffected individuals in the same maternal line, and there was no evidence of major deletion of mt DNA in patients. This study provides no positive evidence of mitochondrial inheritance in mitochondrial myopathy, but this has not been excluded.

  15. Cobalt chloride-induced estrogen receptor alpha down-regulation involves hypoxia-inducible factor-1alpha in MCF-7 human breast cancer cells.

    PubMed

    Cho, Jungyoon; Kim, Dukkyung; Lee, SeungKi; Lee, YoungJoo

    2005-05-01

    The estrogen receptor (ER) is down-regulated under hypoxia via a proteasome-dependent pathway. We studied the mechanism of ERalpha degradation under hypoxic mimetic conditions. Cobalt chloride-induced ERalpha down-regulation was dependent on the expression of newly synthesized protein(s), one possibility of which was hypoxia-inducible factor-1alpha (HIF-1alpha). To examine the role of HIF-1alpha expression in ERalpha down-regulation under hypoxic-mimetic conditions, we used a constitutively active form of HIF-1alpha, HIF-1alpha/herpes simplex viral protein 16 (VP16), constructed by replacing the transactivation domain of HIF-1alpha with that of VP16. Western blot analysis revealed that HIF-1alpha/VP16 down-regulated ERalpha in a dose-dependent manner via a proteasome-dependent pathway. The kinase pathway inhibitors PD98059, U0126, wortmannin, and SB203580 did not affect the down-regulation. A mammalian two-hybrid screen and immunoprecipitation assays indicated that ERalpha interacted with HIF-1alpha physically. These results suggest that ERalpha down-regulation under hypoxia involves protein-protein interactions between the ERalpha and HIF-1alpha.

  16. Mitochondrial Dynamics and Mitochondrial Dysfunction in Diabetes.

    PubMed

    Wada, Jun; Nakatsuka, Atsuko

    2016-06-01

    The mitochondria are involved in active and dynamic processes, such as mitochondrial biogenesis, fission, fusion and mitophagy to maintain mitochondrial and cellular functions. In obesity and type 2 diabetes, impaired oxidation, reduced mitochondrial contents, lowered rates of oxidative phosphorylation and excessive reactive oxygen species (ROS) production have been reported. Mitochondrial biogenesis is regulated by various transcription factors such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors (NRFs). Mitochondrial fusion is promoted by mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), while fission is governed by the recruitment of dynamin-related protein 1 (DRP1) by adaptor proteins such as mitochondrial fission factor (MFF), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51), and fission 1 (FIS1). Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARKIN promote DRP1-dependent mitochondrial fission, and the outer mitochondrial adaptor MiD51 is required in DRP1 recruitment and PARKIN-dependent mitophagy. This review describes the molecular mechanism of mitochondrial dynamics, its abnormality in diabetes and obesity, and pharmaceuticals targeting mitochondrial biogenesis, fission, fusion and mitophagy.

  17. Mitochondrial Aging: Is There a Mitochondrial Clock?

    PubMed

    Zorov, Dmitry B; Popkov, Vasily A; Zorova, Ljubava D; Vorobjev, Ivan A; Pevzner, Irina B; Silachev, Denis N; Zorov, Savva D; Jankauskas, Stanislovas S; Babenko, Valentina A; Plotnikov, Egor Y

    2017-09-01

    Fragmentation (fission) of mitochondria, occurring in response to oxidative challenge, leads to heterogeneity in the mitochondrial population. It is assumed that fission provides a way to segregate mitochondrial content between the "young" and "old" phenotype, with the formation of mitochondrial "garbage," which later will be disposed. Fidelity of this process is the basis of mitochondrial homeostasis, which is disrupted in pathological conditions and aging. The asymmetry of the mitochondrial fission is similar to that of their evolutionary ancestors, bacteria, which also undergo an aging process. It is assumed that mitochondrial markers of aging are recognized by the mitochondrial quality control system, preventing the accumulation of dysfunctional mitochondria, which normally are subjected to disposal. Possibly, oncocytoma, with its abnormal proliferation of mitochondria occupying the entire cytoplasm, represents the case when segregation of damaged mitochondria is impaired during mitochondrial division. It is plausible that mitochondria contain a "clock" which counts the degree of mitochondrial senescence as the extent of flagging (by ubiquitination) of damaged mitochondria. Mitochondrial aging captures the essence of the systemic aging which must be analyzed. We assume that the mitochondrial aging mechanism is similar to the mechanism of aging of the immune system which we discuss in detail. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Relationship of surface epithelium concentrations of IL-1 alpha and IL-1 beta to clinical inflammation during experimental gingivitis.

    PubMed

    Biesbrock, A; Yeh, C H

    2000-01-01

    Experimental evidence has clearly demonstrated that IL-1 cytokine levels increase in experimental gingivitis (EG) models in response to plaque accumulation following the cessation of oral hygiene. These changes in cytokine production are reported to occur prior to visible signs of clinical inflammation, and as such may represent early markers of gingival inflammation. This clinical study used a novel dermal sampling tape as a method to collect cytokines from gingival epithelium, as opposed to the more commonly sampled gingival crevicular fluid. The primary objective of the study was to examine the relationship between changes in cytokine levels and clinical inflammation. Ten subjects participated in a 14-day EG model, where 5 days following a dental prophylaxis subjects refrained from all oral hygiene measures for 14-days. Clinical measures including the Löe-Silness Gingival Index (GI), a bleeding index derived from the GI, and the inflammation index (II) were made at baseline prior to the initiation of the EG period and following 14 days of EG. Dermal tape samples were collected from the right posterior buccal quadrant of each subject at both baseline and day 14. The tapes were extracted and the extracts analyzed for both IL-1 alpha and IL-1 beta by ELISA. Results of this study indicate that over a 14-day EG period statistically significant (p < 0.05) increases in GI, gingival bleeding, II, and IL-1 alpha were observed (tested by matched-pairs t test and Wilcoxon signed ranks test). A directional increase in IL-1 beta was also observed. Linear regression analyses demonstrated a strong positive correlation between the number of gingival bleeding sites from the region of gingiva sampled with Sebutape and IL-1 alpha (r = 0.93), as well as IL-1 beta (r = 0.90). In addition, linear regression analyses also demonstrated a strong positive correlation between the mean II score from the region of gingiva sampled with Sebutape and IL-1 alpha (r = 0.93), as well as IL-1

  19. Characterization of the cytokine pattern of porcine bone marrow-derived cells treated with 1alpha,25(OH)D.

    PubMed

    Sipos, W; Duvigneau, J C; Schmoll, F; Exel, B; Hofbauer, G; Baravalle, G; Hartl, R T; Dobretsberger, M; Pietschmann, P

    2005-10-01

    The biologically active form of vitamine D(3) [1alpha,25(OH)(2)D(3)] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1alpha,25(OH)(2)D(3) on the cytokine pattern of porcine bone marrow-derived cells from piglets aged 1-3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell-derived mRNA was subjected to semiquantitative RT-PCR specific for a panel of porcine cytokines (IL-1alpha, IL-6, IL-8, IL-10, and TNF-alpha). In addition, an immunofluorescence analysis using anti-porcine mAbs specific for IL-1beta, IL-2, IL-4, IL-6, IL-12, TNF-alpha, and IFN-gamma was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF-kappaB ligand (RANKL) bone marrow cell- as well as porcine white blood cell-derived mRNA was investigated by RT-PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT-PCR, expression of IL-1alpha, IL-6, IL-8, IL-10 and TNF-alpha mRNA could be found in cells cultured with and without 1alpha,25(OH)(2)D(3). Immunofluorescence analysis revealed that IL-1, IL-6, and TNF-alpha were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1alpha,25(OH)(2)D(3) tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL-specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.

  20. [Sequential expression of hypoxia-inducible factor 1alpha and its significance in secondary spinal cord injury].

    PubMed

    Niu, Qingfei; Jia, Changqing; Wang, Nan; Chen, Xiaochun; Chi, Renjie; Bai, Shuling

    2011-01-01

    To investigate the expression pattern of hypoxia-inducible factor 1alpha (HIF-1alpha) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI. A total of 66 SD rats (female or male) with weight (250 +/- 20) g were randomly divided into 3 groups: normal control group (group A, n = 6), pseudo injury group (group B, n = 6), and spinal cord injury (SCI) group (group C, n = 54). In group A, no treatment was given as normal control. In group B, only laminectomy was applied. In group C, laminectomy was applied and static compression model of SCI was built at T10 level. The expression of HIF-1alpha was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with light staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of glial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-la was poorly expressed in group A and increased a little in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of ganglion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then declined. At 14 days, it appeared only in a small amount of ganglion cells of white matter. There was no significant difference in the number of HIF-1alpha positive cells between groups A and B (t = 1.325, P = 0.137). The number of HIF-1alpha

  1. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    SciTech Connect

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan; Fan, Guo-Huang; Richmond, Ann

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP-2 or

  2. Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters

    PubMed Central

    Suhandono, Sony; Apriyanto, Ardha; Ihsani, Nisa

    2014-01-01

    In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family. Three promoters MeEF1A3, MeEF1A4 and MeEF1A5 were successfully isolated. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5′UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants. PMID:24404183

  3. Activation of the endothelium by IL-1 alpha and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a.

    PubMed Central

    Coulpier, M; Andreev, S; Lemercier, C; Dauchel, H; Lees, O; Fontaine, M; Ripoche, J

    1995-01-01

    Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinflammatory cytokine IL-1 alpha has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL-1 alpha and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL-1 alpha, significant potentialization of IL-1 alpha-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10(-7) to 10(-5) M for dexamethasone and 50-200 U for IL-1 alpha. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL-1 alpha on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-1 alpha was not observed for another marker of endothelial activation, IL-1 alpha-induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-1 alpha therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL-1 alpha, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-1 alpha and endogenous glucocorticoids coexist, such as in septic shock. Images Fig. 4 Fig. 6 PMID:7621583

  4. Stimulus specific changes of energy metabolism in hypertrophied heart.

    PubMed

    Rimbaud, S; Sanchez, H; Garnier, A; Fortin, D; Bigard, X; Veksler, V; Ventura-Clapier, R

    2009-06-01

    Cardiac energy metabolism is a determinant of the response to hypertrophic stimuli. To investigate how it responds to physiological or pathological stimuli, we compared the energetic status in models of hypertrophy induced by physiological stimuli (pregnancy or treadmill running) and by pathological stimulus (spontaneously hypertensive rats, SHR) in 15 week-old female rats, leading to a 10% cardiac hypertrophy. Late stage of compensated hypertrophy was also studied in 25 week-old SHR (35% of hypertrophy). Markers of cardiac remodelling did not follow a unique pattern of expression: in trained rats, only ANF was increased; in gravid rats, calcineurin activation and BNP expression were reduced while beta-MHC expression was enhanced; all markers were clearly up-regulated in 25 week-old SHR. Respiration of permeabilized fibers revealed a 17% increase in oxidative capacity in trained rats only. Mitochondrial enzyme activities, expression of the master regulator PGC-1alpha and mitochondrial transcription factor A, and content of mitochondrial DNA were not consistently changed, suggesting that compensated hypertrophy does not involve alterations of mitochondrial biogenesis. Mitochondrial fatty acid utilization tended to increase in trained rats and decreased by 14% in 15 week-old SHR. Expression of markers of lipid oxidation, PPARalpha and its down-stream targets MCAD and CPTI, was up-regulated after training and tended to decrease in gravid and 15 week-old SHR rats. Taken together these results show that there is no univocal pattern of cardiac adaptation in response to physiological or pathological hypertrophic stimuli, suggesting that other factors could play a role in determining adaptation of energy metabolism to increased workload.

  5. In-vivo interleukin-1-alpha induction following chlorin-e6 photodynamic therapy in Balb/c mice

    NASA Astrophysics Data System (ADS)

    Ziolkowski, Piotr P.; Symonowicz, Krzysztof; Milach, Jerzy

    1996-01-01

    Photodynamic therapy may induce in in-vivo conditions the cytokine Interleukin-1-alpha in Balb/c mice. The sensitizer, i.e. chlorin e6, in the doses 2.5 and 5.0 mg/kg of body weight followed by light treatment with doses 50 and 100 J/sq.cm resulted in the increase in serum levels of the cytokine. The levels of Interleukin-1-alpha have been determined at different time points using enzyme-linked immunosorbent assay (ELISA). These levels in control animals did not exceed the mean value of 15 pg/ml, whereas in photodynamically treated mice the levels were almost 3 - 4 times higher. The entire experiment has been carried out on healthy animals.

  6. PfEMP1-DBL1alpha amino acid motifs in severe disease states of Plasmodium falciparum malaria.

    PubMed

    Normark, Johan; Nilsson, Daniel; Ribacke, Ulf; Winter, Gerhard; Moll, Kirsten; Wheelock, Craig E; Bayarugaba, Justus; Kironde, Fred; Egwang, Thomas G; Chen, Qijun; Andersson, Björn; Wahlgren, Mats

    2007-10-02

    An infection with Plasmodium falciparum may lead to severe malaria as a result of excessive binding of infected erythrocytes in the microvasculature. Vascular adhesion is mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP1), which is encoded for by highly polymorphic members of the var-gene family. Here, we profile var gene transcription in fresh P. falciparum trophozoites from Ugandan children with malaria through var-specific DBL1alpha-PCR amplification and sequencing. A method for subsectioning region alignments into homology areas (MOTIFF) was developed to examine collected sequences. Specific PfEMP1-DBL1alpha amino acid motifs correlated with rosetting and severe malaria, with motif location corresponding to distinct regions of receptor interaction. The method is potentially applicable to other families of variant proteins and may be useful in identifying sequence-phenotype relationships. The results suggest that certain PfEMP1 sequences are predisposed to inducing severe malaria.

  7. Nutrition-induced catch-up growth increases hypoxia inducible factor 1alpha RNA levels in the growth plate.

    PubMed

    Even-Zohar, N; Jacob, J; Amariglio, N; Rechavi, G; Potievsky, O; Phillip, M; Gat-Yablonski, G

    2008-03-01

    Although catch-up growth is a well-known phenomenon, the local pathways at the epiphyseal growth plate that govern this process remain poorly understood. To study the mechanisms governing catch-up growth in the growth plate, we subjected prepubertal rats to 10 days of 40% food restriction, followed by a renewal of the regular food supply to induce catch-up growth. The animals were weighed daily, and their humeral length was measured at sacrifice. The proximal tibial epiphyseal growth plates (EGPs) were studied, and findings were compared with EGPs from animals fed ad libitum and animals under food restriction. The gene expression profile in the growth plates was examined using DNA microarrays, and the expression levels of selected genes were validated by real-time polymerase chain reaction. To localize gene expression in different growth plate zones, microdissection was used. Protein levels and localization were examined using immunohistochemistry. We showed that the expression level of 550 genes decreased during food restriction and increased during catch-up growth, starting already one day after refeeding. HIF-1alpha, as well as several of its downstream targets, was found among these genes. Immunohistochemistry showed a similar pattern for HIF-1alpha protein abundance. Additionally, HIF-1alpha mRNA and protein levels were higher in the proliferating than in the hypertrophic zone, and this distribution was unaffected by nutritional status. These findings indicate that nutrition has a profound effect on gene expression level during growth plate growth, and suggest an important role for HIF-1alpha in the growth plate and its response to nutritional manipulation.

  8. Differential expression of hypoxia inducible factor-1 alpha and tumor cell proliferation between squamous cell carcinomas and adenocarcinomas among operable non-small cell lung carcinomas.

    PubMed Central

    Lee, Chang Hun; Lee, Min Ki; Kang, Chi Duk; Kim, Young Dae; Park, Do Youn; Kim, Jee Yeon; Sol, Mee Young; Suh, Kang Suek

    2003-01-01

    This study aimed to evaluate whether the elevated level of hypoxia-inducible factor-1alpha (HIF-1alpha) correlated with histologic types, angiogenesis, tumor cell proliferation, and clinical parameters in common non-small cell lung carcinomas (NSCLCs). We performed immunohistochemical stains using paraffin-embedded tissue blocks from 84 cases of operable NSCLC [No. of squamous cell carcinoma (SCC), 45; No. of adenocarcinoma (AC), 39]. HIF-1alpha expression was related with histologic types (66.7% in SCCs vs 20.5% in ACs, p<0.001), but not with lymph node status, tumor stage, vascular endothelial growth factor expression, microvessel density (MVD), and proliferating cell nuclear antigen (PCNA) index (p>0.05, respectively). As for the histologic types, MVD and PCNA index were significantly higher in SCCs than in ACs (p=0.009 and p=0.016, respectively). Among HIF-1alpha positive carcinomas, MVD was significantly higher in HIF-1alpha positive SCCs than in HIF-1alpha positive ACs (p=0.023). The overall survival curves were not associated with HIF-1alpha expression or any other histologic parameters (p>0.05). These findings suggest that HIF-1alpha expression in NSCLCs may play a differential role according to histologic types, but its prognostic significance is indeterminate. PMID:12692416

  9. Molecular architecture of leishmania EF-1alpha reveals a novel site that may modulate protein translation: a possible target for drug development.

    PubMed

    Lopez, Martin; Cherkasov, Artem; Nandan, Devki

    2007-05-18

    Elongation factor-1alpha plays an essential role in eukaryotic protein biosynthesis. Recently, we have shown by protein structure modeling the presence of a hairpin-loop of 12 amino acids in mammalian EF-1alpha that is absent in the leishmania homologue [D. Nandan, A. Cherkasov, R. Sabouti, T. Yi, N.E. Reiner, Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue, Biochem. Biophys. Res. Commun. 302 (2003) 646-652]. As a consequence of this deletion, an exposed region is available on the main body of leishmania EF-1alpha. Here we report the generation of an anti-EF-1alpha antibody (DN-3) which bound selectively to the exposed region of leishmania EF-1alpha, with no reactivity with human EF-1alpha. In a leishmania cell-free protein translation system, DN-3 substantially inhibited protein translation. A similar inhibitory effect was observed when a specific peptide based on the exposed region was used in the cell-free protein translation assay. The application of structure-based in silico methods to identify potential ligands to target the exposed region identified a small molecule that selectively attenuated in vitro translation using leishmania extracts. Moreover, this small molecule showed selective suppressive effect on multiplication of leishmania in culture. Taken together, these findings identify a novel, exposed region in leishmania EF-1alpha that may be involved in protein synthesis and a potential site for drug targeting.

  10. 15-Deoxy-Delta(12,14)-prostaglandin-J(2) reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1alpha degradation.

    PubMed

    Olmos, Gemma; Arenas, María I; Bienes, Raquel; Calzada, María Jose; Aragonés, Julián; Garcia-Bermejo, Maria Laura; Landazuri, Manuel O; Lucio-Cazaña, Javier

    2009-07-01

    Hypoxia-inducible factor-1alpha (HIF-1alpha) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) accumulate HIF-1alpha in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ(2) induced an over-accumulation of HIF-1alpha in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1alpha degradation as a target for 15d-PGJ(2) based on: (1) HIF-1alpha colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ(2) inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1alpha in 15d-PGJ(2)-treated cells. Therefore, expression of HIF-1alpha is also modulated by lysosomal degradation.

  11. Differential association of SMC1alpha and SMC3 proteins with meiotic chromosomes in wild-type and SPO11-deficient male mice.

    PubMed

    James, Rosalina D; Schmiesing, John A; Peters, Antoine H F M; Yokomori, Kyoko; Disteche, Christine M

    2002-01-01

    SMC proteins are components of cohesin complexes that function in chromosome cohesion. We determined that SMC1alpha and SMC3 localized to wild-type mouse meiotic chromosomes, but with distinct differences in their patterns. Anti-SMC3 coincided with axial elements of the synaptonemal complex, while SMC1alpha was observed mainly in regions where homologues were synapsed. This pattern was especially visible in pachytene sex vesicles where SMC1alpha localized only weakly to the asynapsed regions. At diplotene, SMC3, but not SMC1alpha, remained bound along axial elements of desynapsed chromosomes. SMC1alpha and SMC3 were also found to localize along meiotic chromosome cores of Spo11 null spermatocytes, in which double-strand break formation required for DNA recombination and homologous pairing were disrupted. In Spo11 -/- cells, SMC1alpha localization differed from SMC3 again, confirming that SMC1alpha is mainly associated with homologous or non-homologous synapsed regions, whereas SMC3 localized throughout the chromosomes. Our results suggest that the two cohesin proteins may not always be associated in a dimer and may function as separate complexes in mammalian meiosis, with SMC1alpha playing a more specific role in synapsis. In addition, our results indicate that cohesin cores can form independently of double-strand break formation and homologous pairing.

  12. Diagnosis of HNF-1alpha mutations on a PNA zip-code microarray by single base extension.

    PubMed

    Song, Jae Yang; Park, Hyun Gyu; Jung, Sung-Ouk; Park, JaeChan

    2005-02-01

    In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3' complementarity to the specific mutation site and 5' complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1alpha with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.

  13. Overexpression of IL-1alpha in skin differentially modulates the immune response to scarification with vaccinia virus.

    PubMed

    Tian, Tian; Liu, Luzheng; Freyschmidt, Eva-Jasmin; Murphy, George F; Kupper, Thomas S; Fuhlbrigge, Robert C

    2009-01-01

    Transepidermal inoculation of vaccinia virus (VV), or scarification, has been used effectively for the induction of specific and long-lasting immunity to smallpox and is superior to other routes of immunization. Scarification of individuals with atopic skin disease or immune deficiency, however, can lead to persistent viral replication and result in significant morbidity and mortality. These effects of scarification presumably reflect the unique immunological properties of skin and the immune cells resident in, or recruited to, the site of inoculation. To explore these phenomena, we utilized transgenic mice engineered to overexpress IL-1alpha, a critical mediator of cutaneous inflammation, in the epidermis. Following scarification with VV, both transgenic and wild-type mice develop local pox. At high doses of VV, IL-1alpha transgenic mice recruited immune cells to the inoculation site more rapidly and demonstrated enhanced T-cell and humoral immune responses. At limiting doses, however, IL-1alpha transgenic mice could effectively control virus replication without formation of pox lesions or activation of a memory response. This study suggests that IL-1 might be useful as an adjuvant to enhance antiviral immunity and promote safer vaccination strategies; however, understanding the balance of IL-1 effects on innate and adaptive immune functions will be critical to achieve optimal results.

  14. Increased intestinal protein synthesis during sepsis and following the administration of tumour necrosis factor alpha or interleukin-1 alpha.

    PubMed Central

    von Allmen, D; Hasselgren, P O; Higashiguchi, T; Frederick, J; Zamir, O; Fischer, J E

    1992-01-01

    The influence of sepsis on intestinal protein synthesis was studied in rats. Sepsis was induced by caecal ligation and puncture (CLP); control rats were sham-operated. Protein synthesis was measured in vivo in the jejunum and ileum following a flooding dose of [14C]leucine. At 8 h after CLP the protein synthesis rate was increased by approx. 15% in jejunal mucosa, and at 16 h after CLP, the protein synthesis rate was increased by 50-60% in the mucosa and seromuscular layer of both jejunum and ileum. In a second series of experiments, rats were treated with recombinant tumour necrosis factor alpha (rTNF alpha) or recombinant interleukin-1 alpha (rIL-1 alpha) administered at a total dose of 300 micrograms/kg body weight over 16 h. Control rats received corresponding volumes of solvent. Treatment with rTNF alpha resulted in an approx. 25% increase in mucosal protein synthesis in jejunum. Following treatment with rIL-1 alpha, protein synthesis increased by 25% in jejunal mucosa and almost doubled in ileal mucosa. The results suggest that sepsis stimulates intestinal protein synthesis and that this response may, at least in part, be mediated by TNF and/or IL-1. PMID:1530589

  15. In vitro stimulation of stage-specific deoxyribonucleic acid synthesis in rat seminiferous tubule segments by interleukin-1. alpha

    SciTech Connect

    Parvinen, M.; Soeder, O.M.; Mali, P.; Froeysa, B.R.; Ritzen, E.M. )

    1991-09-01

    Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 {alpha}, and labeled with (3H)thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiated DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 {alpha} stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 {alpha} seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.

  16. [Expression of elongation factor-1 alpha-A and beta-actin promoters in embryos of transgenic Medaka (Oryzias latipes)].

    PubMed

    Long, Hua

    2003-06-01

    Two expression vectors with the promoter of either Medaka (Oryzias latipes) elongation factor gene or beta-actin gene were constructed based on pBluescript SK+. Both of them are linked with green-fluorescent protein (GFP) gene. And they are named as pB-EF and pB-BA, respectively. The microinjection experiments were conducted with fertilized Medaka eggs at one-cell stage. The expression of two vectors, pB-EF and pB-BA, was observed under stereo-fluorescence microscope. The detection results showed that both EF-1 alpha-A promoter and beta-actin promoter are strong. In the process of embryo development, the activity of beta-actin promoter became stronger while that of EF-1 alpha-A promoter weaker gradually. beta-actin promoter was but EF-1 alpha-A promoter distributed throughout fish body uniformly. The expression rate of two vectors, pB-EF and pB-BA, are 8.23% and 6.10%, respectively.

  17. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

    PubMed Central

    Weis, K; Ryder, U; Lamond, A I

    1996-01-01

    Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay. While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery. Images PMID:8617227

  18. Studies on the 1alpha, 25-dihydroxycholecalciferol-like activity in a calcinogenic plant. Cestrum diurnum, in the chick.

    PubMed

    Wasserman, R H; Corradino, R A; Krook, L; Hughes, M R; Haussler, M R

    1976-04-01

    Cestrum diurnum (day-blooming jessamine) has been proposed to cause calcinosis in horses and cattle in Florida. The present studies investigated some physiological properties of the plant, using the chick as the experimental animal. The inclusion of dried leaf powder in a rachitogenic diet restored intestinal calcium-binding protein synthesis (CaBP) and increased calcium absorption in the cholecalciferol-deficient chick. The estimated level of cholecalciferol-equivalents in the dried leaf was about 30,000 to 35,000 IU/kg. Most of the activity was extractable with methanol:chloroform (2:1), indicating that the major cholecalciferol-like component in C. diurnum was different from the water soluble factor(s) in Solanum malacoxylon. The time course of effect of C. diurnum extract in rachitic chicks was similar to that ot 1,25-dihydroxycholecalciferol but the former had a longer lag time. The strontium fed chick, in which the kidney 25-hydroxycholecalciferol-1alpha-hydroxylase is inhibited, responded to C. diurnum extract, confirming the 1alpha,25-dihydroxycholecalciferol-like character of the Cestrum factor. The extract also appeared to interact with the intestinal 1 alpha,25-dihydroxycholecalciferol cytosol receptor although this observation is preliminary. These findings indicate that the l alpha,25-dihydroxycholecalciferol-like principle in C. diurnum many cause excessive calcium and phosphate absorption leading to calcinosis.

  19. Mitochondrial redox signaling and cancer invasiveness

    PubMed Central

    Enns, Linda

    2014-01-01

    The concept that invasive cancer is associated with increased levels of reactive oxygen species (ROS) generated by mitochondria is consistent with an ROS-mediated signaling mechanism. As a tumor grows, it encounters adverse microenvironments, one of which is low oxygen (hypoxia), which selects tumor cells with characteristics of increased invasiveness. Hypoxic environments select for tumor cells with stabilized HIF1 apha, a transcription factor that regulates genes coding for pro-tumor cytokines that signal stromal cells such as macrophages and fibroblasts to support an invasive tumor cell phenotype. HIF1 alpha-mediated switches in the energy production of tumor cells from OXPHOS to glycolysis, as well as age-associated decreases in the metabolic rate of the host, enhance invasive qualities of tumor cells. An increase in environmental oxygen in combination with a mitochondrial targeted catalase mimetic and a metabolism booster may be of interest to investigate as a treatment strategy for invasive cancer. PMID:22886605

  20. Mitochondrial inheritance in a mitochondrially mediated disease.

    PubMed

    Egger, J; Wilson, J

    1983-07-21

    Mendelian inheritance involves the transmission to successive generations of DNA contained in genes in the nucleus, but DNA is also contained in mitochondria, where it is believed to be responsible for the encoding of certain mitochondrial enzymes. Since nearly all mitochondrial DNA is maternally transmitted, one might expect a nonmendelian pattern of inheritance in mitochondrial cytopathy, a syndrome in which there are abnormalities in mitochondrial structure and deficiencies in a variety of mitochondrial enzymes. We studied the pedigrees of 6 affected families whose members we had examined personally and of 24 families described in the literature. In 27 families, exclusively maternal transmission occurred; in 3 there was also paternal transmission in one generation. Altogether, 51 mothers but only 3 fathers had transmitted the condition. These results are consistent with mitochondrial transmission of mitochondrial cytopathy; the inheritance and enzyme defects of mitochondrial cytopathy can be considered in the light of recent evidence that subunits of respiratory-enzyme complexes are encoded solely by mitochondrial DNA. The occasional paternal transmission may be explained if certain enzyme subunits that are encoded by nuclear DNA are affected.

  1. Macrophage inflammatory protein 1alpha enhances in a different manner adhesion of hematopoietic progenitor cells from bone marrow, cord blood, and mobilized peripheral blood.

    PubMed

    Suehiro, Y; Muta, K; Umemura, T; Abe, Y; Nishimura, J; Nawata, H

    1999-11-01

    Regulatory mechanisms governing adhesion of hematopoietic progenitor cells to the stromal nische are poorly understood. Growth factors such as stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor, and thrombopoietin were reported to upregulate the adhesion of hematopoietic progenitors to immobilized fibronectin through activation of integrin alpha4beta1 and alpha5beta1. Macrophage inflammatory protein (MIP)-1alpha is a C-C chemokine that suppresses colony formation by stem/progenitor cells in vitro. We asked if MIP-1alpha would modulate the adhesive phenotype of colony-forming cells (CFCs) obtained from healthy donor bone marrow (BM), cord blood (CB), and mobilized peripheral blood (mPB) CD34+ cells, in comparison with SCF, using immobilized fibronectin. SCF significantly increased the level of adhesion of CFCs from BM, CB, and mPB. On the other hand, MIP-1alpha significantly increased the level of adhesion of CFCs from BM and CB, but less so from mPB. The effects of MIP-1alpha were inhibited by blocking antibodies to integrin alpha4, alpha5, or beta1, and polymerization plus rearrangement of F-actin were observed in affected cells by labeling with rhodamine-conjugated phalloidine. These data indicate that the effect of MIP-1alpha on the adhesive phenotype of CFCs is mediated by modulation of the organization of integrin. The amount of MIP-1alpha receptor on mPB was less than for BM or CB, which may explain the distinct characteristics in the adhesive response induced by MIP-1alpha. We suggest that hematopoietic progenitor cells from different sources may be heterogeneous with respect to maturation, integrin affinity, MIP-1alpha receptor expression, and regulation of MIP-1alpha signaling. Our data indicate that MIP-1alpha may affect migration, homing, and mobilization of hematopoietic progenitors by modulating the adhesive phenotype of these cells.

  2. Phytase and 1alpha-hydroxycholecalciferol supplementation of broiler chickens during the starting and growing/finishing phases.

    PubMed

    Driver, J P; Pesti, G M; Bakalli, R I; Edwards, H M

    2005-10-01

    Supplemental 1alpha-hydroxycholecalciferol (1alpha-OHD3) has been shown to have qualitatively similar and quantitatively additive effects to exogenous phytase. Two experiments were conducted from 0 to 35 d in floor pens to determine the additive effect of phytase and 1alpha-OHD3 when supplemented to Ca- and P-deficient diets. In both experiments, at least 4 replicates per treatment (50 chicks per replicate) were used. Corn-soybean-meal-and soybean-oil-based diets were fed and birds were raised in a house impervious to ultraviolet light. During the starter phase (ST), from 0 to 18 d, chicks were fed a 23% CP diet containing 0.60% Ca and 0.47% total P (tP). During the grower/finisher phase (GF), from 19 to 35 d, birds were fed a 19% CP diet containing 0.30% Ca and 0.37% tP. A combination of 1,000 phytase units/kg of Natuphos phytase and 5 microg/kg of 1alpha-OHD3 (P+1A) was supplemented to some of the feed during the ST and GF. Diets containing adequate Ca and P were also fed during the ST (0.90% Ca, 0.68% tP) and GF (0.80% Ca, 0.67% tP). Performance characteristics and the incidence of rickets and tibial dyschondroplasia were measured at 18 and 35 d. In experiment 1, unsupplemented chicks performed well but had considerable leg problems. Chicks fed P+1A during the ST or GF did not perform as well as birds fed P+1A throughout. Birds fed P+1A throughout performed as well birds fed the adequate diets without any indication of leg problems. In experiment 2, unsupplemented birds performed similarly to unsupplemented birds in experiment 1. However, chicks fed the supplements or the control diets did not perform as well or accumulate as much bone ash as birds in experiment 1, although the diets were formulated identically in both experiments. Diets with as little as 0.30% Ca and 0.37% tP appear to be adequate for broilers older than 18 d if supplemented with the correct amounts of phytase and 1alpha-OHD3. However, there are unknown variables that may limit the potential of

  3. Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

    SciTech Connect

    Gu Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Takeda, Jun; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke . E-mail: jinkan@tom.life.h.kyoto-u.ac.jp

    2006-08-04

    Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2 mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.

  4. The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

    SciTech Connect

    Serasinghe, Madhavika N.; Yoon, Yisang

    2008-11-15

    Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.

  5. Mitochondrial Disease: Possible Symptoms

    MedlinePlus

    ... Mitochondrial Medical & Scientific Meetings Grand Rounds Researcher Education Research Grants Funded Projects Patient Evaluation for Professionals Energy Metabolism Review Mitochondrial Structure, Function and Diseases Review Cell Biology of Diagnosis ...

  6. Isolation of Mitochondrial Ribosomes.

    PubMed

    Carroll, Adam J

    2017-01-01

    Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.

  7. [Recent advances in the study of AMPK and inflammatory pulmonary disease].

    PubMed

    Xuan, Ling-Ling; Hou, Qi

    2014-08-01

    AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis. Recent studies demonstrated that AMPK is a novel signaling molecule modulating inflammatory responses and oxidative stress which are involved in inflammatory pulmonary diseases, such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary infectious diseases and pulmonary fibrosis. AMPK attenuates inflammatory lung injury by phosphorylating its downstream targets, such as sirtuin1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), p53 and forkhead box O3a (FoxO3a). This review summarized the relationship between AMPK and the development of inflammatory pulmonary diseases.

  8. Nitrate-containing beetroot enhances myocyte metabolism and mitochondrial content.

    PubMed

    Vaughan, Roger A; Gannon, Nicholas P; Carriker, Colin R

    2016-01-01

    Beetroot ( tián cài) juice consumption is of current interest for improving aerobic performance by acting as a vasodilator and possibly through alterations in skeletal muscle metabolism and physiology. This work explored the effects of a commercially available beetroot supplement on metabolism, gene expression, and mitochondrial content in cultured myocytes. C2C12 myocytes were treated with various concentrations of the beetroot supplement for various durations. Glycolytic metabolism and oxidative metabolism were quantified via measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured using quantitative reverse transcription-polymerase chain reaction, and mitochondrial content was assessed with flow cytometry and confocal microscopy. Cells treated with beetroot exhibited significantly increased oxidative metabolism, concurrently with elevated metabolic gene expression including peroxisome proliferator-activated receptor gamma coactivator-1 alpha, nuclear respiratory factor 1, mitochondrial transcription factor A, and glucose transporter 4, leading to increased mitochondrial biogenesis. Our data show that treatment with a beetroot supplement increases basal oxidative metabolism. Our observations are also among the first to demonstrate that beetroot extract is an inducer of metabolic gene expression and mitochondrial biogenesis. These observations support the need for further investigation into the therapeutic and pharmacological effects of nitrate-containing supplements for health and athletic benefits.

  9. Interleukin 1. alpha. inhibits prostaglandin E sub 2 release to suppress pulsatile release of luteinizing hormone but not follicle-stimulating hormone

    SciTech Connect

    Rettori, V.; McCann, S.M. ); Gimeno, M.F. ); Karara, A. ); Gonzalez, M.C. )

    1991-04-01

    Interleukin 1{alpha} (IL-1{alpha}), a powerful endogenous pyrogen released from monocytes and macrophages by bacterial endotoxin, stimulates corticotropin, prolactin, and somatotropin release and inhibits thyrotropin release by hypothalamic action. The authors injected recombinant human IL-1{alpha} into the third cerebral ventricle, to study its effect on the pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in conscious, freely moving, ovariectomized rats. Intraventricular injection of 0.25 pmol of IL-1{alpha} caused an almost immediate reduction of plasma LH concentration. To determine the mechanism of the suppression of LH release, mediobasal hypothalamic fragments were incubated in vitro with IL-1{alpha} (10 pM) and the release of LH-releasing hormone (LHRH) and prostaglandin E{sub 2} into the medium was measured by RIA in the presence or absence of nonrepinephrine. 1{alpha} reduced basal LHRH release and blocked LHRH release induced by nonrepinephrine. In conclusion, IL-1{alpha} suppresses LH but not FSH release by an almost complete cessation of pulsatile release of LH in the castrated rat. The mechanism of this effect appears to be by inhibition of prostaglandin E{sub 2}-mediated release of LHRH.

  10. Functional defect of truncated hepatocyte nuclear factor-1{alpha} (G554fsX556) associated with maturity-onset diabetes of the young

    SciTech Connect

    Kooptiwut, Suwattanee; Sujjitjoon, Jatuporn; Plengvidhya, Nattachet; Boonyasrisawat, Watip; Chongjaroen, Nalinee; Jungtrakoon, Prapapron; Semprasert, Namoiy; Furuta, Hiroto; Nanjo, Kishio; Banchuin, Napatawn; Yenchitsomanus, Pa-thai

    2009-05-22

    A novel frameshift mutation attributable to 14-nucleotide insertion in hepatocyte nuclear factor-1{alpha} (HNF-1{alpha}) encoding a truncated HNF-1{alpha} (G554fsX556) with 76-amino acid deletion at its carboxyl terminus was identified in a Thai family with maturity-onset diabetes of the young (MODY). The wild-type and mutant HNF-1{alpha} proteins were expressed by in vitro transcription and translation (TNT) assay and by transfection in HeLa cells. The wild-type and mutant HNF-1{alpha} could similarly bind to human glucose-transporter 2 (GLUT2) promoter examined by electrophoretic mobility shift assay (EMSA). However, the transactivation activities of mutant HNF-1{alpha} on human GLUT2 and rat L-type pyruvate kinase (L-PK) promoters in HeLa cells determined by luciferase reporter assay were reduced to approximately 55-60% of the wild-type protein. These results suggested that the functional defect of novel truncated HNF-1{alpha} (G554fsX556) on the transactivation of its target-gene promoters would account for the {beta}-cell dysfunction associated with the pathogenesis of MODY.

  11. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  12. Inhibition of oxygen-induced hypoxia-inducible factor-1alpha degradation unmasks estradiol induction of vascular endothelial growth factor expression in ECC-1 cancer cells in vitro.

    PubMed

    Molitoris, Kristin Happ; Kazi, Armina A; Koos, Robert D

    2009-12-01

    Estradiol (E(2)) rapidly and strongly induces vascular endothelial growth factor (VEGF) transcription in uterine endometrial epithelial cells in vivo. We have shown that this is mediated by both the estrogen receptor-alpha and hypoxia-inducible factor (HIF)-1alpha. By contrast, E(2) induces little or no VEGF expression in cultured breast or endometrial cancer cells, which lack HIF-1alpha due to the abnormally high concentration of oxygen ( approximately 20%) to which they are exposed. To test the hypothesis that restoring HIF-1alpha in cultured cells would restore the ability of E(2) to induce VEGF expression, we treated human endometrial cancer cells (ECC-1) with cobalt chloride (CoCl(2);100 microm), which prevents oxygen-induced HIF-1alpha degradation. HIF-1alpha was absent in untreated ECC-1 cells but detectable by 4 h after treatment with CoCl(2) alone, as was a significant increase in VEGF mRNA. E(2) plus CoCl(2) induced detectable HIF-1alpha expression at 2 h and an even higher level than that induced by CoCl(2) alone at 4 h; this HIF-1alpha was localized in the nuclei. This was accompanied by increasing VEGF expression, with the increase at 4 h severalfold higher than that induced by CoCl(2) alone and was concurrent with recruitment of both HIF-1alpha and estrogen receptor-alpha to the VEGF promoter. These results confirm that HIF-1alpha plays an essential role in E(2)-induced expression of VEGF. Through the induction of increased microvascular permeability and the consequent exudation of plasma growth factors, VEGF in turn may play an essential role in cancer cell proliferation in vivo.

  13. Double immunohistochemical staining method for HIF-1alpha and its regulators PHD2 and PHD3 in formalin-fixed paraffin-embedded tissues.

    PubMed

    Vaughan, Mary M; Toth, Karoly; Chintala, Sreenivasulu; Rustum, Youcef M

    2010-07-01

    Hypoxia-inducible factor (HIF-1alpha) is expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). As HIF-1alpha is selectively expressed in tumor cells, inhibitors are being developed for cancer therapy. Although methods for the detection of HIF-1alpha and PHDs are available, an immunohistochemical double staining method for these markers in individual tumor cells is not available. For method development a human squamous cell carcinoma (SCC) xenograft, A253, was used as a known positive control tissue for HIF-1alpha in well-differentiated areas without microvessels. This laboratory showed that tumor cells in these areas are strongly positive for hypoxia markers. Another human, poorly differentiated SCC xenograft, FaDu, without hypoxic areas, was used as a negative control. PHD2 and 3 immunostaining was optimized individually using the human kidney. To optimize HIF-1alpha detection the pressure cooker time for antigen retrieval, concentration of the primary antibody, amplification reagent, and DAB development time were decreased. Casein blocking further decreased the background. Double staining resulted in brown nuclei for HIF-1alpha (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype-matched controls were negative. Normal human tissues had no detectable HIF-1alpha, but expressed PHD2, 3. The potential use of this new and improved method was confirmed by analyzing 15 surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1alpha. This new method defined the optimal conditions for detection of HIF-1alpha and PHDs in individual tumor cells and could have a diagnostic and therapeutic potential.

  14. 1-alpha-Hydroxyvitamin D3 derivatives in the treatment of renal bone diseases: justification and optimal modalities of administration.

    PubMed

    Fournier, A; Morinière, P H; Oprisiu, R; Yverneau-Hardy, P; Westeel, P F; Mazouz, H; el Esper, N; Ghazali, A; Boudailliez, B

    1995-01-01

    The use of 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3] derivatives in a uremic patient is justified only in the treatment of hyperparathyroidism (i.e. when plasma intact parathyroid hormone - PTH - levels are above five or three times the upper limit of normal according to whether the patient is on continuous ambulatory peritoneal dialysis or on hemodialysis and between 0.5-1.5, 1-2 and 2-3 times the upper limit of normal for a creatinine clearance of, respectively, 30, between 30 and 10, or below 10 ml/min/1.73 m2). The following prerequisites have however to be satisfied: (1) a good vitamin D3 repletion should be secured by plasma 25(OH(D) levels of 20-30 ng/ml (if necessary by administration of native vitamin D or 25(OH)D3), and (2) phosphate retention (which is aggravated by the increased phosphate intestinal absorption induced by the 1 alpha (OH)D derivatives) and the consequent possible hyperphosphatemia should be prevented or corrected by the oral administration of alkaline salts of calcium given before the meals as phosphate binders without inducing hypercalcemia. These prerequisites explain the narrow therapeutical margin of 1 alpha (OH)D3 derivatives in uremic patients before dialysis (more so in the adult than in the child) and the possible broadening of this margin in the patients on dialysis by the use of low dialysate calcium concentrations (1.25-1.00 mmol/l) in order to prevent hypercalcemia by inducing a negative perdialytic calcium balance. Once hyperphosphatemia is prevented by oral calcium, 1 alpha (OH)D3 derivatives have the advantage to suppress the transcription of the prepro PTH gene by a mechanism independent of an increase in plasma calcium. Controlled randomized trials have not confirmed the claimed advantage in efficacy and safety of the parenteral versus the oral route nor of the intermittent versus the daily mode of their administration. The advantages of using the so called 'nonhypercalcemic hyperphosphatemic' vitamin D3 derivatives in

  15. HNF-1alpha plays an important role in IL-6-induced expression of the human angiotensinogen gene.

    PubMed

    Jain, Sudhir; Li, Yanna; Patil, Sai; Kumar, Ashok

    2007-07-01

    Angiotensinogen (AGT) is the precursor of one of the most important vasoactive hormone angiotensin II and this gene locus is associated with human essential hypertension. AGT is an acute phase protein and its gene expression is regulated by IL-6. Previous studies have identified three potential STAT-3 binding sites (APREs) located between -160 and -280 of the hAGT gene promoter but only APRE-1 (located between -271 and -279) was shown to be a bonafide enhancer for IL-6-induced promoter activity. We show here that APRE-2, located between -236 and -247, is indeed an HNF-1alpha-binding site and plays an important role in basal and IL-6 induced promoter activity of this gene. Our chromatin immunoprecipitation (ChIP) assay shows that HNF-1alpha binds to this region of the hAGT gene promoter and its recruitment is increased in the presence of IL-6 in Hep3B cells. We also show that the promoter activity of a deletion construct containing only 223 bp of the hAGT gene promoter (that contains only APRE-3) is increased after IL-6 treatment. Our ChIP assay shows that IL-6 treatment recruits STAT-3 to APRE-3 and suggests that this is also an IL6 responsive element. We have previously shown that GR binds to the proximal promoter of the hAGT gene. Since GR physically interacts with STAT-3, we propose that transcription factors GR, STAT-3, and HNF-1alpha that bind to the nucleotide sequence located between -160 and -280 of the hAGT gene promoter are responsible for IL-6 induced promoter activity of this gene.

  16. Relation of oral 1alpha-hydroxy vitamin D3 to the progression of aortic arch calcification in hemodialysis patients.

    PubMed

    Ogawa, Tetsuya; Ishida, Hideki; Akamatsu, Mayuko; Matsuda, Nami; Fujiu, Ayuko; Ito, Kyoko; Ando, Yoshitaka; Nitta, Kosaku

    2010-01-01

    The role of decreased active vitamin D levels on vascular calcification has not been elucidated in hemodialysis (HD) patients. The aim of the present study was to evaluate the relationship between progression of aortic arch calcification (AoAC) and prescribed dose of 1alpha-hydroxy vitamin D. The enrolled study subjects were 65 (40 men and 25 women) HD patients. Calcification of the aortic arch was semiquantitatively estimated with a score (AoACS) on plain chest radiology. Change in AoACS (DeltaAoACS) was obtained by subtracting the baseline AoACS value from the follow-up AoACS value. The second assessment was performed from 2 years after the first determination. The nonprogressors (63.2 +/- 14.5 years) were significantly younger than the progressors (68.2 +/- 10.8 years) (P = 0.0419). In addition, prescribed dose of 1alpha-hydroxy vitamin D3 was significantly higher in the nonprogressors (125.5 +/- 109.1 microg) than progressors (84.8 +/- 81.1 microg) (P = 0.0371). Multiple regression analysis revealed prescribed dose of 1alpha-hydroxy vitamin D(3) (beta value = -0.324, P = 0.0051) as well as DBP (beta value = -0.418, P = 0.007), serum levels of P (beta value = 0.333, P = 0.006) and C-reactive protein (beta value = 0.237, P = 0.0048) to be significant independent determinants of DeltaAoACS. In conclusion, the evaluation of AoACS on chest radiography is a very simple tool in HD patients. Active vitamin D therapy seems to protect patients from developing vascular calcification.

  17. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  18. Noscapine inhibits hypoxia-mediated HIF-1alpha expression andangiogenesis in vitro: a novel function for an old drug.

    PubMed

    Newcomb, Elizabeth W; Lukyanov, Yevgeniy; Schnee, Tona; Ali, M Aktar; Lan, Li; Zagzag, David

    2006-05-01

    Overexpression of hypoxia-inducible factor-1 (HIF-1) is a common feature in solid malignancies related to oxygen deficiency. Since increased HIF-1 expression correlates with advanced disease stage, increased angiogenesis and poor prognosis, HIF-1 and its signaling pathway have become targets for cancer chemotherapy. In this study, we identified noscapine to be a novel small molecule inhibitor of the HIF-1 pathway based on its structure-function relation-ships with HIF-1 pathway inhibitors belonging to the benzylisoquinoline class of plant metabolites and/or to microtubule binding agents. We demonstrate that noscapine treatment of human glioma U87MG and T98G cell lines exposed to the hypoxic mimetic agent, CoCl2, inhibits hypoxia-mediated HIF-1alpha expression and transcriptional activity as measured by decreased secretion of VEGF, a HIF-1 target gene. Inhibition of hypoxia-mediated HIF-1alpha expression was due, in part, to its ability to inhibit accumulation of HIF-1alpha in the nucleus and target it for degradation via the proteasome. One mechanism of action of microtubule binding agents is their antiangiogenic activity associated with disruption of endothelial tubule formation. We show that noscapine has similar properties in vitro. Thus, noscapine may possess novel antiangiogenic activity associated with two broad mechanisms of action: first, by decreasing HIF-1alpha expression in hypoxic tumor cells, upregulation of target genes, such as VEGF, would be decreased concomitant with its associated angiogenic activity; second, by inhibiting endothelial cells from forming blood vessels in response to VEGF stimulation, it may limit the process of neo-vascularization, correlating with antitumor activity in vivo. For more than 75 years, noscapine has traditionally been used as an oral cough suppressant with no known toxic side effects in man. Thus, the studies reported here have found a novel function for an old drug. Given its low toxicity profile, its demonstrated

  19. Mitochondrial Diseases and Cardiomyopathies.

    PubMed

    Brunel-Guitton, Catherine; Levtova, Alina; Sasarman, Florin

    2015-11-01

    Mitochondrial cardiomyopathies are clinically and genetically heterogeneous. An integrative approach encompassing clinical, biochemical, and molecular investigations is required to reach a specific diagnosis. In this review we summarize the clinical and genetic aspects of mitochondrial disorders associated with cardiomyopathy, including disorders of oxidative phosphorylation. It also describes groups of disorders that, although not usually classified as mitochondrial disorders, stem from defects in mitochondrial function (eg, disorders of β-oxidation and the carnitine cycle), are associated with secondary mitochondrial impairment (eg, organic acidurias), and are important diagnostically because they are treatable. Current biochemical and molecular techniques for the diagnosis of mitochondrial cardiomyopathies are described, and a diagnostic algorithm is proposed, to help clinicians in their approach to cardiomyopathies in the context of mitochondrial diseases.

  20. Effect of doxercalciferol (1alpha-hydroxyvitamin D2) on PTH, bone turnover and bone mineral density in a hemodialysis patient with persistent secondary hyperparathyroidism post parathyroidectomy.

    PubMed

    Parisi, M S; Oliveri, B; Somoza, J; Mautalen, C

    2003-06-01

    The efficacy and safety of the vitamin D analog, doxercalciferol (1alpha-hydroxyvitamin D2, 1alphaD2) in the treatment of secondary hyperparathyroidism in hemodialysis patients has been previously reported. We report these effect of 16-week 1alphaD2 treatment on mineral metabolism and bone mineral density (BMD) in a hemodialysis patient with persistent secondary hyperparathyroidism post parathyroidectomy, resistant to previous calcitriol treatment. Levels of iPTH, bone-specific alkaline phosphatase and serum type I collagen C telopeptide were above normal at baseline and were substantially decreased with 1alphaD2 treatment (-92%, -63% and -53%, respectively). BMD increased in all areas: total skeleton (+6.5%), lumbar spine (+6.9%) and total femur (+4.3%). The patient showed no hypercalcemia, and phosphorus levels remained between 3.3 and 6.2 mg/dl.

  1. Bacterial cell wall products increases stabilization of HIF-1 alpha in an oligodendrocyte cell line preconditioned by cobalt chloride or desferrioxamine.

    PubMed

    Yao, Song-yi; Soutto, Mohammed; Sriram, Subramaniam

    2008-08-30

    We examined the effect of lipopolysaccharide (LPS) or lipotechoic acid (LTA) on the regulation of hypoxia inducible factor (HIF-1) alpha on the MO3.13 cells, a human oligodendroglial cell line. Our study shows that MO3.13 cells express the toll like receptors (TLR's) but do not increase cellular levels of HIF-1 alpha following exposure to bacterial cell wall products. When MO3.13 cells were preconditioned by desferrioxamine (DFO) or cobalt chloride (CoCl(2)) and then treated with either LPS or LTA, HIF-1 alpha levels were higher than that induced by DFO or CoCl(2) alone. The increase in HIF-1 alpha was due to increased protein stability that was mediated by activation of the ERK-MAP kinase pathway.

  2. Evolution of translational elongation factor (EF) sequences: reliability of global phylogenies inferred from EF-1 alpha(Tu) and EF-2(G) proteins.

    PubMed Central

    Creti, R; Ceccarelli, E; Bocchetta, M; Sanangelantoni, A M; Tiboni, O; Palm, P; Cammarano, P

    1994-01-01

    The EF-2 coding genes of the Archaea Pyrococcus woesei and Desulfurococcus mobilis were cloned and sequenced. Global phylogenies were inferred by alternative tree-making methods from available EF-2(G) sequence data and contrasted with phylogenies constructed from the more conserved but shorter EF-1 alpha(Tu) sequences. Both the monophyly (sensu Hennig) of Archaea and their subdivision into the kingdoms Crenarchaeota and Euryarchaeota are consistently inferred by analysis of EF-2(G) sequences, usually at a high bootstrap confidence level. In contrast, EF-1 alpha(Tu) phylogenies tend to be inconsistent with one another and show low bootstrap confidence levels. While evolutionary distance and DNA maximum parsimony analyses of EF-1 alpha(Tu) sequences do show archaeal monophyly, protein parsimony and DNA maximum-likelihood analyses of these data do not. In no case, however, do any of the tree topologies inferred from EF-1 alpha(Tu) sequence analyses receive significant bootstrap support. PMID:8159735

  3. 1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid.

    PubMed

    Schwartz, Z; Shaked, D; Hardin, R R; Gruwell, S; Dean, D D; Sylvia, V L; Boyan, B D

    2003-05-01

    1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of

  4. Anti-interleukin-1 alpha autoantibodies in humans: Characterization, isotype distribution, and receptor-binding inhibition--higher frequency in Schnitzler's syndrome (urticaria and macroglobulinemia)

    SciTech Connect

    Saurat, J.H.; Schifferli, J.; Steiger, G.; Dayer, J.M.; Didierjean, L. )

    1991-08-01

    Since autoantibodies (Abs) to cytokines may modify their biologic activities, high-affinity binding factors for interleukin-1 alpha (IL-1 alpha BF) were characterized in human sera. IL-1 alpha BF was identified as IgG (1) by sucrose density-gradient centrifugation followed by immunodiffusion autoradiography, (2) by ligand-blotting method, (3) by ligand binding to affinity-immobilized serum IgG, and (4) by IgG affinity purification followed by sucrose density-gradient centrifugation. IL-1 alpha binding activity resided in the F(ab)2 fragment. The apparent equilibrium constant was in the range of IgG found after immunization with conventional antigens (i.e., 10(-9) to 10(-10) mol/L). Anti-IL-1 alpha IgG auto-Abs represented only an extremely small fraction of total IgG (less than 1/10(-5)). Some sera with IL-1 alpha BF and purified IgG thereof were able to inhibit by 96% to 98% the binding of human recombinant IL-1 alpha to its receptor on murine thymoma EL4-6.1 cells, whereas other sera did not. When 125I-labeled anti-IL-1 alpha IgG complexes were injected into rats, they prolonged the plasma half-life of 125I-labeled IL-1 alpha several fold and altered its tissue distribution. The predominant class was IgG (12/19), mainly IgG4 (9/19), but in five of the sera, anti-IL-1 alpha IgA was also detected. In a screening of 271 sera, IL-1 alpha BF was detected in 17/98 normal subjects and was not more frequent in several control groups of patients, except in patients with Schnitzler's syndrome (fever, chronic urticaria, bone pain, and monoclonal IgM paraprotein) (6/9; p less than 0.005). The pathologic significance of these auto-Abs remains to be determined.

  5. A Pak- and Pix-dependent branch of the SDF-1alpha signalling pathway mediates T cell chemotaxis across restrictive barriers.

    PubMed

    Volinsky, Natalia; Gantman, Anna; Yablonski, Deborah

    2006-07-01

    Pak (p21-activated kinase) serine/threonine kinases have been shown to mediate directional sensing of chemokine gradients. We hypothesized that Pak may also mediate chemokine-induced shape changes, to facilitate leucocyte chemotaxis through restrictive barriers, such as the extracellular matrix. A potent inhibitor, Pak(i), was characterized and used to probe the role of Pak-family kinases in SDF-1alpha (stromal-cell derived factor-1alpha/CXCL12)-induced chemotaxis in a T cell model. Pak(i) potently inhibited SDF-1alpha-induced Pak activation by a bivalent mechanism, as indicated by its complete inactivation upon point mutation of two binding sites, but partial inactivation upon mutation of either site alone. Importantly, Pak(i) was not toxic to cells over the time frame of our experiments, since it did not substantially affect cell surface expression of CXCR4 (CXC chemokine receptor 4) or integrins, cell cycle progression, or a number of ligand-induced responses. Pak(i) produced dose-dependent inhibition of SDF-1alpha-induced migration through rigid filters bearing small pores; but unexpectedly, did not substantially affect the magnitude or kinetics of chemotaxis through filters bearing larger pores. SDF-1alpha-induced Pak activation was partly dependent on PIX (Pak-interactive exchange factor); correspondingly, an allele of beta-PIX that cannot bind Pak inhibited SDF-1alpha-induced chemotaxis through small, but not large pores. By contrast, other key players in chemotaxis: G(i), PI3K (phosphoinositide 3-kinase), and the Rho-family G-proteins, Rac and Cdc42 (cell division cycle 42), were required for SDF-1alpha-induced migration regardless of the barrier pore-size. These studies have revealed a distinct branch of the SDF-1alpha signalling pathway, in which the Rac/Cdc42 effector, Pak, and its partner, PIX, specifically regulate the cellular events required for chemokine-induced migration through restrictive barriers.

  6. Preparation and biological activity of 24-epi-26,26,26,27,27,27-hexafluoro- 1 alpha,25-dihydroxyvitamin D2.

    PubMed

    Iseki, K; Oishi, S; Namba, H; Taguchi, T; Kobayashi, Y

    1995-11-01

    A new fluorinated analog of vitamin D2, 24-epi-26,26,26,27,27,27-hexafluoro- 1 alpha,25-dihydroxyvitamin D2, was efficiently synthesized starting from (R)-4-isopropyl-3-propionyl-2- oxazolidinone with high stereochemical control. In all four physiological test systems, the fluorinate vitamin D2 analog was found to be slightly less active than 1 alpha,25-dihydroxyvitamin D3.

  7. Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

    PubMed Central

    Moon, Sung-Kyun; Lee, Haa-Yung; Pan, Huiqi; Takeshita, Tamotsu; Park, Raekil; Cha, Kiweon; Andalibi, Ali; Lim, David J

    2006-01-01

    Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway. PMID:16433908

  8. Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function.

    PubMed

    Escuin, Daniel; Kline, Erik R; Giannakakou, Paraskevi

    2005-10-01

    We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1alpha. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1alpha protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1alpha transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1alpha protein in the human ovarian cancer cell line 1A9 and its beta-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1alpha protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1alpha in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1alpha nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link beta-tubulin drug binding with HIF-1alpha protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients.

  9. Stem cell homing and angiomyogenesis in transplanted hearts are enhanced by combined intramyocardial SDF-1alpha delivery and endogenous cytokine signaling.

    PubMed

    Zhao, Tiemin; Zhang, Dongsheng; Millard, Ronald W; Ashraf, Muhammad; Wang, Yigang

    2009-04-01

    We used a heterotopic transplanted working heart model to probe the collaborative role of bone marrow-derived progenitor cells (BPCs) and stromal cell-derived factor (SDF)-1alpha in attenuating tissue remodeling in recipient and transplanted hearts. BPCs from male transgenic rats expressing green fluorescent protein (GFP(+) BPCs, 2 x 10(6) cells) were injected intravenously into myeloablated female rats. One month later, heterotopic heart transplantation was performed. The left anterior descending coronary artery (LAD) of the recipient heart was occluded permanently. Mesenchymal stem cells (MSCs; 2 x 10(6) cells) with a null gene (null group) or overexpressing SDF-1alpha (SDF-1alpha group) were injected intramyocardially in the LAD perfusion region of both recipient and transplanted hearts. Recipient and transplanted hearts (n = 10 hearts/group) were harvested 21 days later for analysis. The survival of transplanted hearts was assessed daily by palpation in additional animals (n = 7). Five days after LAD occlusion, subpopulations of GFP(+) BPCs in the circulation were significantly higher in the SDF-1alpha group. Y chromosome, 5-bromo-2'-deoxyuridine, Ki67-positive nuclei, newly formed vessels, and GFP(+) cells significantly increased in transplanted hearts of the SDF-1alpha group at 21 days after the injection of MSCs overexpressing SDF-1alpha, whereas fewer TUNEL-positive nuclei were found. The survival of transplanted hearts was also markedly increased in the SDF-1alpha group (P < 0.05). Supplementation of endogenous cytokines released from the ischemic myocardium with exogenous MSCs overexpressing SDF-1alpha significantly increased BPC homing to acutely ischemic recipient and progressively ischemic transplanted hearts. BPC recruitment resulted in the regeneration of new cardiomyocytes and blood vessels and extended survival of the transplanted hearts.

  10. Follicular development and expression of nuclear respiratory factor-1 and peroxisome proliferator-activated receptor γ coactivator-1 alpha in ovaries of fetal and neonatal doelings.

    PubMed

    Zhou, Z; Wan, Y; Zhang, Y; Wang, Z; Jia, R; Fan, Y; Nie, H; Ying, S; Huang, P; Wang, F

    2012-11-01

    In livestock, the ovarian reserve of follicles is established during the fetal stage. However, at least two-thirds of the oocytes present in the reserve die because of apoptosis before birth. Notably, mitochondria have been reported to play a crucial role in the fate (life/death) of oocytes. In this study, mitochondrial regulators nuclear respiratory factor-1 (NRF-1) and PPAR γ coactivator-1 alpha (PGC-1α) were examined during this period of follicle development to investigate their effects on follicular development and apoptosis. Fetal and neonatal Capra haimen were used, ranging in age from 60 d postcoitum (dpc) to 30 d postpartum (dpp). Our data demonstrated that egg nests were the earliest recognizable gamete cells in ovaries of fetal and neonatal doelings. Proportions of egg nests decreased from 92.68 to 25.08% whereas single follicles increased from 7.32 to 74.92% between 60 and 120 dpc. Subsequently, between 90 and 120 dpc, the proportion of primordial follicles increased from 9.98 to 61.56% (P < 0.01). However, it did not change between 1 and 30 dpp (P = 0.12). The proportion of primary follicles increased from 1.23 to 37.93% between 90 dpc to 1 dpp (P = 0.01) but did not change between 1 and 30 dpp (P = 0.11). Meanwhile, proportions of secondary and tertiary follicles increased in an age-dependent manner. In addition, results of this study suggested that NRF-1 and PGC-1α proteins are mainly localized in germ cells of egg nests, cytoplasm of oocytes, and granulosa cells of follicles ranging from primordial to tertiary follicles. The transcript abundance of NRF-1 mRNA was up-regulated in 60-dpc-old ovaries compared with 1-dpp-old ovaries (P < 0.05), but the PGC-1α mRNA expression pattern did not change (P = 0.05). Nevertheless, the number of terminal deoxynucleotidyltransferase UTP nick-end labeling (TUNEL) positive cells and caspase-3 activity in 60-dpc-old ovaries was less than those in 1-dpp-old ovaries (P < 0.01, P = 0.01). In conclusion, our results

  11. Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1.

    PubMed

    Osada-Oka, Mayuko; Ikeda, Takako; Akiba, Satoshi; Sato, Takashi

    2008-08-01

    The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.

  12. Role for macrophage inflammatory protein (MIP)-1alpha and MIP-1beta in the development of osteolytic lesions in multiple myeloma.

    PubMed

    Abe, Masahiro; Hiura, Kenji; Wilde, Javier; Moriyama, Keiji; Hashimoto, Toshihiro; Ozaki, Shuji; Wakatsuki, Shingo; Kosaka, Masaaki; Kido, Shinsuke; Inoue, Daisuke; Matsumoto, Toshio

    2002-09-15

    Multiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1alpha and MIP-1beta correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1alpha and MIP-1beta. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1alpha and MIP-1beta, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1alpha and MIP-1beta induce expression of receptor activator of nuclear factor-kappaB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1alpha and MIP-1beta may be major osteoclast-activating factors produced by MM cells.

  13. Effects of medicinal cake-separated moxibustion on plasma 6-keto-PGF1alpha and TXB2 contents in the rabbit of hyperlipemia.

    PubMed

    Xiaorong, Chang; Jie, Yan; Zenghui, Yue; Jing, Shen; Yaping, Lin; Shouxiang, Yi; Xiangping, Cao

    2005-06-01

    Hyperlipemia rabbit models established with high cholesterol and fat diet were treated with direct moxibustion and medicinal cake-separated moxibustion. The post-treatment plasma 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and thromboxane B2 (TXB2) contents were determined by radioimmunoassay. Results indicated that the plasma 6-keto-PGF1alpha content significantly increased, the TXB2 level decreased (P < 0.05) and the TXB2 /6-keto-PGF1alpha ratio also decreased (P < 0.01) in the medicinal cake-separated moxibustion group as compared with those in the model group respectively, but there was no significant difference between the medicinal cake-separated moxibustion group and the direct moxibustion group (P > 0.05), suggesting that both the medicinal cake-separated moxibustion and direct moxibustion can regulate the plasma 6-keto-PGF1alpha and TXB2 contents, and the TXB2/6-keto-PGF1alpha ratio with similar actions, and have a certain protective action on endothelial cells of the aorta in the rabbit of hyperlipemia.

  14. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  15. Mitochondrial helicases and mitochondrial genome maintenance

    PubMed Central

    de Souza-Pinto, Nadja C.; Aamann, Maria D.; Kulikowicz, Tomasz; Stevnsner, Tinna V.; Bohr, Vilhelm A.

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases have been studied in detail; however, the roles of specific helicases in mitochondrial biology remain poorly characterized. This review presents important recent advances in identifying and characterizing mitochondrial helicases, some of which also operate in the nucleus. PMID:20576512

  16. Mitochondrial lipids in neurodegeneration.

    PubMed

    Aufschnaiter, Andreas; Kohler, Verena; Diessl, Jutta; Peselj, Carlotta; Carmona-Gutierrez, Didac; Keller, Walter; Büttner, Sabrina

    2017-01-01

    Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.

  17. Increased intrinsic mitochondrial function in humans with mitochondrial haplogroup H.

    PubMed

    Larsen, Steen; Díez-Sánchez, Carmen; Rabøl, Rasmus; Ara, Ignacio; Dela, Flemming; Helge, Jørn W

    2014-02-01

    It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present. © 2013.

  18. Mitochondrial antigens, molecular mimicry and autoimmune disease.

    PubMed

    Baum, H

    1995-05-24

    The immune system is normally tolerant to mitochondrial self-antigens, but responsive against bacteria. Low-titre anti-mitochondrial antibodies (AMA) might be involved in this discrimination. Tolerance is broken in diseases characterised by high titre AMA. Some of these AMA, against cardiolipin, cross-react with DNA. The best studied AMA are those characterising primary biliary cirrhosis (PBC). These are directed against E2 subunits of the oxo-acid dehydrogenase complexes, and also against subunits E1 alpha, E1 beta and X of the pyruvate dehydrogenase complex. AMA of PBC patients also react with bacterial E2s. Reactivities are primarily peptide-specific but with cross-reactivity between mitochondrial and microbial antigens and between E2s of respective complexes. Immunodominant epitopes, for anti E2 AMA, include the conserved sequence flanking the site of lipoyl attachment. It is proposed that the initial stimulus for antibody production is chronic urinary tract infection. AMA themselves are not pathogenic, but CD4+ T-cells would be primed, recognising the lipoyl domain epitope in association with class II HLA. Inappropriate expression of class II antigens on bile duct epithelia, (as found in PBC), might lead to presentation of a particular fragment of HLA-DR alpha, known to be a major MHC presented self-peptide in the mouse. That sequence strongly mimics the lipoyl domain and might be recognised by primed T-cells, initiating the autoimmune cascade. In the mouse, a peptide of ND1 of Complex I is presented in association with class I MHC. Cells exhibiting somatic mutation of such a peptide might thus be subject to attack by CD8+ T-cells. If such peptides were presented by class II HLA, autoimmune diseases might arise, related to mimicry between such peptides and microbial sequences and/or self-antigens. These considerations might apply in Leber's disease and in age-related pathology.

  19. Decreased mitochondrial copy numbers in oral squamous cell carcinoma.

    PubMed

    Takeda, Daisuke; Hasegawa, Takumi; Ueha, Takeshi; Sakakibara, Akiko; Kawamoto, Teruya; Minamikawa, Tsutomu; Sakai, Yoshitada; Komori, Takahide

    2016-08-01

    Mitochondrial dysfunction and altered respiration have long been suspected to affect the development and progression of cancer. Although quantitative changes in mitochondrial DNA (mtDNA) have been reported in head and neck squamous cell carcinoma (SCC), differences in mtDNA copy numbers between normal and cancerous tissues from same patients have not been assessed. We compared mtDNA copy numbers and expressions of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) between normal mucous membrane and cancerous tissues resected from 35 patients with oral SCC, using TaqMan quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining. We found mtDNA copy numbers and expressions of PGC-1α and TFAM were decreased in cancerous tissues compared with normal tissues from the same patients. The PGC-1α-TFAM mitochondrial pathway may be associated with malignant potential in human oral SCC, and could be an attractive therapeutic target. © 2016 Wiley Periodicals, Inc. Head Neck 38:1170-1175, 2016. © 2016 Wiley Periodicals, Inc.

  20. G(s)alpha deficiency in skeletal muscle leads to reduced muscle mass, fiber-type switching, and glucose intolerance without insulin resistance or deficiency.

    PubMed

    Chen, Min; Feng, Han-Zhong; Gupta, Divakar; Kelleher, James; Dickerson, Kathryn E; Wang, Jie; Hunt, Desmond; Jou, William; Gavrilova, Oksana; Jin, Jian-Ping; Weinstein, Lee S

    2009-04-01

    The ubiquitously expressed G protein alpha-subunit G(s)alpha is required for receptor-stimulated intracellular cAMP responses and is an important regulator of energy and glucose metabolism. We have generated skeletal muscle-specific G(s)alpha-knockout (KO) mice (MGsKO) by mating G(s)alpha-floxed mice with muscle creatine kinase-cre transgenic mice. MGsKO mice had normal body weight and composition, and their serum glucose, insulin, free fatty acid, and triglyceride levels were similar to that of controls. However, MGsKO mice were glucose intolerant despite the fact that insulin sensitivity and glucose-stimulated insulin secretion were normal, suggesting an insulin-independent mechanism. Isolated muscles from MGsKO mice had increased basal glucose uptake and normal responses to a stimulator of AMP-activated protein kinase (AMPK), which indicates that AMPK and its downstream pathways are intact. Compared with control mice, MGsKO mice had reduced muscle mass with decreased cross-sectional area and force production. In addition, adult MGsKO mice showed an increased proportion of type I (slow-twitch, oxidative) fibers based on kinetic properties and myosin heavy chain isoforms, despite the fact that these muscles had reduced expression of peroxisome proliferator-activated receptor coactivator protein-1alpha (PGC-1alpha) and reduced mitochondrial content and oxidative capacity. Therefore G(s)alpha deficiency led to fast-to-slow fiber-type switching, which appeared to be dissociated from the expected change in oxidative capacity. MGsKO mice are a valuable model for future studies of the role of G(s)alpha signaling pathways in skeletal muscle adaptation and their effects on whole body metabolism.

  1. Deletion of inducible nitric-oxide synthase in leptin-deficient mice improves brown adipose tissue function.

    PubMed

    Becerril, Sara; Rodríguez, Amaia; Catalán, Victoria; Sáinz, Neira; Ramírez, Beatriz; Collantes, María; Peñuelas, Iván; Gómez-Ambrosi, Javier; Frühbeck, Gema

    2010-06-04

    Leptin and nitric oxide (NO) on their own participate in the control of non-shivering thermogenesis. However, the functional interplay between both factors in this process has not been explored so far. Therefore, the aim of the present study was to analyze the impact of the absence of the inducible NO synthase (iNOS) gene in the regulation of energy balance in ob/ob mice. Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated, and the expression of molecules involved in the control of brown fat cell function was analyzed by real-time PCR, western-blot and immunohistochemistry. Twelve week-old DBKO mice exhibited reduced body weight (p<0.05), decreased amounts of total fat pads (p<0.05), lower food efficiency rates (p<0.05) and higher rectal temperature (p<0.05) than ob/ob mice. Ablation of iNOS also improved the carbohydrate and lipid metabolism of ob/ob mice. DBKO showed a marked reduction in the size of brown adipocytes compared to ob/ob mutants. In this sense, in comparison to ob/ob mice, DBKO rodents showed an increase in the expression of PR domain containing 16 (Prdm16), a transcriptional regulator of brown adipogenesis. Moreover, iNOS deletion enhanced the expression of mitochondria-related proteins, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1alpha), sirtuin-1 (Sirt-1) and sirtuin-3 (Sirt-3). Accordingly, mitochondrial uncoupling proteins 1 and 3 (Ucp-1 and Ucp-3) were upregulated in brown adipose tissue (BAT) of DBKO mice as compared to ob/ob rodents. Ablation of iNOS improved the energy balance of ob/ob mice by decreasing food efficiency through an increase in thermogenesis. These effects may be mediated, in part, through the recovery of the BAT phenotype and brown fat cell function improvement.

  2. Carbohydrate feeding during recovery alters the skeletal muscle metabolic response to repeated sessions of high-intensity interval exercise in humans.

    PubMed

    Cochran, Andrew J R; Little, Jonathan P; Tarnopolsky, Mark A; Gibala, Martin J

    2010-03-01

    Exercise training under conditions of reduced carbohydrate (CHO) availability has been reported to augment gains in skeletal muscle oxidative capacity; however, the underlying mechanisms are unclear. We examined the effect of manipulating CHO intake on the acute metabolic response to high-intensity interval exercise, including signaling cascades linked to mitochondrial biogenesis. Ten men performed two trials in random order separated by >or=1 wk. Each trial consisted of a morning (AM) and afternoon (PM) training session (5 x 4 min cycling at approximately 90-95% of heart rate reserve) separated by 3 h of recovery during which subjects ingested a high-CHO drink (HI-HI) or nonenergetic placebo (HI-LO) before PM exercise. Biopsies (vastus lateralis) revealed that muscle phosphocreatine and ATP content were similar after AM exercise but decreased to a greater extent during PM exercise in HI-LO vs. HI-HI. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) increased approximately 4-fold and 2-fold, respectively, during AM exercise with no difference between conditions. After PM exercise, p38 MAPK phosphorylation was higher in HI-LO vs. HI-HI, whereas AMPK was not different between conditions. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 alpha) gene expression increased approximately 8-fold during recovery from AM exercise and remained elevated during PM exercise with no differences between conditions. Cytochrome oxidase subunit 4 (COXIV) mRNA was also elevated 3 h after AM exercise, with no difference between conditions. These data provide evidence that p38 MAPK is a nutrient-sensitive signaling molecule that could be involved in the altered skeletal muscle adaptive response reported after exercise training under conditions of restricted CHO intake, but further research is required to confirm this hypothesis.

  3. Impaired oxygen-dependent reduction of HIF-1alpha and -2alpha proteins in pre-eclamptic placentae.

    PubMed

    Rajakumar, A; Doty, K; Daftary, A; Harger, G; Conrad, K P

    2003-01-01

    Pre-eclamptic (PE) placentae overexpress hypoxia inducible transcription factors-1alpha and -2alpha proteins (Biol. Repro. 64: 499-506, 2001; Ibid 1019-1020). Possible explanations include (a) impaired oxygen-dependent reduction, and/or (b) enhanced sensitivity to reduced oxygen. After 18 h equilibration under 21 per cent O(2) atmosphere, we subjected villous explants prepared from placentae of normal pregnant (NP) and pre-eclamptic (PE) women (n=8 each) to 4h of hypoxia (2 per cent oxygen), and then studied the disappearance of HIF-1alpha and -2alpha proteins during subsequent oxygenation over 90 min (21 per cent oxygen). The disappearance of these HIF proteins as assessed by Western analysis was significantly impaired in the pre-eclamptic tissues. Even after 18h equilibration under a 21 per cent O(2) atmosphere, and then a further 4h at 21 per cent O(2), HIF-1alpha and -2alpha protein expression remained increased in villous explants from PE women (both P< 0.04 vs NP). To address whether chronic hypoxia per se (which is believed to exist in the pre-eclamptic placenta) might contribute to these findings, we subjected villous explants from normal placentae (n=6) to 18 h preincubation under 2 per cent or 21 per cent oxygen prior to subsequent incubation for 4h at 2 per cent oxygen and then 90 min at 21 per cent oxygen. The time course of disappearance of HIF proteins during oxygenation was similar irrespective of the 2 per cent or 21 per cent preconditioning. To evaluate oxygen sensitivity, we exposed villous explants from NP and PE women (n=6 each) to different oxygen atmospheres for 4h and measured HIF protein induction. Although the data showed a significant inverse relationship between HIF expression and oxygen concentration, there was no significant difference between the slopes of this relationship for the two groups of women. We conclude that villous explants from PE placentae fail to adequately downregulate HIF protein expression upon oxygenation. This

  4. Progress in mitochondrial epigenetics.

    PubMed

    Manev, Hari; Dzitoyeva, Svetlana

    2013-08-01

    Mitochondria, intracellular organelles with their own genome, have been shown capable of interacting with epigenetic mechanisms in at least four different ways. First, epigenetic mechanisms that regulate the expression of nuclear genome influence mitochondria by modulating the expression of nuclear-encoded mitochondrial genes. Second, a cell-specific mitochondrial DNA content (copy number) and mitochondrial activity determine the methylation pattern of nuclear genes. Third, mitochondrial DNA variants influence the nuclear gene expression patterns and the nuclear DNA (ncDNA) methylation levels. Fourth and most recent line of evidence indicates that mitochondrial DNA similar to ncDNA also is subject to epigenetic modifications, particularly by the 5-methylcytosine and 5-hydroxymethylcytosine marks. The latter interaction of mitochondria with epigenetics has been termed 'mitochondrial epigenetics'. Here we summarize recent developments in this particular area of epigenetic research. Furthermore, we propose the term 'mitoepigenetics' to include all four above-noted types of interactions between mitochondria and epigenetics, and we suggest a more restricted usage of the term 'mitochondrial epigenetics' for molecular events dealing solely with the intra-mitochondrial epigenetics and the modifications of mitochondrial genome.

  5. Mitochondrial threshold effects.

    PubMed Central

    Rossignol, Rodrigue; Faustin, Benjamin; Rocher, Christophe; Malgat, Monique; Mazat, Jean-Pierre; Letellier, Thierry

    2003-01-01

    The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases. PMID:12467494

  6. [Mitochondrial and oocyte development].

    PubMed

    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  7. Autoantibodies to mitochondrial 2-oxo-acid dehydrogenase complexes in localized scleroderma.

    PubMed

    Fujimoto, M; Sato, S; Ihn, H; Tamaki, T; Kikuchi, K; Soma, Y; Tamaki, K

    1996-08-01

    Sera from patients with localized scleroderma frequently produce cytoplasmic staining by indirect immunofluorescence, although the antigen remains to be determined. We studied the prevalence, antigen specificity and associated clinical characteristics of anti-cytoplasmic antibodies in localized scleroderma. Serum samples from 60 patients with localized scleroderma were examined by indirect immunofluorescence analysis and immunoblotting. By immunofluorescence analysis on HEp-2 cell substrate, seven of 60 (12%) patients were shown to be positive for anti-cytoplasmic antibodies. Among these, six patients with generalized morphea had anti-mitochondrial antibodies as shown by immunoblotting: they showed reactivity with the E2 component of pyruvate dehydrogenase complex (PDC), with protein X, and with the E2 component of alpha-oxo-glutarate dehydrogenase complex, while two of them showed reactivity with PDC-E1 alpha. One of these patients who was positive for anti-PDC-E1 alpha antibody showed laboratory abnormalities, suggesting the presence of primary biliary cirrhosis. The age of disease onset was significantly higher in these six patients than in those without anti-mitochondrial antibodies. Furthermore, five of them were classified into generalized morphea with multiple plaque lesions but without linear lesions (multiple plaque type). These observations suggest that major antigens for anti-cytoplasmic antibodies in patients with localized scleroderma are mitochondrial enzymes, 2-oxo-acid dehydrogenase complexes. Patients with anti-mitochondrial antibodies may comprise a unique subset of localized scleroderma designated multiple plaque type of generalized morphea of older onset.

  8. Testosterone replacement attenuates mitochondrial damage in a rat model of myocardial infarction.

    PubMed

    Wang, Fengyue; Yang, Jing; Sun, Junfeng; Dong, Yanli; Zhao, Hong; Shi, Hui; Fu, Lu

    2015-05-01

    Testosterone can affect cardiovascular disease, but its effects on mitochondrial dynamics in the post-infarct myocardium remain unclear. To observe the effects of testosterone replacement, a rat model of castration-myocardial infarction (MI) was established by ligating the left anterior descending coronary artery 2 weeks after castration with or without testosterone treatment. Expression of mitochondrial fission and fusion proteins was detected by western blot and immunofluorescence 14 days after MI. Cardiac function, myocardial inflammatory infiltration and fibrosis, cardiomyocyte apoptosis, mitochondrial microstructure, and ATP levels were also assessed. Compared with MI rats, castrated rats showed aggravated mitochondrial and myocardial insults, including mitochondrial swelling and disordered arrangement; loss of cristae, reduced mitochondrial length; decreased ATP levels; cardiomyocyte apoptosis; and impaired cardiac function. Results of western blotting analyses indicated that castration downregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1A) and mitofusin 2, but upregulated dynamin-related protein 1. The results were also supported by results obtained using immunofluorescence. However, these detrimental effects were reversed by testosterone supplementation, which also elevated the upstream AMP-activated protein kinase (AMPK) activation of PGC1A. Thus, testosterone can protect mitochondria in the post-infarct myocardium, partly via the AMPK-PGC1A pathway, thereby decreasing mitochondrial dysfunction and cardiomyocyte apoptosis. The effects of testosterone were confirmed by the results of ELISA analyses.

  9. Investigating complex I deficiency in Purkinje cells and synapses in patients with mitochondrial disease

    PubMed Central

    Chrysostomou, Alexia; Grady, John P.; Laude, Alex; Taylor, Robert W.; Turnbull, Doug M.

    2015-01-01

    Aims Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus. Methods We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age‐matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses. Results Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses. Conclusions This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia. PMID:26337858

  10. Follicular dendritic cell tumor of the mediastinum: expression of fractalkine and SDF-1alpha as mast cell chemoattractants.

    PubMed

    Guettier, Catherine; Validire, Pierre; Emilie, Dominique; Tricottet, Viviane; Sebagh, Mylène; Anjo, Aurora; Misset, Jean-Louis; Reynes, Michel

    2006-02-01

    Follicular dendritic cell tumor (FDCT) is a rare tumor mainly located in laterocervical lymph nodes. We report one case of mediastinal FDCT associated with a history of bullous skin disease and clinically obvious immunosuppression. This tumor was characterized by heavy mast cell infiltration. Mast cells were in close relationship with tumor cells as demonstrated by ultrastructural examination and their presence are probably related with the strong expression of mast cell chemoattractants as fraktalkine and stromal cell-derived factor-1alpha by tumor cells. The long follow-up period of more than 17 years allowed to us assess the relatively indolent evolution of this tumor characterized by three slowly growing local recurrences without metastasis.

  11. Effects of prenatal malnutrition on GABAA receptor alpha1, alpha3 and beta2 mRNA levels.

    PubMed

    Steiger, Janine L; Alexander, Mark J; Galler, Janina R; Farb, David H; Russek, Shelley J

    2003-09-15

    Exposure of pregnant rats to protein malnutrition throughout pregnancy alters the developing hippocampus, leading to increased inhibition and selective changes in hippocampal-mediated behaviors. Given that GABA mediates most inhibitory neurotransmission, we asked whether selective changes in the levels of GABA receptor subunit mRNAs might result. Quantitative RNase protection profiling of 12 GABAA and GABAB receptor subunit mRNAs show that alpha1 and beta2 decrease in the adult (P90) hippocampal formation of prenatally malnourished rats, while the levels of alpha3 are increased. Moreover, the distribution of alpha1, alpha3 and beta2 mRNAs remains unchanged in CA1 and CA3 hippocampal subfields relative to dentate gyrus. The data suggest that prenatal malnutrition produces global changes of certain GABAA, but not GABAB, receptor mRNAs in the hippocampal formation.

  12. Mitochondrial and Metabolic Gene Expression in the Aged Rat Heart

    PubMed Central

    Barton, Gregory P.; Sepe, Joseph J.; McKiernan, Susan H.; Aiken, Judd M.; Diffee, Gary M.

    2016-01-01

    Aging is associated with a decline in cardiac function. Exercise intervention has been suggested as a way to improve this decrement. Age-related decline in cardiac function is associated with decreases in fatty acid oxidation, mitochondrial function, and AMP-activated protein kinase (AMPK) activity. The molecular mechanisms involved with age-related changes in mitochondrial function and substrate metabolism are poorly understood. We determined gene expression differences in hearts of Young (6 mo), Old (33 mo), and old exercise trained (Old + EXE) (34 mo) FBN rats, using Qiagen PCR arrays for Glucose, Fatty acid, and Mitochondrial metabolism. Old rats demonstrated decreased (p < 0.05) expression for key genes in fatty acid oxidation, mitochondrial function, and AMPK signaling. There were no differences in the expression of genes involved in glucose metabolism with age. These gene expression changes occurred prior to altered protein translation as we found no differences in the protein content of peroxisome proliferator activated receptor gamma, coactivators 1 alpha (PGC-1α), peroxisome proliferator activated receptor alpha (PPARα), and AMPKα2 between young and old hearts. Four months of exercise training did not attenuate the decline in the gene expression in aged hearts. Despite this lack of change in gene expression, exercise-trained rats demonstrated increased exercise capacity compared to their sedentary counterparts. Taken together, our results show that differential expression of genes associated with fatty acid metabolism, AMPK signaling and mitochondrial function decrease in the aging heart which may play a role in age-related declines in fatty acid oxidation, AMPK activity, and mitochondrial function in the heart. PMID:27601998

  13. Intrinsic cardiac neurons involved in cardiac regulation possess alpha 1-, alpha 2-, beta 1- and beta 2-adrenoceptors.

    PubMed

    Armour, J A

    1997-03-01

    To determine whether intrinsic cardiac neurons involved in cardiac regulation possess alpha 1-, alpha 2-, beta 1-, or beta 2-adrenoceptors. The alpha1-adrenoceptor agonist phenylephrine, the alpha 2-adrenoceptor agonist clonidine, the beta 1-adrenoceptor agonist prenaterol and the beta 2-adrenoceptor agonist terbutaline were administered individually to a population of spontaneously active intrinsic cardiac neurons either locally (10 microL of 100 microM solution; eight dogs) or via the local arterial blood supply (0.1 mL of 100 microM solution; 20 dogs) in artificially ventilated, open chest anesthetized dogs. Neuronal and cardiac effects induced by each of the adrenergic agonists were also tested in the presence of an antagonist selective to each adrenoceptor subtype studied. The activity of intrinsic cardiac neurons was modified by at least one of the adrenoceptor agonists tested, and 34% of the spontaneously active neurons were affected by all four agonists. Alpha-adrenoceptor agonists either increased or decreased neuronal activity, depending on the population of neurons studied. On the other hand, the activity generated by intrinsic cardiac neurons was augmented by beta-adrenoceptor agonists. Ventricular contractile force increased when intrinsic cardiac neurons were excited by adrenoceptor agonists. The spontaneous activity generated by neurons was suppressed by beta-adrenoceptor, but not alpha-adrenoceptor, blockade. Neuronal and cardiovascular responses were no longer elicited by an agonist in the presence of its selective antagonist; they were elicited in the presence of antagonists to the other receptor subtypes studied. Intrinsic cardiac neurons involved in cardiac regulation possess alpha 1-, alpha 2-, beta 1- or beta 2-adrenoceptors. Intrinsic cardiac adrenergic neurons receive tonic inputs via beta-, but not alpha-, adrenoceptors. These data indicate that adrenergic blockade may affect cardiac function, in part, via modification of the intrinsic

  14. Differential dependence of hypoxia-inducible factors 1 alpha and 2 alpha on mTORC1 and mTORC2.

    PubMed

    Toschi, Alfredo; Lee, Evan; Gadir, Noga; Ohh, Michael; Foster, David A

    2008-12-12

    Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1 alpha and HIF2 alpha has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2 alpha expression is the more important contributor. Expression of HIF1 alpha is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2 alpha expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1 alpha and HIF2 alpha to rapamycin, both HIF1 alpha and HIF2 alpha expression is dependent on mTOR. HIF1 alpha expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2 alpha was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1 alpha is dependent on both mTORC1 and mTORC2, HIF2 alpha is dependent only on mTORC2. We also examined the dependence of HIF alpha expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2 alpha was dependent on Akt2, whereas that of HIF1 alpha was dependent on Akt3. Because HIF2 alpha is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2 alpha has been implicated.

  15. Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3.

    PubMed

    Sicinski, R R; DeLuca, H F

    1999-12-01

    Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.

  16. 1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases.

    PubMed

    Boyan, Barbara D; Wong, Kevin L; Fang, Mimi; Schwartz, Zvi

    2007-03-01

    Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.

  17. Polymorphisms in the IL-1A gene are correlated with levels of interleukin-1alpha protein in gingival crevicular fluid of teeth with severe periodontal disease.

    PubMed

    Shirodaria, S; Smith, J; McKay, I J; Kennett, C N; Hughes, F J

    2000-11-01

    Interleukin-1 (IL-1) is a potent stimulator of bone resorption and is strongly implicated in the destruction due to bystander damage seen in periodontal disease. Recent studies suggest that polymorphisms of the (IL-1) gene complex may be significant risk factors for a number of chronic inflammatory diseases. The severity of periodontal disease has been positively associated with carriage of allele 2 at position -889 of the IL-1A gene in conjunction with allele 2 of the IL-1B gene at position +3953. In this study, we tested the hypothesis that allele 2 of the IL-1A gene at position -889 might act to elevate levels of IL-1alpha protein in patients with periodontal disease. Since levels of IL-1alpha protein are low in healthy individuals, we used a group of patients with severe periodontal disease to investigate if levels of IL-1alpha protein in gingival crevicular fluid can be correlated to patient genotype. IL-1alpha levels were measured by enzyme immunoassay in 46 patients with severe periodontal disease. These patients were genotyped by PCR and allele-specific restriction digests. The carriage rate for allele 2 in the diseased population was 68%. Overall, the carriage of allele 2 was associated with almost a four-fold increase in IL-1alpha protein levels. Differences were most pronounced in non-smokers, while heavy smokers showed reduced levels of IL-1alpha protein regardless of genotype. These results suggest a mechanism whereby this genetic polymorphism acts to modulate IL-1alpha protein production and may influence the pathogenesis of periodontal disease by affecting the extent of IL-1-associated bystander damage.

  18. SDF-1alpha is expressed in astrocytes and neurons in the AIDS dementia complex: an in vivo and in vitro study.

    PubMed

    Rostasy, Kevin; Egles, Christophe; Chauhan, Ashok; Kneissl, Michelle; Bahrani, Padmanabhan; Yiannoutsos, Constantin; Hunter, Dale D; Nath, Avindra; Hedreen, John C; Navia, Bradford A

    2003-06-01

    Recent in vitro studies suggest that the alpha chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor CXCR-4 may contribute to neuronal apoptosis in HIV infection of the brain. The cellular and regional expression of this chemokine and its relationship to the AIDS dementia complex (ADC), however, have remained undetermined. Using immunohistochemistry and semiquantitative RT-PCR, we examined the expression of SDF-1alpha in the frontal cortex (FC), the adjacent deep white matter (DWM). and the basal ganglia (BG) of 17 patients with ADC and 5 normal controls, and the FC and temporal cortex of 6 patients with Alzheimer disease (AD). Additionally, SDF-1alpha expression was studied in 3 different neuronal cultures: differentiated SK-N-MC cells, primary human fetal neuronal, and mouse hippocampal cultures. SDF-1alpha staining was predominantly localized to astrocytes in all 3 groups in the gray matter of the FC and the BG, often in the vicinity of cortical and basal ganglia neurons, but was generally absent in the DWM. Further, the number of positive neurons was significantly greater in the BG of AIDS subjects with advanced brain disease compared to subjects with lesser disease (p = 0.029). All cultures showed prominent SDF-1alpha staining of neurons within the cytoplasm and in neurites, whereas preferential expression in GABA-ergic neurons was found in hippocampal cultures. This is the first study to show that SDF-1alpha is constitutively expressed in astrocytes of the deep and cortical gray matter as well as in neurons of the human brain. Its increased expression in basal ganglia neurons of patients with advanced HIV CNS disease suggests it may also contribute to pathogenesis.

  19. Nonstructural protein 1{alpha} subunit-based inhibition of NF-{kappa}B activation and suppression of interferon-{beta} production by porcine reproductive and respiratory syndrome virus

    SciTech Connect

    Song Cheng; Krell, Peter; Yoo, Dongwan

    2010-11-25

    Induction of type I interferon (IFN-{alpha}/{beta}) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1{alpha} (Nsp1{alpha}) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-{beta} production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1{alpha} suppressed the activation of nuclear factor (NF)-{kappa}B when stimulated with dsRNA or tumor necrosis factor (TNF)-{alpha}, and NF-{kappa}B suppression was RIG-I-dependent. The suppression of NF-{kappa}B activation was associated with the poor production of IFN-{beta} during PRRSV infection. The C-terminal 14 amino acids of the Nsp1{alpha} subunit were critical in maintaining immunosuppressive activity of Nsp1{alpha} for both IFN-{beta} and NF-{kappa}B, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1{alpha} inhibited I{kappa}B phosphorylation and as a consequence NF-{kappa}B translocation to the nucleus was blocked, leading to the inhibition of NF-{kappa}B stimulated gene expression. Our results suggest that PRRSV Nsp1{alpha} is a multifunctional nuclear protein participating in the modulation of the host IFN system.

  20. Transcutaneous application of carbon dioxide (CO2) induces mitochondrial apoptosis in human malignant fibrous histiocytoma in vivo.

    PubMed

    Onishi, Yasuo; Kawamoto, Teruya; Ueha, Takeshi; Kishimoto, Kenta; Hara, Hitomi; Fukase, Naomasa; Toda, Mitsunori; Harada, Risa; Minoda, Masaya; Sakai, Yoshitada; Miwa, Masahiko; Kurosaka, Masahiro; Akisue, Toshihiro

    2012-01-01

    Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH) cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO(2)) to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO(2) exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA) was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO(2) induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO(2) treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO(2) treated tumors, highlighting the involvement of mitochondria in apoptosis. These

  1. Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

    SciTech Connect

    Souza, Sandra C.; Chau, Mary D.L.; Yang, Qing; Gauthier, Marie-Soleil; Clairmont, Kevin B.; Wu, Zhidan; Gromada, Jesper; Dole, William P.

    2011-07-08

    peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) by 1.4-fold. Treatment of human adipocytes with fatty acids and tumor necrosis factor {alpha} (TNF{alpha}) induced insulin resistance and down-regulation of mitochondrial genes, which was restored by ANP treatment. These results show that ANP regulates lipid catabolism and enhances energy dissipation through AMPK activation in human adipocytes.

  2. Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.

    PubMed

    Venter, P A; Naudé, R J; Oelofsen, W; Swan, G E

    1997-01-01

    The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.

  3. Mitochondrial oxidative stress and mitochondrial DNA.

    PubMed

    Kang, Dongchon; Hamasaki, Naotaka

    2003-10-01

    Mitochondria produce reactive oxygen species (ROS) under physiological conditions in association with activity of the respiratory chain in aerobic ATP production. The production of ROS is essentially a function of O2 consumption. Hence, increased mitochondrial activity per se can be an oxidative stress to cells. Furthermore, production of ROS is markedly enhanced in many pathological conditions in which the respiratory chain is impaired. Because mitochondrial DNA, which is essential for execution of normal oxidative phosphorylation, is located in proximity to the ROS-generating respiratory chain, it is more oxidatively damaged than is nuclear DNA. Cumulative damage of mitochondrial DNA is implicated in the aging process and in the progression of such common diseases as diabetes, cancer, and heart failure.

  4. Mitochondrial Dysfunction in Depression

    PubMed Central

    Bansal, Yashika; Kuhad, Anurag

    2016-01-01

    Abstract: Background Depression is the most debilitating neuropsychiatric disorder with significant impact on socio-occupational and well being of individual. The exact pathophysiology of depression is still enigmatic though various theories have been put forwarded. There are evidences showing that mitochondrial dysfunction in various brain regions is associated with depression. Recent findings have sparked renewed appreciation for the role of mitochondria in many intracellular processes coupled to synaptic plasticity and cellular resilience. New insights in depression pathophysiology are revolving around the impairment of neuroplasticity. Mitochondria have potential role in ATP production, intracellular Ca2+ signalling to establish membrane stability, reactive oxygen species (ROS) balance and to execute the complex processes of neurotransmission and plasticity. So understanding the various concepts of mitochondrial dysfunction in pathogenesis of depression indubitably helps to generate novel and more targeted therapeutic approaches for depression treatment. Objective The review was aimed to give a comprehensive insight on role of mitochondrial dysfunction in depression. Result Targeting mitochondrial dysfunction and enhancing the mitochondrial functions might act as potential target for the treatment of depression. Conclusion Literature cited in this review highly supports the role of mitochondrial dysfunction in depression. As impairment in the mitochondrial functions lead to the generation of various insults that exaggerate the pathogenesis of depression. So, it is useful to study mitochondrial dysfunction in relation to mood disorders, synaptic plasticity, neurogenesis and enhancing the functions of mitochondria might show promiscuous effects in the treatment of depressed patients. PMID:26923778

  5. Clinical mitochondrial genetics

    PubMed Central

    Chinnery, P.; Howell, N.; Andrews, R.; Turnbull, D.

    1999-01-01

    The last decade has been an age of enlightenment as far as mitochondrial pathology is concerned. Well established nuclear genetic diseases, such as Friedreich's ataxia,12 Wilson disease,3 and autosomal recessive hereditary spastic paraplegia,4 have been shown to have a mitochondrial basis, and we are just starting to unravel the complex nuclear genetic disorders which directly cause mitochondrial dysfunction (table 1). However, in addition to the 3 billion base pair nuclear genome, each human cell typically contains thousands of copies of a small, 16.5 kb circular molecule of double stranded DNA (fig 1). Mitochondrial DNA (mtDNA) accounts for only 1% of the total cellular nucleic acid content. It encodes for 13 polypeptides which are essential for aerobic metabolism and defects of the mitochondrial genome are an important cause of human disease.9293 Since the characterisation of the first pathogenic mtDNA defects in 1988,513 over 50 point mutations and well over 100 rearrangements of the mitochondrial genome have been associated with human disease9495 (http://www.gen.emory.edu/mitomap.html). These disorders form the focus of this article.


Keywords: mitochondrial DNA; mitochondrial disease; heteroplasmy; genetic counselling PMID:10874629

  6. Activation of the oxidative stress pathway by HIV-1 Vpr leads to induction of hypoxia-inducible factor 1alpha expression.

    PubMed

    Deshmane, Satish L; Mukerjee, Ruma; Fan, Shongshan; Del Valle, Luis; Michiels, Carine; Sweet, Thersa; Rom, Inna; Khalili, Kamel; Rappaport, Jay; Amini, Shohreh; Sawaya, Bassel E

    2009-04-24

    The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with human immunodeficiency virus, type 1 (HIV)-associated dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS. Although the biological importance of oxidative stress on events involved in AIDS neuropathogenesis and the HIV-1 proteins responsible for oxidative stress remain to be elucidated, our results point to the activation of hypoxia-inducible factor 1 (HIF-1) upon HIV-1 infection and its elevation in brain cells of AIDS patients with dementia. HIF-1 is a transcription factor that is responsive to oxygen. Under hypoxic conditions, HIF-1alpha becomes stable and translocates to the nucleus where it dimerizes with aryl hydrocarbon receptor nuclear translocator and modulates gene transcription. Activation of HIF-1 can also be mediated by the HIV-1 accessory protein Vpr. In addition, cellular components, including reactive oxygen species, contribute to the induction of HIF-1alpha. Our results show that Vpr induces reactive oxygen species by increasing H(2)O(2) production, which can contribute to HIF-1alpha accumulation. Interestingly, increased levels of HIF-1alpha stimulated HIV-1 gene transcription through HIF-1 association with HIV-1 long terminal repeat. These observations point to the existence of a positive feedback interplay between HIF-1alpha and Vpr and that, by inducing oxidative stress via activation of HIF-1, Vpr can induce HIV-1 gene expression and dysregulate multiple host cellular pathways.

  7. Effect of elongation factor 1alpha promoter and SUMF1 over in vitro expression of N-acetylgalactosamine-6-sulfate sulfatase.

    PubMed

    Alméciga-Díaz, Carlos J; Rueda-Paramo, Maria A; Espejo, Angela J; Echeverri, Olga Y; Montaño, Adriana; Tomatsu, Shunji; Barrera, Luis A

    2009-09-01

    Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.

  8. HIF-1alpha mediates the induction of IL-8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT-like behaviour.

    PubMed

    Cane, Gaëlle; Ginouvès, Amandine; Marchetti, Sandrine; Buscà, Roser; Pouysségur, Jacques; Berra, Edurne; Hofman, Paul; Vouret-Craviari, Valérie

    2010-05-01

    Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro-angiogenic and pro-inflammatory genes. Here, we demonstrate that exposure of cryptic-like intestinal epithelial cells to C1845 bacteria induces HIF-1alpha protein levels. This effect depends on the binding of F1845 adhesin to the membrane-associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI-3K decreases HIF-1alpha protein levels and blocks C1845-induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria-induced HIF-1alpha regulates the expression of IL-8, VEGF and Twist1, thereby pointing to a role for HIF-1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E-cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition-like phenotype. Since HIF-1alpha silencing results in reversion of bacteria-induced EMT markers, we speculate that HIF-1alpha plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.

  9. Kaposi's sarcoma-associated herpesvirus viral IFN regulatory factor 3 stabilizes hypoxia-inducible factor-1 alpha to induce vascular endothelial growth factor expression.

    PubMed

    Shin, Young C; Joo, Chul-Hyun; Gack, Michaela U; Lee, Hye-Ra; Jung, Jae U

    2008-03-15

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of both developmental and pathologic angiogenesis, composed of an oxygen-sensitive alpha-subunit and a constitutively expressed beta-subunit. HIF-1 activity in tumors depends on the availability of the HIF-1 alpha subunit, the levels of which are increased under hypoxic conditions. Recent studies have shown that HIF-1 plays an important role in KSHV reactivation from latency and pathogenesis. Here, we report a novel mechanism by which KSHV activates HIF-1 activity. Specific interaction between KSHV viral IFN regulatory factor 3 (vIRF3) and the HIF-1 alpha subunit led to the HIF-1 alpha stabilization and transcriptional activation, which induced vascular endothelial growth factor expression and ultimately facilitated endothelial tube formation. Remarkably, the central domain of vIRF3, containing double alpha-helix motifs, was sufficient not only for binding to HIF-1 alpha but also for blocking its degradation in normoxic conditions. This indicates that KSHV has developed a unique mechanism to enhance HIF-1 alpha protein stability and transcriptional activity by incorporating a viral homologue of cellular IRF gene into its genome, which may contribute to viral pathogenesis.

  10. The protein level of hypoxia-inducible factor-1alpha is increased in the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

    PubMed

    Li, Hong-Ge; Ren, Yong-Ming; Guo, Song-Chang; Cheng, Long; Wang, De-Peng; Yang, Jie; Chang, Zhi-Jie; Zhao, Xin-Quan

    2009-02-01

    The plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant species living only at 3,000-5,000 m above sea-level on the Qinghai-Tibetan plateau. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. To investigate how the plateau pika adapts to a high-altitude hypoxic environment at the molecular level, we examined the expression pattern of the HIF-1alpha protein in the pika by Western blot and immunohistochemical analysis. We found that HIF-1alpha protein is expressed at a significantly high level in the pika, which is higher in most tissues (particularly in the lung, liver, spleen and kidney) of the plateau pika than that of mice living at sea-level. Importantly, we found that the protein levels of HIF-1alpha in the lung, liver, spleen and kidney of the pika were increased with increased habitat altitudes. We observed that the plateau pika HIF-1alpha localized to the nucleus of cells by an immunostaining analysis, and enhanced HRE-driven gene expression by luciferase reporter assays. Our study suggests that the HIF-1alpha protein levels are related to the adaptation of the plateau pika to the high-altitude hypoxic environment.

  11. Duodenal active transport of calcium and phosphate in vitamin D-deficient rats: effects of nephrectomy, Cestrum diurnum, and 1alpha,25-dihydroxyvitamin D3.

    PubMed

    Walling, M W; Kimberg, D V; Wasserman, R H; Feinberg, R R

    1976-05-01

    Both the methanol:chloroform extractable material from the leaves of the Solanaceous plant, Cestrum diurnum (C.d.), and a 270 ng dose of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) increased the active absorption of calcium and phosphate across the proximal duodenum, studied in vitro, from sham-operated and nephrectomized (NPX) vitamin D-deficient rats. In these studies, conducted 24 h after surgery, the uremic state in the NPX animals markedly diminished the intestinal transport response to 1alpha,25-(OH)2D3 and also lowered baseline transport values across duodenum from the NPX vitamin D-deficient controls. Both C.d. and 1alpha, 25-(OH)2D3 elevated plasma Ca levels equally well in the sham-operated and NPX groups. The stimulation of intestinal Ca absorption in NPX animals indicates that, like the leaves of the South American plant, Solanum glaucophyllum, C.d. contains materials which can function in an analogous manner to compounds in the vitamin D group that have either a 1alpha hydroxyl group or its steric equivalent.

  12. Panaxynol protects cortical neurons from ischemia-like injury by up-regulation of HIF-1alpha expression and inhibition of apoptotic cascade.

    PubMed

    Yang, Zhi-Hui; Sun, Ke; Yan, Zhong-Hong; Suo, Wen-Hao; Fu, Guo-Hui; Lu, Yang

    2010-01-05

    Apoptosis is one of the major characteristics of delayed neuronal degeneration in neuronal injury following cerebral ischemia. Hypoxia-induced apoptosis may be co-regulated by HIF-1alpha as well as many other factors. In recent years, numerous studies concerning panaxynol (PNN) have been reported. However, whether PNN can show anti-hypoxia properties is still unknown. In this study, the protective effects of PNN on OGD-induced neuronal apoptosis and potential mechanisms were investigated. Pretreatment of the cells with PNN for 24h following exposure to OGD resulted in a significant elevation of cell survival determined by MTT assay, LDH assay, Hoechst staining and flow cytometric assessment. In addition to enhancing the expression of HIF-1alpha, PNN also normalized the caspase-3 expression/activation and increased the Bcl-2/Bax ratio. In our study, the increased level of HIF-1alpha with decreased cellular apoptosis suggested an important role for HIF-1alpha in hypoxic neurons. These results indicated that the neuroprotective effects of PNN on hypoxic neurons were at least partly due to up-regulation of HIF-1alpha and raised the possibility that PNN might reduce neurodegenerative disorders and ischemic brain diseases.

  13. Mitochondrial inheritance in yeast.

    PubMed

    Westermann, Benedikt

    2014-07-01

    Mitochondria are the site of oxidative phosphorylation, play a key role in cellular energy metabolism, and are critical for cell survival and proliferation. The propagation of mitochondria during cell division depends on replication and partitioning of mitochondrial DNA, cytoskeleton-dependent mitochondrial transport, intracellular positioning of the organelle, and activities coordinating these processes. Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism to study the mechanisms that drive segregation of the mitochondrial genome and determine mitochondrial partitioning and behavior in an asymmetrically dividing cell. Here, I review past and recent advances that identified key components and cellular pathways contributing to mitochondrial inheritance in yeast. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  14. Mitochondrial Dynamics in Diabetes

    PubMed Central

    Galloway, Chad A.; Jhun, Bong Sook; Yu, Tianzheng

    2011-01-01

    Abstract Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emerging evidence suggests that mitochondrial dynamics plays an important role in metabolism–secretion coupling in pancreatic β-cells as well as complications of diabetes. This review describes an overview of mechanistic and functional aspects of mitochondrial fission and fusion, and comments on the recent advances connecting mitochondrial dynamics with diabetes and diabetic complications. Antioxid. Redox Signal. 14, 439–457. PMID:20518704

  15. Mitochondrial trafficking in neurons.

    PubMed

    Schwarz, Thomas L

    2013-06-01

    Neurons, perhaps more than any other cell type, depend on mitochondrial trafficking for their survival. Recent studies have elucidated a motor/adaptor complex on the mitochondrial surface that is shared between neurons and other animal cells. In addition to kinesin and dynein, this complex contains the proteins Miro (also called RhoT1/2) and milton (also called TRAK1/2) and is responsible for much, although not necessarily all, mitochondrial movement. Elucidation of the complex has permitted inroads for understanding how this movement is regulated by a variety of intracellular signals, although many mysteries remain. Regulating mitochondrial movement can match energy demand to energy supply throughout the extraordinary architecture of these cells and can control the clearance and replenishing of mitochondria in the periphery. Because the extended axons of neurons contain uniformly polarized microtubules, they have been useful for studying mitochondrial motility in conjunction with biochemical assays in many cell types.

  16. Mitochondrial shaping cuts.

    PubMed

    Escobar-Henriques, Mafalda; Langer, Thomas

    2006-01-01

    A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.

  17. Fish oil diet modulates epididymal and inguinal adipocyte metabolism in mice.

    PubMed

    Bargut, Thereza Cristina Lonzetti; Souza-Mello, Vanessa; Mandarim-de-Lacerda, Carlos Alberto; Aguila, Marcia Barbosa

    2016-03-01

    We aimed to investigate the impact of different high-fat diets containing fish oil on adiposity and white adipose tissue (WAT) function in mice, comparing the effects on epididymal (eWAT) and subcutaneous (sWAT) depots. For this, we used C57BL/6 male mice fed four types of diets for eight weeks: standard chow (SC), high-fat lard (HF-L), high-fat lard plus fish oil (HF-L + FO), and high-fat fish oil (HF-FO). The HF-L group had a greater body mass (BM) gain, insulin resistance, increased gene expression related to lipogenesis (CD36, aP2, SREBP1c, and FAS), decreased gene expression of perilipin in both eWAT and sWAT, and reduced expression of genes related to beta-oxidation (CPT-1a) and to mitochondrial biogenesis (PGC1alpha, NRF1, and TFAM) in eWAT and sWAT. On the other hand, the HF-L + FO and HF-FO groups showed a smaller BM gain and adiposity, and normalization of insulin resistance and lipogenic genes in both eWAT and sWAT. These animals also showed decreased perilipin gene expression and elevated expression of beta-oxidation and mitochondrial biogenesis genes in eWAT and sWAT. 'Beige' adipocytes were identified in sWAT of the HF-FO animals. In conclusion, fish oil intake has anti-obesity effects through modulation of both eWAT and sWAT metabolism in mice and is relevant in diminishing the BM gain, adiposity, and insulin resistance even in combination with a high-fat lard diet in mice.

  18. Effects of antirheumatic drugs on the interleukin-1 alpha induced synthesis and activation of proteinases in articular cartilage explants in culture.

    PubMed

    Arsenis, C; McDonnell, J

    1989-06-01

    Three human cytokines (interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha), added into the medium of bovine or rabbit articular cartilage explant cultures, stimulated the synthesis and activation of various proteinases. Proteoglycan degradation, measured by assaying for sulfated glycosaminoglycans released into the medium, was correlated with the proteinase stimulation. Several antirheumatic drugs were tested in a similar tissue culture system as potential inhibitors of the interleukin-1 alpha mediated stimulation of proteinase and PGE2 syntheses. Arteparon, Dexamethasone, Ibuprofen, Indomethacin, Levamisole, Naproxen, Phenylbutazone, Prednisolone, Piroxicam, Rumalon, Tamoxifen and Diclofenac were essentially ineffective in inhibiting the interleukin-1 alpha mediated induction of proteinase synthesis and sulfated glycosaminoglycan release, although some of them inhibited PGE2 synthesis. Two antimalarial drugs showed some inhibition, but only at higher concentrations.

  19. IL-1. alpha. increases arachidonyl-CoA: Lysophospholipid acyltransterase activity and stimulates ( sup 3 H) arachidonate incorporation into phospholipids in rat mesangial cells

    SciTech Connect

    Nakazato, Y.; Sedor, J.R. )

    1992-01-01

    The proinflammatory cytokine interleukin-1{alpha} is a potent stimulus of prostaglandin synthesis. The authors have previously shown that IL-1 amplifies