Sample records for 1alpha pgc-1alpha mitochondrial

  1. 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:


    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.

  2. Transcription Factor Tfe3 Directly Regulates Pgc-1alpha in Muscle

    PubMed Central



    The microphthalmia (MiT) family of transcription factors is an important mediator of metabolism. Family members Mitf and Tfeb directly regulate the expression of the master regulator of metabolism, peroxisome-proliferator activated receptor gamma coactivator-1 alpha (Pgc-1alpha), in melanomas and in the liver, respectively. Pgc-1alpha is enriched in tissues with high oxidative capacity and plays an important role in the regulation of mitochondrial biogenesis and cellular metabolism. In skeletal muscle, Pgc-1alpha affects many aspects of muscle functionally such as endurance, fiber-type switching, and insulin sensitivity. Tfe3 also regulates muscle metabolic genes that enhance insulin sensitivity in skeletal muscle. Tfe3 has not yet been shown to regulate Pgc-1alpha expression. Our results reported here show that Tfe3 directly regulates Pgc-1alpha expression in myotubes. Tfe3 ectopic expression induces Pgc-1alpha, and Tfe3 silencing suppresses Pgc-1alpha expression. This regulation is direct, as shown by Tfe3’s binding to E-boxes on the Pgc-1alpha proximal promoter. We conclude that Tfe3 is a critical transcription factor that regulates Pgc-1alpha gene expression in myotubes. Since Pgc-1alpha coactivates numerous biological programs in diverse tissues, the regulation of its expression by upstream transcription factors such Tfe3 implies potential opportunities for the treatment of diseases where modulation of Pgc-1alpha expression may have important clinical outcomes. PMID:25736533

  3. 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.


    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.

  4. 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


    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.

  5. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.


    Lin, Jiandie; Wu, Hai; Tarr, Paul T; Zhang, Chen-Yu; Wu, Zhidan; Boss, Olivier; Michael, Laura F; Puigserver, Pere; Isotani, Eiji; Olson, Eric N; Lowell, Bradford B; Bassel-Duby, Rhonda; Spiegelman, Bruce M


    The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination. PMID:12181572

  6. 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


    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.

  7. 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


    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.

  8. 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.


    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.

  9. Defects in energy homeostasis in Leigh syndrome French Canadian variant through PGC-1alpha/LRP130 complex.


    Cooper, Marcus P; Qu, Lishu; Rohas, Lindsay M; Lin, Jiandie; Yang, Wenli; Erdjument-Bromage, Hediye; Tempst, Paul; Spiegelman, Bruce M


    Leigh syndrome French Canadian variant (LSFC) is an autosomal recessive neurodegenerative disorder due to mutation in the LRP130 (leucine-rich protein 130 kDa) gene. Unlike classic Leigh syndrome, the French Canadian variant spares the heart, skeletal muscle, and kidneys, but severely affects the liver. The precise role of LRP130 in cytochrome c oxidase deficiency and hepatic lactic acidosis that accompanies this disorder is unknown. We show here that LRP130 is a component of the PGC-1alpha (peroxisome proliferator-activated receptor coactivator 1-alpha) transcriptional coactivator holocomplex and regulates expression of PEPCK (phosphoenolpyruvate carboxykinase), G6P (glucose-6-phosphatase), and certain mitochondrial genes through PGC-1alpha. Reduction of LRP130 in fasted mice via adenoviral RNA interference (RNAi) vector blocks the induction of PEPCK and G6P, and blunts hepatic glucose output. LRP130 is also necessary for PGC-1alpha-dependent transcription of several mitochondrial genes in vivo. These data link LRP130 and PGC-1alpha to defective hepatic energy homeostasis in LSFC, and reveal a novel regulatory mechanism of glucose homeostasis. PMID:17050673

  10. 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


    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.

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

    SciTech Connect

    Summermatter, Serge; Santos, Gesa


    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

  12. Dietary Fucoxanthin Increases Metabolic Rate and Upregulated mRNA Expressions of the PGC-1alpha Network, Mitochondrial Biogenesis and Fusion Genes in White Adipose Tissues of Mice

    PubMed Central

    Wu, Meng-Ting; Chou, Hong-Nong; Huang, Ching-jang


    The mechanism for how fucoxanthin (FX) suppressed adipose accumulation is unclear. We aim to investigate the effects of FX on metabolic rate and expressions of genes related to thermogenesis, mitochondria biogenesis and homeostasis. Using a 2 × 2 factorial design, four groups of mice were respectively fed a high sucrose (50% sucrose) or a high-fat diet (23% butter + 7% soybean oil) supplemented with or without 0.2% FX. FX significantly increased oxygen consumption and carbon dioxide production and reduced white adipose tissue (WAT) mass. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), cell death-inducing DFFA-like effecter a (CIDEA), PPARα, PPARγ, estrogen-related receptor α (ERRα), β3-adrenergic receptor (β3-AR) and deiodinase 2 (Dio2) were significantly upregulated in inguinal WAT (iWAT) and epididymal WAT (eWAT) by FX. Mitochondrial biogenic genes, nuclear respiratory factor 1 (NRF1) and NRF2, were increased in eWAT by FX. Noticeably, FX upregulated genes of mitochondrial fusion, mitofusin 1 (Mfn1), Mfn2 and optic atrophy 1 (OPA1), but not mitochondrial fission, Fission 1, in both iWAT and eWAT. In conclusion, dietary FX enhanced the metabolic rate and lowered adipose mass irrespective of the diet. These were associated with upregulated genes of the PGC-1α network and mitochondrial fusion in eWAT and iWAT. PMID:24534841

  13. 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


    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.

  14. PGC-1alpha induces dynamic protein interactions on the ERRalpha gene multi-hormone response element nucleosome in kidney cells.


    Wang, Liangli; Li, Yin; Hu, Peng; Teng, Christina T


    ERR (oestrogen-related receptor)-alpha modulates the oestrogen signalling pathway and regulates genes participating in the physiological energy balance programme. Oestrogen and PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator-1alpha), the master regulator of the energy homoeostasis programme, both regulate the expression of ERRalpha through the MHRE (multi-hormone response element) of the ERRalpha gene. Although the molecular mechanism of oestrogen action on ERRalpha regulation is well characterized, the mechanism of PGC-1alpha induction is unclear. In this study, we examine chromatin structural changes and protein interactions at the MHRE nucleosome in response to PGC-1alpha expression in HK2 human kidney cells. We mapped the nucleosome positions of the ERRalpha gene promoter and examined the changes of histone acetylation in response to PGC-1alpha expression. The interactions of DNA-binding proteins, ERRalpha and ERRgamma, co-activators {CBP [CREB (cAMP-response-element-binding protein)-binding protein], p300, PCAF (p300/CBP-associated factor)}, co-repressor [RIP140 (receptor-interacting protein of 140 kDa)] and RNA polymerase II at the MHRE nucleosome region were investigated over time before and after PGC-1alpha expression in the HK2 cells. We found a dynamic cyclic interaction of these proteins shortly after PGC-1alpha expression and a slower cycling interaction, with fewer proteins involved, 20 h later. By using the siRNA (small interfering RNA) knockdown approach, we discovered that ERRgamma was involved in the initial phase, but not in the later phase, of PGC-1alpha-induced ERRalpha expression. PMID:18673300

  15. 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


    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.

  16. 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


    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

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


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


    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. PMID:17640976

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


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


    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. PMID:27271583

  19. 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


    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.

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

    SciTech Connect

    Nitz, Inke . E-mail:; Ewert, Agnes; Klapper, Maja; Doering, Frank


    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.

  1. Detrimental effects of oxidative losses in parkin activity in a model of sporadic Parkinson's disease are attenuated by restoration of PGC1alpha.


    Siddiqui, Almas; Rane, Anand; Rajagopalan, Subramanian; Chinta, Shankar J; Andersen, Julie K


    Loss of parkin E3 ligase activity as a result of parkin gene mutation in rare familial forms of Parkinson's disease (PD) has been shown to be detrimental to mitochondrial function and to contribute to ensuing neurodegeneration. This has been shown by ourselves and others to be in part due to reductions in parkin-mediated ubiquitination of the transcriptional repressor PARIS, limiting the protein's subsequent degradation by the proteasome. Subsequent elevations in PARIS protein levels result in reduced expression of the master mitochondrial regulator PGC-1α, impacting in turn on mitochondrial function. Here, we report that oxidatively-mediated reductions in parkin solubility and function in a mouse model of age-related sporadic PD coincides with increased PARIS levels and reduced PGC-1α signaling. Furthermore, restoration of PGC-1α expression was found to abrogate losses in mitochondrial function and degeneration of dopaminergic (DAergic) neurons within the substantia nigra pars compacta (SNpc) associated with this particular model. These findings suggest that the PGC-1α signaling pathway constitutes a viable therapeutic target for the treatment of not only familial PD, but also more common sporadic forms of the disorder. PMID:27185595

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


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


    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. PMID:19797682

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

    SciTech Connect

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


    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.

  4. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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


    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.

  6. Acute {beta}-adrenergic stimulation does not alter mitochondrial protein synthesis or markers of mitochondrial biogenesis in adult men.


    Robinson, Matthew M; Richards, Jennifer C; Hickey, Matthew S; Moore, Daniel R; Phillips, Stuart M; Bell, Christopher; Miller, Benjamin F


    Exercise-induced expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is dramatically inhibited in mice pretreated with a beta-adrenergic receptor (beta-AR) antagonist, suggesting that beta-ARs play an important role in the regulation of skeletal muscle PGC-1alpha expression, and potentially, mitochondrial biogenesis. Accordingly, we hypothesized that acute beta-AR stimulation would induce transcriptional pathways involved in skeletal muscle mitochondrial biogenesis in humans. Whole body protein turnover (WBPT), myofibrillar protein synthesis (MyPS), skeletal muscle mitochondrial protein synthesis (MiPS), and mitochondrial biogenic signaling were determined in samples of vastus lateralis obtained on two separate occasions in 10 young adult males following 1 h of continuous intravenous administration of saline (CON) or a nonspecific beta-AR agonist [isoproterenol (ISO): 12 fat free mass(-1).min(-1)], combined with coinfusion of [1,2](13)C-leucine. beta-AR stimulation induced appreciable increases in heart rate and systolic blood pressure (both P < 0.001) but did not affect mitochondrial biogenic signaling (no change in PGC-1alpha, TFAM, NRF-1, NRF-2, COX, or NADHox expression via RT-PCR; P > 0.05). Additionally, MiPS [CON: 0.099 +/- 0.028, ISO: 0.074 +/- 0.046 (mean +/- SD); P > 0.05] and MyPS (CON: 0.059 +/- 0.008, ISO: 0.055 +/- 0.009; P > 0.05), as well as measures of WBPT were unaffected. On the basis of this investigation, we conclude that acute intravenous beta-AR stimulation does not increase mitochondrial protein synthesis or biogenesis signals in skeletal muscle. PMID:19907002

  7. Development of a stable cell line with an intact PGC-1alpha/ERRalpha axis for screening environmental chemicals

    PubMed Central

    Teng, Christina T.; Beames, Burton; Merrick, B. Alex; Martin, Negin; Romeo, Charles; Jetten, Anton M.


    The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway. PMID:24457025

  8. 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.


    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.

  9. The impact of mitochondrial DNA and nuclear genes related to mitochondrial functioning on the risk of Parkinson's disease.


    Gaweda-Walerych, Katarzyna; Zekanowski, Cezary


    Mitochondrial dysfunction and oxidative stress are the major factors implicated in Parkinson's disease (PD) pathogenesis. The maintenance of healthy mitochondria is a very complex process coordinated bi-genomically. Here, we review association studies on mitochondrial haplogroups and subhaplogroups, discussing the underlying molecular mechanisms. We also focus on variation in the nuclear genes (NDUFV2, PGC-1alpha, HSPA9, LRPPRC, MTIF3, POLG1, and TFAM encoding NADH dehydrogenase (ubiquinone) flavoprotein 2, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, mortalin, leucine-rich pentatricopeptide repeat containing protein, translation initiation factor 3, mitochondrial DNA polymerase gamma, and mitochondrial transcription factor A, respectively) primarily linked to regulation of mitochondrial functioning that recently have been associated with PD risk. Possible interactions between mitochondrial and nuclear genetic variants and related proteins are discussed. PMID:24532986

  10. The Impact of Mitochondrial DNA and Nuclear Genes Related to Mitochondrial Functioning on the Risk of Parkinson’s Disease

    PubMed Central

    Gaweda-Walerych, Katarzyna; Zekanowski, Cezary


    Mitochondrial dysfunction and oxidative stress are the major factors implicated in Parkinson’s disease (PD) pathogenesis. The maintenance of healthy mitochondria is a very complex process coordinated bi-genomically. Here, we review association studies on mitochondrial haplogroups and subhaplogroups, discussing the underlying molecular mechanisms. We also focus on variation in the nuclear genes (NDUFV2, PGC-1alpha, HSPA9, LRPPRC, MTIF3, POLG1, and TFAM encoding NADH dehydrogenase (ubiquinone) flavoprotein 2, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, mortalin, leucine-rich pentatricopeptide repeat containing protein, translation initiation factor 3, mitochondrial DNA polymerase gamma, and mitochondrial transcription factor A, respectively) primarily linked to regulation of mitochondrial functioning that recently have been associated with PD risk. Possible interactions between mitochondrial and nuclear genetic variants and related proteins are discussed. PMID:24532986

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

    SciTech Connect

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


    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.

  12. Mitochondrial pyruvate dehydrogenase. Molecular cloning of the E1 alpha subunit and expression analysis.

    PubMed Central

    Grof, C P; Winning, B M; Scaysbrook, T P; Hill, S A; Leaver, C J


    A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1 alpha subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-length clones were identified on the basis of similarity to E1 alpha sequences from nonplant sources. Southern blot analysis revealed a small family of genes in potato (Solanum tuberosum L.), whereas in cucumber (Cucumis sativus) there are only one or two genes. Tissue-specific variation in the relative amounts of E1 alpha mRNA was observed in northern blot analysis of different potato tissues, with the highest steady-state transcript levels found in floral tissue. Measurement of pyruvate dehydrogenase activity in cucumber cotyledons showed that there is a transient increase to a maximum at 4 to 5 d postimbibition. Western blot analysis revealed that the amount of E1 alpha protein also peaks at this time. Steady-state transcript levels in germinating cucumber cotyledons also show transient accumulation, peaking 2 d postimbibition. These data are consistent with regulation of E1 alpha at the level of transcription and/or mRNA stability in postgerminative cucumber cotyledons. PMID:7659754

  13. 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.


    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.

  14. RhTFAM treatment stimulates mitochondrial oxidative metabolism and improves memory in aged mice

    PubMed Central

    Thomas, Ravindar R.; Khan, Shaharyar M.; Smigrodzki, Rafal M.; Onyango, Isaac G.; Dennis, Jameel; Khan, Omer M.; Portell, Francisco R.; Bennett, James P.


    Mitochondrial function declines with age in postmitotic tissues such as brain, heart and skeletal muscle. Despite weekly exercise, aged mice showed substantial losses of mtDNA gene copy numbers and reductions in mtDNA gene transcription and mitobiogenesis signaling in brain and heart. We treated these mice with weekly intravenous injections of recombinant human mitochondrial transcription factor A (rhTFAM). RhTFAM treatment for one month increased mitochondrial respiration in brain, heart and muscle, POLMRT expression and mtDNA gene transcription in brain, and PGC-1 alpha mitobiogenesis signaling in heart. RhTFAM treatment reduced oxidative stress damage to brain proteins, improved memory in Morris water maze performance and increased brain protein levels of BDNF and synapsin. Microarray analysis showed co-expression of multiple Gene Ontology families in rhTFAM-treated aged brains compared to young brains. RhTFAM treatment reverses age-related memory impairments associated with loss of mitochondrial energy production in brain, increases levels of memory-related brain proteins and improves mitochondrial respiration in brain and peripheral tissues. PMID:23075607

  15. Phosphorylation of ferredoxin and regulation of renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase activity in vitro.


    Nemani, R; Ghazarian, J G; Moorthy, B; Wongsurawat, N; Strong, R; Armbrecht, H J


    The kidney is the principal physiologic site of production of biologically active 1,25-dihydroxyvitamin D. The 25-hydroxyvitamin D-1 alpha-hydroxylase (1-OHase) activity found in renal mitochondria is under tight hormonal control. Parathyroid hormone stimulates the renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D in young animals, which is accompanied by dephosphorylation of ferredoxin (Fx), a component of the mitochondrial 1-OHase enzyme complex (Siegel, N., Wongsurawat, N., and Armbrecht, H. J. (1986) J. Biol. Chem. 261, 16998-17003). The present study investigates the capacity of Fx to be phosphorylated in vitro and to modulate the 1-OHase activity of a reconstituted system. Fx was phosphorylated by renal mitochondrial type II protein kinase. Phosphorylation did not alter Fx mobility on sodium dodecyl sulfate gels but did decrease the pI as measured by isoelectric focusing. Amino acid analysis demonstrated that 1 mol of serine and 1 mol of threonine were phosphorylated per mol of Fx. Peptide mapping of phosphorylated Fx was consistent with phosphorylation of serine 88 and threonine 85 or 97. Fx was selectively dephosphorylated by rabbit skeletal muscle protein phosphatase C2 but not C1. Phosphorylation of Fx significantly inhibited the 1-OHase activity of a reconstituted system consisting of Fx reductase, Fx, and renal mitochondrial cytochrome P-450. These findings suggest that phosphorylation/dephosphorylation of Fx may play a role in modulating renal 1,25-dihydroxyvitamin D production. PMID:2768268

  16. Methionine restriction decreases endogenous oxidative molecular damage and increases mitochondrial biogenesis and uncoupling protein 4 in rat brain.


    Naudí, Alba; Caro, Pilar; Jové, Mariona; Gómez, José; Boada, Jordi; Ayala, Victoria; Portero-Otín, Manuel; Barja, Gustavo; Pamplona, Reinald


    Aging plays a central role in the occurrence of neurodegenerative diseases. Caloric restriction (CR) mitigates oxidative stress by decreasing the rate of generation of endogenous damage, a mechanism that can contribute to the slowing of the aging rate induced by this intervention. Various reports have recently linked methionine to aging, and methionine restriction (MetR) without energy restriction also increases life span. We have thus hypothesized that MetR can be responsible, at least in part, for the decrease in endogenous oxidative damage in CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their life span. We have found that MetR: (1) decreases the mitochondrial complex I content and activity, as well as complex III content, while the complex II and IV, the mitochondrial flavoprotein apoptosis-inducing factor (AIF) and ATP content are unchanged; (2) increases the mitochondrial biogenesis factor PGC-1alpha; (3) increases the resistance of brain to metabolic and oxidative stress by increasing mitochondrial uncoupling protein 4 uncoupling protein 4 (UCP4); and (4) decreases mitochondrial oxidative DNA damage and all five different markers of protein oxidation measured and lowers membrane unsaturation in rat brain. No changes were detected for protein amino acid composition. These beneficial MetR-induced changes likely derived from metabolic reprogramming at the cellular and tissue level can play a key role in the protection against aging-associated neurodegenerative disorders. PMID:17716000

  17. Methyl-Arginine Profile of Brain from Aged PINK1-KO+A53T-SNCA Mice Suggests Altered Mitochondrial Biogenesis

    PubMed Central

    Auburger, Georg; Gispert, Suzana


    Hereditary Parkinson's disease can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) or the autosomal recessive deficiency of PINK1. We recently showed that the combination of PINK1-knockout with overexpression of A53T-SNCA in double mutant (DM) mice potentiates phenotypes and reduces survival. Now we studied brain hemispheres of DM mice at age of 18 months in a hypothesis-free approach, employing a quantitative label-free global proteomic mass spectrometry scan of posttranslational modifications focusing on methyl-arginine. The strongest effects were documented for the adhesion modulator CMAS, the mRNA decapping/deadenylation factor PATL1, and the synaptic plasticity mediator CRTC1/TORC1. In addition, an intriguing effect was observed for the splicing factor PSF/SFPQ, known to interact with the dopaminergic differentiation factor NURR1 as well as with DJ-1, the protein responsible for the autosomal recessive PARK7 variant of PD. CRTC1, PSF, and DJ-1 are modulators of PGC1alpha and of mitochondrial biogenesis. This pathway was further stressed by dysregulations of oxygen sensor EGLN3 and of nuclear TMPO. PSF and TMPO cooperate with dopaminergic differentiation factors LMX1B and NURR1. Further dysregulations concerned PRR18, TRIO, HNRNPA1, DMWD, WAVE1, ILDR2, DBNDD1, and NFM. Thus, we report selective novel endogenous stress responses in brain, which highlight early dysregulations of mitochondrial homeostasis and midbrain vulnerability. PMID:27034888

  18. Oxysterols induce mitochondrial impairment and hepatocellular toxicity in non-alcoholic fatty liver disease.


    Bellanti, Francesco; Mitarotonda, Domenica; Tamborra, Rosanna; Blonda, Maria; Iannelli, Giuseppina; Petrella, Antonio; Sanginario, Vittorio; Iuliano, Luigi; Vendemiale, Gianluigi; Serviddio, Gaetano


    Non-alcoholic fatty liver disease (NAFLD) is a chronic hepatic disorder affecting up to 25% of the general population. Several intracellular events leading to NAFLD and progression to non-alcoholic steatohepatitis (NASH) have been identified, including lipid accumulation, mitochondrial dysfunction and oxidative stress. Emerging evidence links both hepatic free fatty acids (FFAs) and cholesterol (FC) accumulation in NAFLD development; in particular oxysterols, the oxidative products of cholesterol, may contribute to liver injury. We performed a targeted lipidomic analysis of oxysterols in the liver of male Wistar rats fed a high-fat (HF), high-cholesterol (HC) or high-fat/high-cholesterol (HF/HC) diet. Both HF and HC diets caused liver steatosis, but the HF/HC diet resulted in steatohepatitis with associated mitochondrial dysfunction. Above all, the oxysterol cholestane-3beta,5alpha,6beta-triol (triol) was particularly increased in the liver of rats fed diets rich in cholesterol. To verify the molecular mechanism involved in mitochondrial dysfunction and hepatocellular toxicity, Huh7 and primary rat hepatocytes were exposed to palmitic acid (PA) and/or oleic acid (OA), with or without triol. This compound induced apoptosis in cells co-exposed to both PA and OA, and this was associated with impaired mitochondrial respiration as well as down-regulation of PGC1-alpha, mTFA and NRF1.In conclusion, our data show that hepatic free fatty acid or oxysterols accumulation per se induce low hepatocellular toxicity. On the contrary, hepatic accumulation of both fatty acids and toxic oxysterols such as triol are determinant in the impairment of mitochondrial function and biogenesis, contributing to liver pathology in NAFLD. PMID:26461297

  19. Enhanced mitochondrial biogenesis contributes to Wnt induced osteoblastic differentiation of C3H10T1/2 cells.


    An, Jee Hyun; Yang, Jae-Yeon; Ahn, Byung Yong; Cho, Sun Wook; Jung, Ju Yeon; Cho, Hwa Young; Cho, Young Min; Kim, Sang Wan; Park, Kyong Soo; Kim, Seong Yeon; Lee, Hong Kyu; Shin, Chan Soo


    Mitochondria play a key role in cell physiology including cell differentiation and proliferation. We investigated the changes of mitochondrial biogenesis during Wnt-induced osteoblastic differentiation of murine mesenchymal C3H10T1/2 cells. Scanning electron microscopy demonstrated that activation of Wnt signaling by Wnt-3A conditioned medicum (CM) resulted in significant increase in the number of mitochondria in C3H10T1/2 cells. In addition, the induction of alkaline phosphatase (ALP) activities by Wnt-3A CM was accompanied by significant increase in mitochondrial mass (p<0.05), mitochondrial membrane potential (p<0.05), intracellular reactive oxygen species production (p<0.05), resting oxygen consumption rate (p<0.05), cellular ATP content (p< or =0.05) and mtDNA copy number (p<0.05) compared to the cells with control CM (L292-CM) treatment. Moreover, co-treatment with Dkk-1 or WIF-1, both of which are Wnt inhibitors, abrogated the Wnt-3A-induced ALP activities as well as mitochondrial biogenesis markers. Upregulation of mitochondrial biogenesis by overexpression of mitochondrial transcription factor A (Tfam) significantly enhanced Wnt-induced osteogenesis as measured by ALP activities. In contrast, inhibition of mitochondrial biogenesis by treatment with Zidovudine (AZT) resulted in significant inhibition of ALP activities. Finally, ALP activities in human osteosarcoma cell line devoid of mitochondrial DNA (rho(0) cells) was significantly suppressed both in basal and Wnt-3A stimulated state compared to those from mitochondria-intact cells (rho+ cells). As a mechanism for Wnt-mediated mitochondrial biogenesis, we found that Wnt increased the expression of PGC-1alpha, a critical molecules in mitochondrial biogenesis, through Erk and p38 MAPK pathway independent of beta-catenin signaling. We also found that increased mitochondrial biogenesis is in turn positively regulating TOPflash reporter activity as well as beta-catenin levels. To summarize, mitochodrial

  20. A pilot study on the molecular phylogeny of Drepanoidea (Insecta: Lepidoptera) inferred from the nuclear gene EF-1alpha and the mitochondrial gene COI.


    Wu, C G; Han, H X; Xue, D Y


    A molecular phylogenetic study of the Drepanoidea based on the EF-1alpha sequences and combined EF-1alpha and COI sequences was carried out in order to infer higher classification at and above the subfamily level. The sample contained 14 taxa representing 13 genera recognized in the Drepanoidea. The results revealed that the Drepaninae, Thyatirinae and Cyclidiinae respectively form monophyletic groups. The sister relationship between the Drepaninae and the Thyatirinae was validated. The monophyly of the Cyclidiinae with the Drepaninae+Thyatirinae was supported robustly. Hypsomadius insignis and Oreta vatama within the traditional definition of the Drepaninae formed an individual clade with robust support (100%) and constitutes a sister relationship to a clade containing the rest of the Drepaninae in all the topologies, which means that the subfamily Oretinae of the Drepanidae should be restored. The family Drepanidae is divided into four subfamilies: Drepaninae, Oretinae, Thyatirinae and Cyclidiinae in this work. The family Epicopeiidae formed a monophyly with high bootstrap values. The result of combined analysis of EF-1alpha and COI showed that the Epicopeiidae have a closer phylogenetic relationship with the Geometridae than with the Drepanidae and belong to neither the Drepanoidea nor the Geometroidea. PMID:19580687

  1. Characterization of the human, mouse and rat PGC1 beta (peroxisome-proliferator-activated receptor-gamma co-activator 1 beta) gene in vitro and in vivo.

    PubMed Central

    Meirhaeghe, Aline; Crowley, Vivion; Lenaghan, Carol; Lelliott, Christopher; Green, Kath; Stewart, Abigail; Hart, Kevin; Schinner, Sven; Sethi, Jaswinder K; Yeo, Giles; Brand, Martin D; Cortright, Ron N; O'Rahilly, Stephen; Montague, Carl; Vidal-Puig, Antonio J


    PGC1 alpha is a co-activator involved in adaptive thermogenesis, fatty-acid oxidation and gluconeogenesis. We describe the identification of several isoforms of a new human PGC1 alpha homologue, cloned independently and named PGC1 beta. The human PGC1 beta gene is localized to chromosome 5, has 13 exons and spans more than 78 kb. Two different 5' and 3' ends due to differential splicing were identified by rapid amplification of cDNA ends PCR and screening of human cDNA libraries. We show that PGC1 beta variants in humans, mice and rats are expressed predominantly in heart, brown adipose tissue, brain and skeletal muscle. PGC1 beta expression, unlike PGC1 alpha, is not up-regulated in brown adipose tissue in response to cold or obesity. Fasting experiments showed that PGC1 alpha, but not PGC1 beta, is induced in liver and this suggests that only PGC1 alpha is involved in the hepatic gluconeogenesis. No changes in PGC1 beta gene expression were observed associated with exercise. Human PGC1 beta-1a and -2a isoforms localized to the cell nucleus and, specifically, the isoform PGC1 beta-1a co-activated peroxisome-proliferator-activated receptor-gamma, -alpha and the thyroid hormone receptor beta1. Finally, we show that ectopic expression PGC1 beta leads to increased mitochondrial number and basal oxygen consumption. These results suggest that PGC1 beta may play a role in constitutive adrenergic-independent mitochondrial biogenesis. PMID:12678921

  2. PDE5 inhibitors protect against post-infarction heart failure.


    Li, Na; Yuan, Yuan; Li, Shuang; Zeng, Cao; Yu, Wenjun; Shen, Mingzhi; Zhang, Rongqing; Li, Congye; Zhang, Yingmei; Wang, Haichang


    Heart failure (HF) is one of the main causes for cardiovascular morbidity and mortality. This study was designed to examine the effect of PDE-5 inhibition on cardiac geometry, function and apoptosis in post-infarct HF. Our data revealed that treatment of the PDE-5 inhibitor sildenafil, beginning 3 days after left anterior descending coronary artery ligation, attenuated LV remodeling, cardiac dysfunction, cardiomyocyte apoptosis and mitochondrial anomalies including ATP production, mitochondrial respiratory defects, decline of mitochondrial membrane potential (MMP) and compromised mitochondrial ultrastructure. Sildenafil partially ameliorated the downregulation of Sirt3 protein and acetylation of PGC-1alpha in peri-infarct myocardial regions. In cultured neonatal mouse ventricular myocytes subjected to hypoxia for 24 hrs, sildenafil suppressed apoptosis, promoted ATP production and elevated MMP, along with the increased Sirt3 protein expression and decreased PGC-1alpha acetylation. Interestingly, knock down of Sirt3 attenuated or nullified sildenafil-offered beneficial effects. Our findings demonstrated that sildenafil exerts its cardioprotective effect against post-infarction injury by improving mitochondrial ultrastructure and function via the Sirt3/PGC-1alpha pathway. This observation should shed some lights towards application of sildenafil in energy-related cardiovascular diseases. PMID:27100500

  3. 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


    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

  4. Role of the PGC-1 family in the metabolic adaptation of goldfish to diet and temperature.


    LeMoine, Christophe M R; Genge, Christine E; Moyes, Christopher D


    In mammals, the peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 (PGC-1) family members and their binding partners orchestrate remodelling in response to diverse challenges such as diet, temperature and exercise. In this study, we exposed goldfish to three temperatures (4, 20 and 35 degrees C) and to three dietary regimes (food deprivation, low fat and high fat) and examined the changes in mitochondrial enzyme activities and transcript levels for metabolic enzymes and their genetic regulators in red muscle, white muscle, heart and liver. When all tissues and conditions were pooled, there were significant correlations between the mRNA for the PGC-1 coactivators (both alpha and beta) and mitochondrial transcripts (citrate synthase), metabolic gene regulators including PPARalpha, PPARbeta and nuclear respiratory factor-1 (NRF-1). PGC-1beta was the better predictor of the NRF-1 axis, whereas PGC-1alpha was the better predictor of the PPAR axis (PPARalpha, PPARbeta, medium chain acyl CoA dehydrogenase). In contrast to these intertissue/developmental patterns, the response of individual tissues to physiological stressors displayed no correlations between mRNA for PGC-1 family members and either the NRF-1 or PPAR axes. For example, in skeletal muscles, low temperature decreased PGC-1alpha transcript levels but increased mitochondrial enzyme activities (citrate synthase and cytochrome oxidase) and transcripts for COX IV and NRF-1. These results suggest that in goldfish, as in mammals, there is a regulatory relationship between (i) NRF-1 and mitochondrial gene expression and (ii) PPARs and fatty acid oxidation gene expression. In contrast to mammals, there is a divergence in the roles of the coactivators, with PGC-1alpha linked to fatty acid oxidation through PPARalpha, and PGC-1beta with a more prominent role in mediating NRF-1-dependent control of mitochondrial gene expression, as well as distinctions between their respective roles in development and

  5. Increased HIF1 alpha in SDH and FH deficient tumors does not cause microsatellite instability.


    Lehtonen, Heli J; Mäkinen, Markus J; Kiuru, Maija; Laiho, Päivi; Herva, Riitta; van Minderhout, Ivonne; Hogendoorn, Pancras C W; Cornelisse, Cees; Devilee, Peter; Launonen, Virpi; Aaltonen, Lauri A


    Germline mutations in nuclear genes encoding mitochondrial enzymes fumarate hydratase (FH) and succinate dehydrogenase (subunits SDHB/C/D) have been implicated in the development of tumor syndromes referred to as hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary paragangliomatosis (HPGL), respectively. FH and SDH are operating in the tricarboxylic acid cycle (the TCA cycle, the Krebs cycle). In the FH and SDH deficient tumors, accumulation of the substrates, fumarate and succinate, has been shown to cause stabilization of hypoxia inducible factor 1 alpha (HIF1 alpha). According to recent studies, HIF1 alpha could contribute to the hypoxia induced genomic instability seen in many cancers, through repression of mismatch repair (MMR) protein MSH2. In this study, in agreement with previous works, we found HIF1 alpha to be moderately or highly stabilized in 67% (16/24) and 77% (48/62) of HLRCC tumors and SDHB/C/D paragangliomas (PGL) and pheochromocytomas (PHEO), respectively. In addition, a set of 54 other familial and nonfamilial PGLs/PHEOs were studied. Moderately or highly stabilized HIF1 alpha was present in 68% (26/38) of the PGLs but in PHEOs (n = 16) no such pattern was observed. We then analyzed the suggested link between HIF1 alpha stabilization and MSH2 repression, in HLRCC and HPGL tumor material. No microsatellite instability (MSI) or lack of MSH2 expression was, however, observed. Thus we failed to provide in vivo evidence for the proposed link between HIF1 alpha stabilization and functional MMR deficiency, in TCAC deficient tumors. PMID:17520677

  6. 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.


    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.

  7. Nuclear receptors, mitochondria and lipid metabolism.


    Alaynick, William A


    Lipid metabolism is a continuum from emulsification and uptake of lipids in the intestine to cellular uptake and transport to compartments such as mitochondria. Whether fats are shuttled into lipid droplets in adipose tissue or oxidized in mitochondria and peroxisomes depends on metabolic substrate availability, energy balance and endocrine signaling of the organism. Several members of the nuclear hormone receptor superfamily are lipid-sensing factors that affect all aspects of lipid metabolism. The physiologic actions of glandular hormones (e.g. thyroid, mineralocorticoid and glucocorticoid), vitamins (e.g. vitamins A and D) and reproductive hormones (e.g. progesterone, estrogen and testosterone) and their cognate receptors are well established. The peroxisome-proliferator activated receptors (PPARs) and liver X receptors (LXRs), acting in concert with PPARgamma Coactivator 1alpha (PGC-1alpha), have been shown to regulate insulin sensitivity and lipid handling. These receptors are the focus of intense pharmacologic studies to expand the armamentarium of small molecule ligands to treat diabetes and the metabolic syndrome (hypertension, insulin resistance, hyperglycemia, dyslipidemia and obesity). Recently, additional partners of PGC-1alpha have moved to the forefront of metabolic research, the estrogen-related receptors (ERRs). Although no endogenous ligands for these receptors have been identified, phenotypic analyses of knockout mouse models demonstrate an important role for these molecules in substrate sensing and handling as well as mitochondrial function. PMID:18375192

  8. Molecular cloning and characterization of the Xenopus hypoxia-inducible factor 1alpha (xHIF1alpha).


    de Beaucourt, Arnaud; Coumailleau, Pascal


    We report the molecular cloning and the characterization of the Xenopus homolog of mammalian hypoxia-inducible factor 1alpha (HIF1alpha), a member of the bHLH/PAS transcription factor family. Searches in Xenopus genome sequences and phylogenetic analysis reveal the existence of HIF1alpha and HIF2alpha paralogs in the Xenopus laevis species. Sequence data analyses indicate that the organization of protein domains in Xenopus HIF1alpha (xHIF1alpha) is strongly conserved. We also show that xHIF1alpha heterodimerizes with the Xenopus Arnt1 protein (xArnt1) with the proteic complex being mediated by the HLH and PAS domains. Subcellular analysis in a Xenopus XTC cell line using chimeric GFP constructs show that over-expression of xHIF1alpha and xArnt1 allows us to detect the xHIF1alpha/xArnt1 complex in the nucleus, but only in the presence of both partners. Further analyses in XTC cell line show that over-producing xHIF1alpha and xArnt1 mediates trans-activation of the hypoxia response element (HRE) reporter. The trans-activation level can be increased in hypoxia conditions. Interestingly such trans-activation properties can be also observed when human Arnt1 is used together with the xHIF1alpha. PMID:17471499

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

    SciTech Connect

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


    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.

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

    SciTech Connect

    Shlomai, Amir; Shaul, Yosef


    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.

  11. 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.


    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.

  12. Biological activity profiles of 1alpha,25-dihydroxyvitamin D2, D3, D4, D7, and 24-epi-1alpha,25-dihydroxyvitamin D2.


    Tsugawa, N; Nakagawa, K; Kawamoto, Y; Tachibana, Y; Hayashi, T; Ozono, K; Okano, T


    We have synthesized several 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D] derivatives and evaluated their biological activity in terms of their binding affinity for the vitamin D receptor (VDR) and vitamin D-binding protein (DBP), antiproliferative or differentiation-inducing effects on human promyelocytic leukemic HL-60 cells, and transcriptional activity on a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), and human osteocalcin gene promoter, including a VDRE in transfected human osteosarcoma MG-63 cells. Furthermore, human VDR- or retinoic acid X receptor alpha (RXR alpha)-mediated luciferase activities of the derivatives were also measured by a one-hybrid system in human epitheloid carcinoma, cervix HeLa cells and African green monkey kidney CV-1 cells. Binding affinity for VDR, bone-resorbing activity, antiproliferative and cell-differentiating effects, transactivation potencies on target genes and VDR- or RXR alpha-mediated gene regulations of 1alpha,25(OH)2D2 and 1alpha,25(OH)2D4 were almost comparable to the effects of 1alpha,25(OH)2D3 while 24-epi-1alpha,25(OH)2D2 and 1alpha,25(OH)2D7 were much less active than 1alpha,25(OH)2D3 in these respects. This is the first report concerning biological assessment of 1alpha,25(OH)2D2, 1alpha,25(OH)2D3, 1alpha,25(OH)2D4, 24-epi-1alpha,25(OH)2D2 and 1alpha,25(OH)2D7 at the molecular level, especially with regards to the structural differences at the 24R- or 24S-methyl group and a double bond between carbons 22 and 23 in the side chain of 1alpha,25(OH)2D derivatives. PMID:10328556

  13. Hypoxia inducible factor-1 alpha and multiple myeloma

    PubMed Central

    Tiwary, Bhupendra Nath


    Rapid tumor growth creates a state of hypoxia in the tumor microenvironment and results in release of hypoxia inducible factor-1 alpha (HiF-1α) in the local milieu. Hypoxia inducible factor activity is deregulated in many human cancers, especially those that are highly hypoxic. In multiple myeloma (MM) in initial stages of disease establishment, the hypoxic bone marrow microenvironment supports the initial survival and growth of the myeloma cells. Hypoxic tumour cells are usually resistant to radiotherapy and most conventional chemotherapeutic agents, rendering them highly aggressive and metastatic. Therefore, HIF is an attractive, although challenging, therapeutic target in MM directly or indirectly in recent years. PMID:26900575

  14. 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:


    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.

  15. Human keratinocyte line HaCaT metabolizes 1alpha-hydroxyvitamin D3 and vitamin D3 to 1alpha,25-dihydroxyvitamin D3 (calcitriol).


    Lehmann, B; Pietzsch, J; Kämpf, A; Meurer, M


    Cultured human keratinocytes have the property to hydroxylate exogenous 25-hydroxyvitamin D3 (25OHD3) at the C-1alpha position thus producing 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). In this study we investigated whether keratinocytes can also hydroxylate vitamin D3 and one of its metabolites at the C-25 position. We could demonstrate that HaCaT keratinocytes can metabolize 1alpha-hydroxyvitamin D3 (1alpha-OHD3) and vitamin D3 to 1alpha,25(OH)2D3. Identification of the generated product as 1alpha,25(OH)2D3 was based on its elution pattern in two different high performance liquid chromatography systems, on its specific binding in a calf thymus receptor assay and on its gas chromatography-mass spectrometry characteristics. The hydroxylation of vitamin D3 to 1alpha,25(OH)2D3 was dose- and time-dependent. Bovine serum albumin added up to 1.5% (w/v) to the culture medium greatly increased the hydroxylation rates. These results show that HaCaT cells have the capacity to hydroxylate vitamin D3 at the C-1/25 positions. The generation of endogenous 1alpha,25(OH)2D3 from vitamin D3 within the skin may indicate a novel pathway which is of importance for the regulation of epidermal cell growth and differentiation. PMID:9833978

  16. Major vault protein forms complexes with hypoxia-inducible factor (HIF)-1alpha and reduces HIF-1alpha level in ACHN human renal adenocarcinoma cells.


    Iwashita, Ken-ichi; Ikeda, Ryuji; Takeda, Yasuo; Sumizawa, Tomoyuki; Furukawa, Tatsuhiko; Yamaguchi, Tatsuya; Akiyama, Shin-ichi; Yamada, Katsushi


    Vaults are evolutionarily highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. Although roles in multidrug resistance and innate immunity have been suggested, the physiological function of vaults remains unclear. Major vault protein (MVP), the main component of the vault particle, has been reported to be induced by hypoxia. However, there are no reports about the effect of vaults on cellular responses to hypoxia. We thus examined whether vaults are implicated in cellular responses to hypoxia. In this study, we focused on hypoxia-inducible factor-1alpha (HIF-1alpha), which is a master regulator of hypoxic responses, and found that: (i) MVP knockdown by RNA interference increases HIF-1alpha protein levels induced by hypoxia and hypoxia mimetics; (ii) MVP knockdown does not affect HIF-1alpha mRNA levels, but decreases the ubiquitination and degradation of HIF-1alpha protein; and (iii) vaults form complexes with HIF-1alpha, PHD2, and pVHL. Taken together, these results suggest that vaults function as scaffolds in HIF-1alpha degradation pathway and promote the ubiquitination and degradation of HIF-1alpha. PMID:20175781

  17. Metabolism of 1alpha-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1alpha,20-dihydroxyvitamin D3.


    Tuckey, Robert C; Janjetovic, Zorica; Li, Wei; Nguyen, Minh N; Zmijewski, Michal A; Zjawiony, Jordan; Slominski, Andrzej


    Cytochrome P450scc (CYP11A1) metabolizes vitamin D3 to 20-hydroxyvitamin D3 as the major product, with subsequent production of dihydroxy and trihydroxy derivatives. The aim of this study was to determine whether cytochrome P450scc could metabolize 1alpha-hydroxyvitamin D3 and whether products were biologically active. The major product of 1alpha-hydroxyvitamin D3 metabolism by P450scc was identified by mass spectrometry and NMR as 1alpha,20-dihydroxyvitamin D3. Mass spectrometry of minor metabolites revealed the production of another dihydroxyvitamin D3 derivative, two trihydroxy-metabolites made via 1alpha,20-dihydroxyvitamin D3 and a tetrahydroxyvitamin D3 derivative. The Km for 1alpha-hydroxyvitamin D3 determined for P450scc incorporated into phospholipid vesicles was 1.4 mol substrate/mol phospholipid, half that observed for vitamin D3. The kcat was 3.0 mol/min/mol P450scc, 6-fold lower than that for vitamin D3. 1alpha,20-Dihydroxyvitamin D3 inhibited DNA synthesis by human epidermal HaCaT keratinocytes propagated in culture, in a time- and dose-dependent fashion, with a potency similar to that of 1alpha,25-dihydroxyvitamin D3. 1alpha,20-Dihydroxyvitamin D3 (10 microM) enhanced CYP24 mRNA levels in HaCaT keratinocytes but the potency was much lower than that reported for 1alpha,25-dihydroxyvitamin D3. We conclude that the presence of the 1-hydroxyl group in vitamin D3 does not alter the major site of hydroxylation by P450scc which, as for vitamin D3, is at C20. The major product, 1alpha,20-dihydroxyvitamin D3, displays biological activity on keratinocytes and therefore might be useful pharmacologically. PMID:19000766

  18. Transformation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains.

    PubMed Central

    Sasaki, J; Mikami, A; Mizoue, K; Omura, S


    To enzymatically synthesize vitamin D derivatives, we screened about 300 Streptomyces sp. strains. Streptomyces sclerotialus FERM BP-1370 and Streptomyces roseoporus FERM BP-1574 were found to have the ability to convert 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, respectively, to 1 alpha, 25-dihydroxyvitamin D3. The average rates of 1 alpha hydroxylation of 25-hydroxyvitamin D3 were 6.9 micrograms liter-1 min-1 with FERM BP-1370 and 7.0 micrograms liter-1 min-1 with FERM BP-1574. The specific cytochrome P-450 inhibitors carbon monoxide, SKF-525-A, and metyrapone inhibited the hydroxylation of 1 alpha- and 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by FERM BP-1370 and FERM BP-1574. The cytochromes P-450 of these strains were detected by reduced CO difference spectra in the whole-cell suspensions. The appearance of cytochrome P-450 suggests that the cytochromes P-450 of FERM BP-1370 and FERM BP-1574 carry out the hydroxylation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3. PMID:1746944

  19. Detection of reactive oxygen species via endogenous oxidative pentose phosphate cycle activity in response to oxygen concentration: implications for the mechanism of HIF-1alpha stabilization under moderate hypoxia.


    Tuttle, Stephen W; Maity, Amit; Oprysko, Patricia R; Kachur, Alexander V; Ayene, Iraimoudi S; Biaglow, John E; Koch, Cameron J


    The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O2. We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O2), whereas hyperoxia (95% O2) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1alpha (HIF-1alpha) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1alpha stabilization under moderate hypoxia. Moreover, the levels of HIF-1alpha that we observed after exposure to moderate hypoxia were comparable between rho0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1alpha in response to the non-toxic levels of H2O2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1alpha stability under moderate as well as severe hypoxia. PMID:17666400

  20. Crystal structure of porcine reproductive and respiratory syndrome virus leader protease Nsp1alpha.


    Sun, Yuna; Xue, Fei; Guo, Yu; Ma, Ming; Hao, Ning; Zhang, Xuejun C; Lou, Zhiyong; Li, Xuemei; Rao, Zihe


    Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV), a positive-strand RNA virus that belongs to the Arteriviridae family of Nidovirales, has been identified as the causative agent of PRRS. Nsp1alpha is the amino (N)-terminal protein in a polyprotein encoded by the PRRSV genome and is reported to be crucial for subgenomic mRNA synthesis, presumably by serving as a transcription factor. Before functioning in transcription, nsp1alpha proteolytically releases itself from nsp1beta. However, the structural basis for the self-releasing and biological functions of nsp1alpha remains elusive. Here we report the crystal structure of nsp1alpha of PRRSV (strain XH-GD) in its naturally self-processed form. Nsp1alpha contains a ZF domain (which may be required for its biological function), a papain-like cysteine protease (PCP) domain with a zinc ion unexpectedly bound at the active site (which is essential for proteolytic self-release of nsp1alpha), and a carboxyl-terminal extension (which occupies the substrate binding site of the PCP domain). Furthermore, we determined the exact location of the nsp1alpha self-processing site at Cys-Ala-Met180 downward arrowAla-Asp-Val by use of crystallographic data and N-terminal amino acid sequencing. The crystal structure also suggested an in cis self-processing mechanism for nsp1alpha. Furthermore, nsp1alpha appears to have a dimeric architecture both in solution and as a crystal, with a hydrophilic groove on the molecular surface that may be related to nsp1alpha's biological function. Compared with existing structure and function data, our results suggest that PRRSV nsp1alpha functions differently from other reported viral leader proteases, such as that of foot-and-mouth disease. PMID:19706710

  1. Effects of 12 metal ions on iron regulatory protein 1 (IRP-1) and hypoxia-inducible factor-1 alpha (HIF-1{alpha}) and HIF-regulated genes

    SciTech Connect

    Li Qin; Chen Haobin; Huang Xi; Costa, Max . E-mail:


    Several metal ions that are carcinogenic affect cellular iron homeostasis by competing with iron transporters or iron-regulated enzymes. Some metal ions can mimic a hypoxia response in cells under normal oxygen tension, and induce expression of HIF-1{alpha}-regulated genes. This study investigated whether 12 metal ions altered iron homeostasis in human lung carcinoma A549 cells as measured by an activation of IRP-1 and ferritin level. We also studied hypoxia signaling by measuring HIF-1{alpha} protein levels, hypoxia response element (HRE)-driven luciferase reporter activity, and Cap43 protein level (an HIF-1{alpha} responsive gene). Our results show the following: (i) Ni(II), Co(II), V(V), Mn(II), and to a lesser extent As(III) and Cu(II) activated the binding of IRP-1 to IRE after 24 h, while the other metal ions had no effect; (ii) 10 of 12 metal ions induced HIF-1{alpha} protein but to strikingly different degrees. Two of these metal ions, Al(III) and Cd(II), did not induce HIF-1{alpha} protein; however, as indicated below, only Ni(II), Co (II), and to lesser extent Mn(II) and V(V) activated HIF-1{alpha}-dependent transcription. The combined effects of both [Ni(II) + As(III)] and [Ni(II) + Cr(VI)] on HIF-1{alpha} protein were synergistic; (iii) Addition of Fe(II) with Ni(II), Co(II), and Cr(VI) attenuated the induction of HIF-1{alpha} after 4 h treatment; (iv) Ni(II), Co(II), and Mn(II) significantly decrease ferritin level after 24 h exposure; (v) Ni(II), Co(II), V(V), and Mn(II) activated HRE reporter gene after 20 h treatment; (vi) Ni(II), Co(II), V(V), and Mn(II) increased the HIF-1-dependent Cap43 protein level after 24 h treatment. In conclusion, only Ni (II), Co (II), and to a lesser extent Mn(II) and V(V) significantly stabilized HIF-1{alpha} protein, activated IRP, decreased the levels of ferritin, induced the transcription of HIF-dependent reporter, and increased the expression of Cap43 protein levels (HIF-dependent gene). The mechanism for the

  2. 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


    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

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

    SciTech Connect

    Auth, Tanja . E-mail:; Kunkel, Elisabeth; Grummt, Friedrich . E-mail:


    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.

  4. 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


    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.

  5. Molecular cloning and phylogenetic analysis of Clonorchis sinensis elongation factor-1alpha.


    Kim, Tae Yun; Cho, Pyo Yun; Na, Jong Won; Hong, Sung-Jong


    Elongation factor-1 (EF-1) plays a primary role in protein synthesis, e.g., in the regulation of cell growth, aging, motility, embryogenesis, and signal transduction. The authors identified a clone CsIH23 by immunoscreening a Clonorchis sinensis cDNA library. The cDNA of CsIH23 was found to have a putative open reading frame containing 461 amino acids with a predicted molecular mass of 50.5 kDa. Its polypeptide sequence was highly homologous with EF-1alpha of parasites and vertebrate animals. CsIH23 polypeptide contained three GTP/GDP-binding sites, one ribosome-binding domain, one actin-binding domain, one tRNA-binding domain, and two glyceryl-phosphoryl-ethanolamine attachment sites. Based on these primary and secondary structural similarities, it was concluded that CsIH23 cDNA encodes C. sinensis EF-1alpha (CsEF-1alpha). In a molecular phylogenic tree, CsEF-1alpha clustered with the EF-1alpha of helminthic parasites. Subsequently, CsEF-1alpha recombinant protein was bacterially overexpressed and purified by Ni-NTA affinity column chromatography. Immunoblotting using CsEF-1alpha recombinant protein produced positive signals for all serum samples tested from clonorchiasis, opisthorchiasis viverinii, and paragonimiasis westermani patients and normal healthy controls. These findings suggest that recombinant CsEF-1alpha is of limited usefulness as serodiagnostic antigen for clonorchiasis. PMID:17674047

  6. 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.


    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.

  7. Desferrioxamine, an iron chelator, enhances HIF-1{alpha} accumulation via cyclooxygenase-2 signaling pathway

    SciTech Connect

    Woo, Kyung Jin; Lee, Tae-Jin; Park, Jong-Wook; Kwon, Taeg Kyu . E-mail:


    Cyclooxygenase-2 (COX-2) is an important inducible enzyme in inflammation and is overexpressed in a variety of cancers. Evidence is rapidly accumulating that chronic inflammation may contribute to carcinogenesis through increase of cell proliferation, angiogenesis, and metastasis in a number of neoplasms, including colorectal carcinoma. In the present study, we investigated some mechanistic aspects of DFX-induced hypoxia-driven COX-2 expression. Desferrioxamine (DFX), an iron chelator, is known to upregulate inflammatory mediators. DFX induced the expression of COX-2 and accumulation of HIF-1{alpha} protein in dose-dependent manners, but hypoxia mimetic agent cobalt chloride (CoCl{sub 2}) induced accumulation of HIF-1{alpha} protein but not increase of COX-2 expression. DFX-induced increase of COX-2 expression and HIF-1{alpha} protein level was attenuated by addition of ferric citrate. This result suggested that the iron chelating function of DFX was important to induce the increase of COX-2 and HIF-1{alpha} protein. PD98059 significantly inhibited the induction of COX-2 protein and accumulation of HIF-1{alpha}, suggesting that DFX-induced increase of HIF-1{alpha} and COX-2 protein was mediated, at least in part, through the ERK signaling pathway. In addition, pretreatment with NS-398 to inhibit COX-2 activity also effectively suppressed DFX-induced HIF-1{alpha} accumulation in human colon cancer cells, providing the evidence that COX-2 plays as a regulator of HIF-1{alpha} accumulation in DFX-treated colon cancer cells. Together, our findings suggest that iron metabolism may regulate stabilization of HIF-1{alpha} protein by modulating cyclooxygenase-2 signaling pathway.

  8. Milk fever controls: comparison of 1-alpha and vitamin D3 in conjunction with induced parturition.


    McMurray, C H; Rice, D A; McBride, P S


    The efficacies of vitamin D3 and its 1-alpha hydroxyl derivative (1-alpha) in controlling clinical milk fever, hypocalcaemia and hypophosphataemia in parturient cows have been compared. A corticosteroid was used in some cases to optimise and control the interval between prophylactic treatment and parturition. Our observations suggest that the combination of 1-alpha and corticosteroid was particularly valuable and could be used in the development of a successful prophylactic regime. This conclusion is supported by both clinical and biochemical measurements. PMID:6255674

  9. [Concept and clinical characteristics of diabetes mellitus in the elderly].


    Yokono, Koichi


    Both decreased insulin secretion and insulin resistance are two major factors of impaired glucose tolerance (IGT) in the elderly. Up to now, decreased lean body mass and relatively increased fat mass, so-called sarcopenia or sarcopenic obesity, contribute to insulin resistance in the elderly. However, recent reports indicate that muscle mitochondrial function is reduced in aging, and this age-associated decline in mitochondrial function contributes to insulin resistance in the elderly. In addition, exercise intervention to IGT in the elderly is more effective to reduce in the incidence of type 2 diabetes than in younger people. Exercise seems to improve insulin resistance through mitochondrial function by activating AMP-activated protein kinase(AMPK) and PPARgamma coactivator-1alpha (PGC-1alpha). Because cognitive impairment is a most crucial factor plunging into frailty in diabetic elderly, diabetic control would be very important in preventing cognitive decline as well as vascular events. However, comprehensive management of diabetes, including dyslipidemia and hypertension, might contribute to the prevention of declines in cognitive function in older diabetic patients. PMID:24397156

  10. USP14 inhibits ER-associated degradation via interaction with IRE1{alpha}

    SciTech Connect

    Nagai, Atsushi; Kadowaki, Hisae; Maruyama, Takeshi; Takeda, Kohsuke; Nishitoh, Hideki Ichijo, Hidenori


    Accumulation of unfolded proteins within the endoplasmic reticulum (ER) lumen induces ER stress. Eukaryotic cells possess the ER quality control systems, the unfolded protein response (UPR), to adapt to ER stress. IRE1{alpha} is one of the ER stress receptors and mediates the UPR. Here, we identified ubiquitin specific protease (USP) 14 as a binding partner of IRE1{alpha}. USP14 interacted with the cytoplasmic region of IRE1{alpha}, and the endogenous interaction between USP14 and IRE1{alpha} was inhibited by ER stress. Overexpression of USP14 inhibited the ER-associated degradation (ERAD) pathway, and USP14 depletion by small interfering RNA effectively activated ERAD. These findings suggest that USP14 is a novel player in the UPR by serving as a physiological inhibitor of ERAD under the non-stressed condition.

  11. 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


    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

  12. Inhibition of GSK3beta by indirubins restores HIF-1alpha accumulation under prolonged periods of hypoxia/anoxia.


    Schnitzer, Steffen E; Schmid, Tobias; Zhou, Jie; Eisenbrand, Gerhard; Brüne, Bernhard


    Hypoxia inducible factor 1 is regulated by the appearance of the HIF-1alpha subunit. HIF-1alpha is subjected to proteasomal destruction or enhanced protein translation, which requires the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. We investigated how PI3K/Akt and glycogen synthase kinase 3beta (GSK3beta) affect HIF-1alpha in human RKO cells under prolonged periods of severe hypoxia/anoxia. 16- to 32-h lasting incubations attenuated Akt activity and decreased HIF-1alpha protein. This was reproduced by blocking PI3K with LY294002. GSK3beta inhibition by indirubins circumvented the effect of hypoxia/anoxia or LY294002 on HIF-1alpha. Ruling stability regulation of HIF-1alpha protein and/or enhanced transcription of HIF-1alpha mRNA via GSK3beta inhibition out is suggestive for translational modulation of HIF-1alpha under the influence of GSK3beta. PMID:15642371

  13. Expression of vascular endothelial growth factor (VEGF), hypoxia inducible factor-1alpha (HIF-1alpha), and microvessel density in endometrial tissue in women with adenomyosis.


    Goteri, Gaia; Lucarini, Guendalina; Montik, Nina; Zizzi, Antonio; Stramazzotti, Daniela; Fabris, Guidalberto; Tranquilli, Andrea Luigi; Ciavattini, Andrea


    Adenomyosis is a disease with a mysterious pathogenesis, defined by an abnormal displacement of the eutopic endometrium deeply and haphazardly inside the myometrium. Angiogenesis has been indicated to play an important role and our aim was to investigate whether vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha (HIF-1alpha) expression and microvessel density (MVD) were different in women with and without adenomyosis. Immunohistochemistry was performed in endometrial tissues in 23 patients who underwent radical hysterectomy for adenomyosis (14) and for ovarian cysts and fibroids (9) at an Academic Hospital. Compared to women without the disease, VEGF expression was increased in endometrium with a normal location in patients with adenomyosis, although not associated to a significant increase of HIF-1alpha and MVD. Moreover, the endometrium with an abnormal location in patients with adenomyosis showed an increased VEGF and HIF-1alpha expression, particularly in the epithelial cells, associated to an increase of MVD, compared with the endometrium in a normal location in the same group of patients. Our present findings suggest that VEGF-mediated angiogenesis might be associated with the development of adenomyosis. In the ectopic foci the abnormal location might contribute to increased HIF-1a expression, stimulation of VEGF production, and increased vessel formation. In endometrium with a normal location, instead, where VEGF increased expression seems not to be correlated with HIF-1alpha increased expression nor with an increased MVD, other mechanisms might be reasonably postulated. Additional studies are required to explore new targeted and more effective treatment modalities. PMID:19188818

  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.


    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. Reduced serum levels of 1 alpha,25-dihydroxyvitamin D during long-term total parenteral nutrition.


    Klein, G L; Horst, R L; Norman, A W; Ament, M E; Slatopolsky, E; Coburn, J W


    Painful bone disease, characterized by patchy osteomalacia and inactive bone, can develop in patients treated with total parenteral nutrition for more than 3 months. Serum levels of 1 alpha,25-dihydroxyvitamin D (1 alpha, 25(OH)2D), 24,25-dihydroxyvitamin D and 25-hydroxyvitamin D were measured in seven adults and five children treated with parenteral nutrition for 9 to 60 months. Serum levels of 1 alpha, 25(OH)2D were markedly reduced, while levels of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were normal. Serum calcium and phosphorus levels were normal or slightly increased, and immunoreactive parathyroid hormone levels were normal or low. Renal function was normal or minimally reduced. Skeletal symptoms disappeared and serum 1 alpha, 25(OH)2D levels rose to normal in one patient when nutrient infusions were discontinued for 6 weeks. Removal of calcium from the nutrient solution for 2 to 4 days was associated with no change in serum 1 alpha, 25(OH)2D in two patients. The cause of the reduction in serum levels of 1 alpha, 25(OH)2D and its role in the pathogenesis of bone disease in these patients remain uncertain. PMID:6786151

  16. Demonstration that circulating 1 alpha, 25-dihydroxyvitamin D is loosely regulated in normal children.

    PubMed Central

    Stern, P H; Taylor, A B; Bell, N H; Epstein, S


    The effects of vitamin D, 2.5 mg (100,000 U)/d for 4 d, on serum calcium, serum 25-hydroxyvitamin D (25-OHD) and serum 1 alpha, 25-dihydroxyvitamin D (1 alpha, 25(OH)2D) were compared in 24 normal adults and 12 normal children. The daily dose of vitamin D was 1,500 U/kg body wt in children weighing less than 45 kg. Vitamin D increased mean serum calcium from 9.5 +/- 0.1 to 9.8 +/- 0.1 mg/dl (P less than 0.05), increased mean serum phosphorus from 4.6 +/- 0.1 to 5.0 +/- 0.1 mg/dl (P less than 0.01), increased mean serum 25-OHD from 25 +/- 3 to 34 +/- 4 ng/ml (P less than 0.001), and increased mean serum 1 alpha, 25(OH)2D from 34 +/- 3 to 42 +/- 4 pg/ml (P less than 0.02) in children. In contrast, vitamin D increased mean serum 25-OHD from 18 +/- 2 to 39 +/- 6 ng/ml (P less than 0.001) and did not change mean serum calcium (9.4 +/- 0.1 vs. 9.5 +/- 0.1 mg/dl), mean serum phosphorus (4.0 +/- 0.1 vs. 4.1 +/- 0.1 mg/dl), or mean serum 1 alpha, 25(OH)2D (31 +/- 2 vs. 29 +/- 3 pg/ml) in adults. Mean serum 1 alpha, 25(OH)2D was significantly higher after vitamin D in children than in adults (P less than 0.02). These results provide evidence that circulating 1 alpha, 25(OH)2D is not as tightly regulated in children as it is in adults. This difference in regulation could account in part for the higher values for serum 1 alpha, 25(OH)2D observed in children. PMID:6975284

  17. 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


    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.

  18. Involvement of net and Hif1alpha in distinct yet intricately linked hypoxia-induced signaling pathways.


    Serchov, Tsvetan; Dubois-Pot-Schneider, Helene; Charlot, Celine; Rösl, Frank; Wasylyk, Bohdan


    The present study compares negative Ets transcription factor (Net) and hypoxia-inducible factor 1alpha (HIF1alpha) regulation by hypoxia. Their protein stabilities are differently regulated by hypoxia, defining three periods in the kinetics: normoxia (high Net levels and low HIF1alpha levels), early hypoxia (high levels of Net and HIF1alpha), and late hypoxia (degradation of Net and HIF1alpha). Modulators of prolyl hydroxylase domain protein (PHD) activity induce a mobility shift of Net, similar to HIF1alpha, suggesting that post-translational modifications of both factors depend on PHD activity. The three PHDs have different roles in the regulation of Net protein levels; PHD1 and PHD3 are involved in the stabilization of Net, whereas PHD2 controls its degradation in late hypoxia. Net physically interacts with PHD2 in hypoxia, whereas PHD1 and PHD3 bind to Net in normoxia and hypoxia. Under the same conditions, PHD2 and PHD3 regulate both HIF1alpha stabilization in early hypoxia and its degradation at late hypoxia, whereas PHD1 is involved in HIF1alpha degradation in late hypoxia. We describe interconnections between the regulation of both Net and HIF1alpha at the protein level. Evidence is provided for a direct physical interaction between Net and HIF1alpha and indirect transcriptional regulation loops that involve the PHDs. Taken together our results indicate that Net and HIF1alpha are components of distinct signaling pathways that are intricately linked. PMID:20427288

  19. Mitochondrial vasculopathy

    PubMed Central

    Finsterer, Josef; Zarrouk-Mahjoub, Sinda


    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

  20. Mitochondrial Cardiomyopathies

    PubMed Central

    El-Hattab, Ayman W.; Scaglia, Fernando


    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.


    Finsterer, Josef; Zarrouk-Mahjoub, Sinda


    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. Mitochondrial Cardiomyopathies.


    El-Hattab, Ayman W; Scaglia, Fernando


    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

  3. Leishmania donovani amastigotes impair gamma interferon-induced STAT1alpha nuclear translocation by blocking the interaction between STAT1alpha and importin-alpha5.


    Matte, Christine; Descoteaux, Albert


    The protozoan parasite Leishmania donovani, the etiological agent of visceral leishmaniasis, is renowned for its capacity to sabotage macrophage functions and signaling pathways stimulated by activators such as gamma interferon (IFN-gamma). Our knowledge of the strategies utilized by L. donovani to impair macrophage responsiveness to IFN-gamma remains fragmentary. In the present study, we investigated the impact of an infection by the amastigote stage of L. donovani on IFN-gamma responses and signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in mouse bone marrow-derived macrophages. The levels of IFN-gamma-induced expression of major histocompatibility complex class II and inducible nitric oxide synthase (iNOS) were strongly reduced in L. donovani amastigote-infected macrophages. As the expression of those genes is mediated by the transcription factors STAT1alpha and IFN regulatory factor 1 (IRF-1), we investigated their activation in amastigote-infected macrophages treated with IFN-gamma. We found that whereas STAT1alpha protein levels and the levels of phosphorylation on Tyr701 and Ser727 were normal, IRF-1 expression was inhibited in infected macrophages. This inhibition of IRF-1 expression correlated with a defective nuclear translocation of STAT1alpha, and further analyses revealed that the IFN-gamma-induced STAT1alpha association with the nuclear transport adaptor importin-alpha5 was compromised in L. donovani amastigote-infected macrophages. Taken together, our results provide evidence for a novel mechanism used by L. donovani amastigotes to interfere with IFN-gamma-activated macrophage functions and provide a better understanding of the strategies deployed by this parasite to ensure its intracellular survival. PMID:20566692

  4. Separate and combined effects of recombinant interleukin-1 alpha and gamma interferon on antibacterial resistance.

    PubMed Central

    Kurtz, R S; Young, K M; Czuprynski, C J


    Our laboratory has previously reported that administration of murine recombinant interleukin 1 alpha (rIL-1 alpha) substantially enhanced the resistance of mice to Listeria monocytogenes infection. Other investigators have reported that gamma interferon (IFN-gamma) plays a pivotal role in antilisteria resistance. In the present study, we have defined doses of human rIL-1 alpha that enhanced the antilisteria resistance of mice. We then addressed the possibility that combined immunotherapy with rIL-1 alpha and recombinant IFN-gamma (rIFN-gamma) might result in an additive or synergistic enhancement of antibacterial resistance. Simultaneous administration of rIL-1 alpha and rIFN-gamma enhanced antilisteria resistance (at 3 days after infection) to a greater extent than did either cytokine alone, although the results did not imply a synergistic action between the two cytokines. Experiments which examined the effects of the timing of cytokine administration indicated that maximal protection was observed when rIL-1 alpha and rIFN-gamma were administered together concomitantly with the L. monocytogenes challenge. When we compared the separate and combined protective effects of rIL-1 alpha and rIFN-gamma throughout the course of a primary L. monocytogenes infection, we observed an additive effect of the two cytokines only at 3 days after challenge, the time at which the peak bacterial burden occurs in the spleens and livers of infected mice. Histopathological comparisons of livers and spleens from cytokine-treated and control listeria-infected mice verified that cytokine treatment reduced the severity of tissue damage in cytokine-treated listeria-infected mice. In an attempt to provide a potential mechanism for the protective effects of rIL-1 alpha and rIFN-gamma administration, we compared levels of colony-stimulating activity in sera from cytokine-treated and control listeria-infected mice. The highest levels of colony-stimulating activity were detected in sera from

  5. Identification of high-affinity anti-IL-1. alpha. autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1. alpha

    SciTech Connect

    Mae, N.; Liberato, D.J.; Chizzonite, R.; Satoh, H. )


    A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 {alpha} (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 {alpha} added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 {alpha} added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 {alpha} in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 {alpha} binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 {alpha}-specific IgG. The binding of 125I-labeled IL-1 {alpha} to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 {times} 10(-11) M) but not by unlabeled IL-1 {beta}. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 {alpha}-specific IgGs was 4.7 {times} 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 {alpha} in human serum by various assay systems and also could be a regulator of circulating IL-1 {alpha}.

  6. 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


    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.

  7. Murine model of otitis media with effusion: immunohistochemical demonstration of IL-1 alpha antigen expression.


    Johnson, M D; Contrino, A; Contrino, J; Maxwell, K; Leonard, G; Kreutzer, D


    Recent studies have suggested that cytokines likely play a central role in the formation and maintenance of otitis media with effusion (OME). Currently, there is no immunologically defined animal model for the study of cytokines as they contribute to the formation of OME. In the present study, a murine model of OME, using eustachian tube blockage via an external surgical approach, was developed. The murine model temporal bone histology appears to mimic the histology found in chronic otitis media with effusion in humans. Additionally, using this murine model, interleukin-1 alpha (IL-1 alpha) expression was detected in the middle ear using standard immunohistochemical techniques. IL-1 alpha seemed localized to the epithelial lining of the middle ear as well as 5% to 10% of inflammatory cells. This model should provide the necessary tool to further study the immunologic aspects of OME. PMID:8072363

  8. 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.


    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.

  9. Evidence for a slightly deleterious effect of intron polymorphisms at the EF1alpha gene in the deep-sea hydrothermal vent bivalve Bathymodiolus.


    Faure, B; Bierne, N; Tanguy, A; Bonhomme, F; Jollivet, D


    A multilocus analysis was initiated in order to infer the general effect of demography and the indirect effect of positive selection on some chromosome segments in Bathymodiolus. Mussels of the genus Bathymodiolus inhabit the very hostile, fragmented and variable environment of deep-sea hydrothermal vents which is thought to cause recurrent population bottlenecks via extinction/colonisation processes and adaptation to new environmental conditions. In the course of this work we discovered that the assumption of neutrality of non-coding polymorphisms usually made in genome scan experiments was likely to be violated at one of the loci we analysed. The direct effect of slight purifying selection on non-coding polymorphisms shares many resemblances with the indirect effect of positive selection through genetic hitchhiking. Combining polymorphism with divergence data for several closely related species allowed us to obtain different expectations for the direct effect of negative selection and the indirect effect of positive selection. We observed a strong excess of rare non-coding polymorphisms at the second intron of the EF1alpha gene in the two species Bathymodiolus azoricus and Bathymodiolus thermophilus, while two other loci, the mitochondrial COI gene and an intron of the Lysozyme gene, did not exhibit such a deviation. In addition, the divergence rate of the EF1alpha intron was estimated to be unexpectedly low when calibrated using the closure of the Panama Isthmus that interrupted gene flow between the two species. The polymorphism to divergence ratio was similar to the one observed for the other two loci, in accordance to the hypothesis of purifying selection. We conclude that slight purifying selection is likely to act on polymorphic intronic mutations of the EF1alpha second intron and discuss the possible relationship with the specific biology of Bathymodiolus mussels. PMID:17707599

  10. Mitochondrial Diseases

    PubMed Central

    Lee, Young-Mock


    Mitochondria contain the respiratory chain enzyme complexes that carry out oxidative phosphorylation and produce the main part of cellular energy in the form of ATP. Although several proteins related with signalling, assembling, transporting, and enzymatic function can be impaired in mitochondrial diseases, most frequently the activity of the respiratory chain protein complexes is primarily or secondarily affected, leading to impaired oxygen utilization and reduced energy production. Mitochondrial diseases usually show a chronic, slowly progressive course and present with multiorgan involvement with varying onset between birth and late adulthood. Neuromuscular system is frequently affected in mitochondrial diseases. Although there is actually no specific therapy and cure for mitochondrial diseases, the understanding of the pathophysiology may further facilitate the diagnostic approach and open perspectives to future in mitochondrial diseases. PMID:24649452

  11. Mitochondrial cytopathies.


    El-Hattab, Ayman W; Scaglia, Fernando


    Mitochondria are found in all nucleated human cells and perform a variety of essential functions, including the generation of cellular energy. Most of mitochondrial proteins are encoded by the nuclear DNA (nDNA) whereas a very small fraction is encoded by the mitochondrial DNA (mtDNA). Mutations in mtDNA or mitochondria-related nDNA genes can result in mitochondrial dysfunction which leads to a wide range of cellular perturbations including aberrant calcium homeostasis, excessive reactive oxygen species production, dysregulated apoptosis, and insufficient energy generation to meet the needs of various organs, particularly those with high energy demand. Impaired mitochondrial function in various tissues and organs results in the multi-organ manifestations of mitochondrial diseases including epilepsy, intellectual disability, skeletal and cardiac myopathies, hepatopathies, endocrinopathies, and nephropathies. Defects in nDNA genes can be inherited in an autosomal or X-linked manners, whereas, mtDNA is maternally inherited. Mitochondrial diseases can result from mutations of nDNA genes encoding subunits of the electron transport chain complexes or their assembly factors, proteins associated with the mitochondrial import or networking, mitochondrial translation factors, or proteins involved in mtDNA maintenance. MtDNA defects can be either point mutations or rearrangements. The diagnosis of mitochondrial disorders can be challenging in many cases and is based on clinical recognition, biochemical screening, histopathological studies, functional studies, and molecular genetic testing. Currently, there are no satisfactory therapies available for mitochondrial disorders that significantly alter the course of the disease. Therapeutic options include symptomatic treatment, cofactor supplementation, and exercise. PMID:26996063


    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elongation factor 1 alpha (EF-1') is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1''promoter has been utilized to drive exogenous gene expression in transfected cells. In this study, we ident...

  13. Separate necdin domains bind ARNT2 and HIF1{alpha} and repress transcription

    SciTech Connect

    Friedman, Eitan R.; Fan Chenming


    PWS is caused by the loss of expression of a set of maternally imprinted genes including NECDIN (NDN). NDN is expressed in post-mitotic neurons and plays an essential role in PWS as mouse models lacking only the Ndn gene mimic aspects of this disease. Patients haploid for SIM1 develop a PW-like syndrome. Here, we report that NDN directly interacts with ARNT2, a bHLH-PAS protein and dimer partner for SIM1. We also found that NDN can interact with HIF1{alpha}. We showed that NDN can repress transcriptional activation mediated by ARNT2:SIM1 as well as ARNT2:HIF1{alpha}. The N-terminal 115 residues of NDN are sufficient for interaction with the bHLH domains of ARNT2 or HIF1{alpha} but not for transcriptional repression. Using GAL4-NDN fusion proteins, we determined that NDN possesses multiple repression domains. We thus propose that NDN regulates neuronal function and hypoxic response by regulating the activities of the ARNT2:SIM1 and ARNT2:HIF1{alpha} dimers, respectively.

  14. Molecular cloning and characterization of ovine IL-1 alpha and IL-1 beta.

    PubMed Central

    Andrews, A E; Barcham, G J; Brandon, M R; Nash, A D


    Interleukin-1 (IL-1) is a cytokine with a wide range of effects on a variety of cell types. By hybridization with human IL-1 alpha and IL-1 beta cDNA probes, the corresponding ovine cDNAs were isolated from a stimulated alveolar macrophage cDNA library. The sequences of these cDNAs showed that ovine IL-1 alpha and IL-1 beta encode proteins of 268 and 266 amino acids, respectively, with both the nucleotide and amino acid sequences showing a high degree of homology with their human, mouse and bovine equivalents. In a mammalian COS cell-expression system these cDNAs produced biologically active IL-1. Further experiments demonstrated the importance of sequences within the 3' untranslated portion of the cDNAs in determining the level of expression of these molecules. The analysis of expression of IL-1 alpha- and IL-1 beta-specific mRNA in response to endotoxin, phorbol myristic acid (PMA) or PMA plus ionomycin revealed a distinct pattern of differential regulation of the two genes. From genomic analysis both IL-1 alpha and IL-1 beta appear to exist as single copies in the ovine genome. Images Figure 4 Figure 5 PMID:1769692

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

    SciTech Connect

    Okamoto, Mika; Wang, Xin; Baba, Masanori . E-mail:


    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.

  16. Interleukin-1 alpha, interleukin-1 beta and interleukin-8 gene expression in human aural cholesteatomas.


    Kim, C S; Lee, C H; Chung, J W; Kim, C D


    Bone destruction is a common characteristic feature of chronic otitis media, especially aural cholesteatoma. A number of immunohistochemical studies have suggested that interleukin-1 (IL-1) may be responsible for cholesteatomatous bone destruction. We designed this study to present the mRNA expression patterns of IL-1 alpha, IL-1 beta, and IL-8, which can induce and activate the leukocyte, the major reservoir of potent proteolytic enzymes. Total RNAs were extracted from aural cholesteatomas, external auditory canal skin (EACS), postauricular skin (PAS), and granulation tissues and transcribed into cDNAs. cDNAs were amplified by using PCR technique with primers for IL-1 alpha, IL-1 beta, IL-8, and beta-actin. Amplified products were hybridized with each internal probe and the relative density was measured. In granulation tissues, the relative density of IL-1 alpha was greater than that of other tissues. The ratio of IL-1 beta and IL-8 of aural cholesteatoma was significantly higher than that of EACS and PAS. We suggest that both of IL-1 alpha and IL-1 beta may play a role in the pathological changes, and that IL-8, which is mainly produced from cholesteatomatous epithelium, may have an important role in the pathological changes of cholesteatomas. PMID:8725537

  17. Stem cell factor induces HIF-1{alpha} at normoxia in hematopoietic cells

    SciTech Connect

    Pedersen, Malin; Loefstedt, Tobias; Sun Jianmin; Holmquist-Mengelbier, Linda; Pahlman, Sven; Roennstrand, Lars


    Signaling by the receptor for stem cell factor (SCF), c-Kit, is of major importance for hematopoiesis, melanogenesis and reproduction, and the biological responses are commonly proliferation and cell survival. Thus, constitutive activation due to c-Kit mutations is involved in the pathogenesis of several forms of cancer, e.g. leukemias, gastrointestinal stromal tumors and testicular tumors. Tumor survival requires oxygen supply through induced neovascularization, a process largely mediated by the vascular endothelial growth factor (VEGF), a prominent target of the transcription factors hypoxia-inducible factor-1 (HIF-1) and HIF-2. Using Affymetrix microarrays we have identified genes that are upregulated following SCF stimulation. Interestingly, many of the genes induced were found to be related to a hypoxic response. These findings were corroborated by our observation that SCF stimulation of the hematopoietic cell lines M-07e induces HIF-1{alpha} and HIF-2{alpha} protein accumulation at normoxia. In addition, SCF-induced HIF-1{alpha} was transcriptionally active, and transcribed HIF-1 target genes such as VEGF, BNIP3, GLUT1 and DEC1, an effect that could be reversed by siRNA against HIF-1{alpha}. We also show that SCF-induced accumulation of HIF-1{alpha} is dependent on both the PI-3-kinase and Ras/MEK/Erk pathways. Our data suggest a novel mechanism of SCF/c-Kit signaling in angiogenesis and tumor progression.

  18. 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.


    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.

  19. Synthesis and secretion of interleukin-1 alpha and interleukin-1 receptor antagonist during differentiation of cultured keratinocytes.


    Corradi, A; Franzi, A T; Rubartelli, A


    Keratinocytes produce interleukin-1 alpha (IL-1 alpha) and the epithelial variant of its inhibitor, interleukin-1 receptor antagonist (icIL-1ra). Both IL-1 alpha and icIL-1ra lack a secretory signal peptide; however, some icIL-1ra is found in the supernatants of cultured keratinocytes. The lack of correlation with the release of the cytosolic enzyme lactate dehydrogenase suggests that icIL-1ra can be actively secreted. Brefeldin A fails to block icIL-1ra release, suggesting that this protein may be externalized by keratinocytes through a leaderless pathway of secretion. Only minute amounts of soluble extracellular IL-1 alpha are detected: however, both IL-1 alpha and icIL-1ra can be released from the external face of the keratinocyte plasma membrane by mild acidic treatment, suggesting that IL-1 alpha can also be secreted by keratinocytes. The observation of membrane-associated IL-1 alpha and icIL-1ra might reflect an autocrine loop of regulation. Support for this hypothesis comes from the finding that keratinocytes, when exposed to exogenous recombinant IL-1 alpha, increase their content in both IL-1 alpha and IL-1ra mRNA. When keratinocytes are subjected to counterflow centrifugal elutriation, three major cell populations are obtained, representing three different degrees of keratinocyte differentiation. Cells from all populations synthesize IL-1 alpha and IL-1ra: however, while IL-1 alpha is uniformly distributed in cells from all maturational stages, IL-1ra accumulates in large, more differentiated keratinocytes. Changes in the ratio of IL-1ra to IL-1 alpha production and secretion by keratinocytes at different degrees of maturation might contribute to the control of growth and differentiation of human skin. PMID:7698236

  20. Modulation of the Bovine Innate Immune Response by Production of 1alpha,25-Dihydroxyvitamin D3 in Bovine Monocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) from 25-hydroxyvitamin D3 25(OH)D3 by 1alpha-hydroxylase (1alpha-OHase). However, recent studies have shown that human monocytes express 1alpha-OHase and produce 1,25(OH)2D3 in response to to...

  1. Genes for the dimerization cofactor of hepatocyte nuclear factor-1[alpha] (DCOH) are on human and murine chromsomes 10

    SciTech Connect

    Milatovich, A.; Mendel, D.B.; Crabtree, G.R.; Francke, U. )


    Hepatocyte nuclear factor-1[alpha] (HNF-1[alpha]; gene symbol, TCF1) forms dimers with itself as well as with HNF-1[beta] and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1[alpha], called DCOH, has been identified. Here, the authors report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids. 25 refs., 1 fig., 2 tabs.

  2. Inhibition of protein synthesis by imexon reduces HIF-1alpha expression in normoxic and hypoxic pancreatic cancer cells.


    Samulitis, Betty K; Landowski, Terry H; Dorr, Robert T


    Hypoxia-inducing factor-1 alpha (HIF-1alpha), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1alpha protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1alpha levels. Imexon did not reduce transcription of new HIF-1alpha mRNA, but did reduce the synthesis of new proteins, including HIF-1alpha, measured by (35)S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1alpha protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1alpha translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2alpha (eIF-2alpha) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug's cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1alpha, and this effect overcomes an increase in the stability of preformed HIF-1alpha due to proteasome inhibition by imexon. Because net HIF-1alpha levels are reduced by imexon, combination studies with other drugs affected by HIF-1alpha survival signaling are warranted. PMID:18607542

  3. Mitochondrial encephalomyopathies.


    Lombes, A; Bonilla, E; Dimauro, S


    Increasingly numerous studies are being devoted to mitochondrial diseases, notably those which involve the neuromuscular system. Our knowledge and understanding of these diseases is progressing rapidly. We owe to Luft et al. (1962) the first description of this type of diseases. Their patient, a woman, presented with clinical symptoms suggestive of mitochondrial dysfunction, major histological abnormalities of skeletal muscle mitochondria and defective oxidative phosphorylation coupling clearly demonstrated in mitochondria isolated from muscle. This clinical, histological and biochemical triad led to the definition of mitochondrial myopathies. Subsequently, the triad was seldom encountered, and most mitochondrial myopathies were primarily defined by the presence of morphological abnormalities of muscle mitochondria. This review deals with the morphological, clinical, biochemical and genetic aspects of mitochondrial encephalomyopathies. The various morphological abnormalities of mitochondria are described. These are not specific of any particular disease. They may be present in some non-mitochondrial diseases and may be lacking in diseases due to specific defects of mitochondrial enzymes (e.g. carnitine palmityl-transferase or pyruvate dehydrogenase). The clinical classification of mitochondrial encephalomyopathies is discussed. There are two main schools of thought: the "lumpers" do not recognize specific syndromes within the spectrum of mitochondrial "cytopathies", the "splitters" try to identify specific syndromes while recognizing the existence of borderline cases. The following syndromes are described: chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), MERRF syndrome (myoclonic epilepsy with ragged-red fibers), MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes) and Leigh and Alpers syndromes. The biochemical classification comprises five types of abnormalities: defects of transport

  4. Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers.


    Thomas, Rusha; Kim, Myoung H


    Hypoxia inducible factor-1 (HIF-1) is central to most adaptation responses of tumors to hypoxia, and consists of a hypoxia inducible HIF-1alpha or -2alpha subunit, and a constitutively expressed HIF-1beta subunit. Previously, mitochondrial uncouplers, rottlerin and FCCP, were shown to increase the rate of cellular O(2 )consumption. In this study, we determined that mitochondrial uncouplers, rottlerin and FCCP, significantly decreased hypoxic as well as normoxic HIF-1 transcriptional activity which was in part mediated by down-regulation of the oxygen labile HIF-1alpha and HIF-2alpha protein levels in PC-3 and DU-145 prostate cancer cells. Our results also revealed that mitochondrial uncouplers decreased the expression of HIF target genes, VEGF and VEGF receptor-2. Taken together, our results indicate that functional mitochondria are important in HIF-1alpha and HIF-2alpha protein stability and transcriptional activity during normoxia as well as in hypoxia, and that mitochondrial uncouplers may be useful in the inhibition of HIF pathway in tumors. PMID:16924414

  5. Modular organization and development activity of an Arabidopsis thaliana EF-1 alpha gene promoter.


    Curie, C; Axelos, M; Bardet, C; Atanassova, R; Chaubet, N; Lescure, B


    The activity of the Arabidopsis thalana A1 EF-1 alpha gene promoter was analyzed in transgenic Arabidopsis plants. The 5' upstream sequence of the A1 gene and several promoter deletions were fused to the beta-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5' non-coding region (5'IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1 alpha cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5'IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1 alpha upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1 alpha upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-alpha promoter but has no significant effect when fused to an enhancer-less 35S promoter. PMID:8492811

  6. Immunoglobulin G3 and immunoglobulin M isotype plasma levels are influenced by interleukin-1alpha genotype.


    Kilpinen, S; Laine, S; Hulkkonen, J; Hurme, M


    The immunoglobulin (Ig) plasma levels are known to be, at least partially, genetically regulated, but all the genes involved are not known. Interleukin-1 (IL-1) is a potent proinflammatory cytokine able to serve as an adjuvant for immune responses. IL-1alpha gene is polymorphic, and at least one of the polymorphisms has been identified in the 5' regulatory region of the promoter, a biallelic base exchange (C-->T) at position -889. We set out to study whether the IL-1alpha genotype might contribute to the genetic component seen in the steady-state antibody levels of healthy individuals. Four hundred healthy blood donors (218 males and 182 females) were genotyped, and the plasma levels of IgM, IgG as well as IgG subclasses were measured. An association was found between IgG3 plasma levels and the IL-1alpha genotype; the 1.1 homozygotes had increased IgG3 levels compared with the 1.2 heterozygotes (P < 0.001 in males and P = 0.04 in females, Mann-Whitney U-test). A similar significant association was also found between IgM plasma levels and the IL-1alpha genotype in males, but it was no longer present in females; the 1.1 homozygotes had higher IgM levels than the 2.2 homozygotes (P = 0.03, Mann-Whitney U-test). The data suggest that IL-1alpha-mediated signals are critical for IgG3 and IgM responses, which are induced by thymus-independent antigens and are important in activating complement. PMID:12641660

  7. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement.


    Fournier, Henri-Noël; Dupé-Manet, Sandra; Bouvard, Daniel; Lacombe, Marie-Lise; Marie, Christiane; Block, Marc R; Albiges-Rizo, Corinne


    Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha. PMID:11919189

  8. 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:; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio . E-mail:; Schettini, Gennaro


    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.

  9. Mitochondrial loss, dysfunction and altered dynamics in Huntington's disease

    PubMed Central

    Kim, Jinho; Moody, Jennifer P.; Edgerly, Christina K.; Bordiuk, Olivia L.; Cormier, Kerry; Smith, Karen; Beal, M. Flint; Ferrante, Robert J.


    Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD. PMID:20660112

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

    SciTech Connect

    Oommen, Deepu; Prise, Kevin M.


    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.

  11. Mitochondrial Myopathies


    ... line and are therefore called the electron transport chain, and complex V actually churns out ATP, so ... coQ10 , is a component of the electron transport chain, which uses oxygen to manufacture ATP. Some mitochondrial ...

  12. Mitochondrial Diseases


    ... in your body tissues. If you have a metabolic disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are small structures that produce energy in ...

  13. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.


    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)

  14. Mitochondrial Myopathy


    ... with ragged-red fibers, and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. The symptoms of ... riboflavin, coenzyme Q, and carnitine (a specialized amino acid) may provide subjective improvement in fatigue and energy ...

  15. Mitochondrial genetics

    PubMed Central

    Chinnery, Patrick Francis; Hudson, Gavin


    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 ( In addition, this review makes use of a growing number of publically available databases including MITOMAP, a human mitochondrial genome database (, the Human DNA polymerase Gamma Mutation Database ( and (, 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

  16. Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

    PubMed Central

    Maier, J A; Statuto, M; Ragnotti, G


    We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function. Images PMID:8114717

  17. Hypoxia-inducible factor-1alpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis.


    Bozova, Sevgi; Elpek, Gülsüm Ozlem


    Angiogenesis progresses together with fibrogenesis during chronic liver injury. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of homeostasis, plays a pivotal role in hypoxia-induced angiogenesis through its regulation of vascular endothelial growth factor (VEGF). The association between hypoxia, angiogenesis and VEGF expression has been demonstrated in experimental cirrhosis. However, expression of HIF-1alpha has yet to be reported. The aim of this study was to investigate the significance of HIF-1alpha expression during experimental liver fibrosis and the relationships between HIF-1alpha expression, VEGF expression and angiogenesis. Cirrhosis was induced in male Wistar rats by intraperitoneal administration of diethyl nitrosamine (DEN) (100 mg/kg, once a week). The serial sections from liver tissues were stained with anti-HIF-1alpha, anti-VEGF and anti-CD34 antibodies before being measured by light microscopy. Our results showed that HIF-1alpha expression gradually increases according to the severity of fibrosis (p<0.01). Moreover, its expression was found to be correlated with angiogenesis (r=0.916) and VEGF expression (r=0.969). The present study demonstrates that HIF-1alpha might have a role in the development of angiogenesis via regulation of VEGF during experimental liver fibrogenesis and suggests that this factor could be a potential target in the manipulation of angiogenesis in chronic inflammatory diseases of the liver. PMID:17614845

  18. The effects of 1alpha,24(S)-dihydroxyvitamin D(2) analog on cancer cell proliferation and cytokine expression.


    Shany, S; Levy, Y; Lahav-Cohen, M


    It is well established that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1alpha,25(OH)(2)D(3) in regulating the immune system. However, therapeutic application of 1alpha,25(OH)(2)D(3) to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1alpha,25(OH)(2)D(3) have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1alpha,24(S)-dihydroxyvitamin D(2) (1alpha,24(S)(OH)(2)D(2)), a vitamin D(2) analog, in restraining cell proliferation was compared to that of 1alpha,25(OH)(2)D(3). In parallel studies, cancer cell lines were grown in increased concentrations (10(-10)-10(-7) M) of each compound for various incubation periods (1-4 days). Growth was assessed by measuring [(3)H]thymidine incorporation. The results revealed that 1alpha,24(S)(OH)(2)D(2) significantly inhibits proliferation to an extent similar to that observed for 1alpha,25(OH)(2)D(3). Moreover, incubating the human leukemia cell line, HL-60, with 1alpha,24(S)(OH)(2)D(2) resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1alpha,25(OH)(2)D(3). Using a Western procedure, it was also shown that 1alpha,24(S)(OH)(2)D(2) like 1alpha,25(OH)(2)D(3) enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-alpha) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be

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

    SciTech Connect

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


    {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.

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


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


    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. PMID:19915160

  1. Potentiation of mitomycin C and porfiromycin antitumor activity in solid tumor models by recombinant human interleukin 1 alpha.


    Braunschweiger, P G; Jones, S A; Johnson, C S; Furmanski, P


    The time- and dose-dependent effects of recombinant human interleukin 1 alpha (IL-1 alpha) on the antitumor activity of mitomycin C (MMC) and porfiromycin (PORF) were studied in RIF-1 and Panc02 solid tumor model systems. IL-1 alpha produced dose-dependent sensitization of clonogenic RIF-1 tumor cells to MMC in vivo. IL-1 alpha chemosensitization was highly schedule dependent, and the most efficacious schedules produced dose-modifying factors of 3.6 and 5.1 for MMC and PORF, respectively. More than additive clonogenic cell kill after IL-1 alpha-chemotherapy combinations reflected increased cellular sensitivity to MMC and PORF. The combinations also produced marked decreases in the yield of viable tumor cells, suggesting that the bioreductive drugs may have also potentiated the microvascular injury and ischemia produced by IL-1 alpha. Dexamethasone inhibited and ketoconazole, an inhibitor of corticosterone biosynthesis, enhanced IL-1 alpha-mediated chemosensitization in these models. IL-1 alpha mediated chemosensitization to MMC, and PORF was also demonstrated by tumor growth inhibition in the RIF-1 model and increased survival of mice in the spontaneously metastasizing Panc02 system. Chemosensitization of bone marrow spleen colony-forming units was not seen. IL-1 alpha (1000 units/ml) had no effect on MMC and PORF cytotoxicity in RIF-1 and PORF cell lines in vitro. The results indicate that the tumor-specific IL-1 alpha-induced pathophysiologies can sensitize solid tumors to agents which are preferentially activated, retained, and cytotoxic to cells under hypoxic conditions. Our results suggest that strategies combining bioreductively activated hypoxic cell cytotoxins and biological agents might offer efficacious alternatives or adjuvants to conventional combination approaches. PMID:1913664

  2. Study on CXCR4/SDF-1alpha axis in lymph node metastasis of cervical squamous cell carcinoma.


    Zhang, J-P; Lu, W-G; Ye, F; Chen, H-Z; Zhou, C-Y; Xie, X


    CXCR4/stromal-cell-derived factor-1alpha (SDF-1alpha) is involved in many cancer metastatic mechanisms. Cervical squamous cell cancer (SCC) tissues (n=35), normal cervical tissues (n=10), metastatic (n=10) and nonmetastatic lymph nodes (n=50), and Hela cells were stained immunohistochemically with CXCR4 monoclonal antibody (mAb). Meanwhile, lymph nodes were stained immunohistochemically with rabbit anti-SDF-1alpha. In vitro invasion of Hela cells was evaluated using Transwell Permeable Supports (Corning, NY), in which Hela cells with/without CXCR4 mAb preincubation were seeded in the upper chambers and medium containing 0-100 ng/mL SDF-1alpha was added to the lower compartments. For evaluating the effect of CXCR4/SDF-1alpha on proliferation of cervical cancer cells, Hela cells were cultured for 72 h exposed to SDF-1alpha with and without CXCR4 mAb. We found that CXCR4 was expressed on SCC cells in all cervical cancer, metastatic lymph node, and Hela cells but not in normal cervix. SDF-1alpha was expressed on lymph cells in all lymph nodes. SDF-1alpha induced the directed migration of Hela cells with a concentration-dependent model, which was inhibited by CXCR4 mAb (P<0.05). SDF-1alpha also stimulated the proliferation of Hela cells mediated by CXCR4 (P<0.05). CXCR4/SDF-1alpha axis probably participates in the metastasis toward lymph nodes in cervical cancer. PMID:17362322

  3. 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.


    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.

  4. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha increased apoptosis of human endometrial cancer HEC-1A cells

    PubMed Central

    Yang, Hui; Yang, Rui; Liu, Hao; Ren, Zhongqian; Wang, Cuicui; Li, Da; Ma, Xiaoxin


    Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) coactivates multiple transcription factors and regulates several metabolic processes. In this study, we focused on the roles of PGC-1α in the apoptosis of endometrial cancer HEC-1A cells. Materials and methods PGC-1a expression in the HEC-1A cells was detected with real-time polymerase chain reaction and Western blot. Small interfering RNA directed against PGC-1α was designed and synthesized, and RNA interference technology was used to knock down PGC-1α mRNA and protein expression. Cell apoptosis, cell cycle, and mitochondrial membrane potential were then analyzed using flow cytometry. The expression of apoptotic proteins, Bcl-2 and Bax, was detected with Western blot. Results The specific downregulation of PGC-1α expression in the HEC-1A cells increased their apoptosis through the mitochondrial apoptotic pathway by reducing the expression of Bcl-2 and increasing the expression of Bax. Conclusion These results suggest that PGC-1α influences the apoptosis of HEC-1A cells and also provides a molecular basis for further investigation of the apoptotic mechanism in human endometrial cancer. PMID:27601924

  5. Enhanced type 1alpha metabotropic glutamate receptor-stimulated phosphoinositide signaling after pertussis toxin treatment.


    Carruthers, A M; Challiss, R A; Mistry, R; Saunders, R; Thomsen, C; Nahorski, S R


    The regulation of phosphoinositide hydrolysis by the type 1alpha metabotropic glutamate receptor (mGluR1alpha) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S, 3R-dicarboxylic acid resulted in a marked accumulation of [3H]inositol monophosphate (InsP1) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1alpha cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [3H]InsP1 and a 2-fold increase in Ins(1,4,5)P3 in the absence of added agonist. Although only very low levels (/=75%, and the EC50 shifted leftward by 65-fold [-log EC50 values (molar), 7.26 +/- 0.23 versus 5.45 +/- 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [3H]InsP1 responses were similar in control and PTX-treated BHK-mGluR1alpha cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1alpha-mediated PIC activity that could be fundamental in

  6. Mitochondrial Evolution

    PubMed Central

    Gray, Michael W.


    Viewed through the lens of the genome it contains, the mitochondrion is of unquestioned bacterial ancestry, originating from within the bacterial phylum α-Proteobacteria (Alphaproteobacteria). Accordingly, the endosymbiont hypothesis—the idea that the mitochondrion evolved from a bacterial progenitor via symbiosis within an essentially eukaryotic host cell—has assumed the status of a theory. Yet mitochondrial genome evolution has taken radically different pathways in diverse eukaryotic lineages, and the organelle itself is increasingly viewed as a genetic and functional mosaic, with the bulk of the mitochondrial proteome having an evolutionary origin outside Alphaproteobacteria. New data continue to reshape our views regarding mitochondrial evolution, particularly raising the question of whether the mitochondrion originated after the eukaryotic cell arose, as assumed in the classical endosymbiont hypothesis, or whether this organelle had its beginning at the same time as the cell containing it. PMID:22952398

  7. Expression and regulation of the macrophage inflammatory protein-1 alpha gene by nicotine in rat alveolar macrophages.


    Chong, Inn-Wen; Lin, Shiu-Ru; Hwang, Jhi-Jhu; Huang, Ming-Shyan; Wang, Tung-Heng; Hung, Jen-Yu; Paulauskis, Joseph D


    Cigarette smoking causes inflammation mainly confined to the airway and lung. Nicotine is one of the primary constituents in cigarette smoke. Alveolar macrophages apparently play a pivotal role in mediating pulmonary inflammation via the production of chemokines. Macrophage inflammatory protein-1 alpha (MIP-1 alpha), a member of CC chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1 alpha mRNA expression in macrophages is induced by a variety of stimuli. In the present study, we further investigate whether nicotine can regulate the gene expression of MIP-1 alpha in macrophages and determine the mechanism leading to increased expression. A rat alveolar macrophage (RAM) cell line, NR8383, was treated with nicotine at a dose of 3.1, 31, 310 microM, or 3.1 mM. Northern blot analysis showed that the induction of MIP-1 alpha mRNA expression was dose-dependent. To define the time course of the inflammatory response, RAM cells were exposed to 31 microM nicotine, MIP-1 alpha mRNA was induced as early as 1 h after treatment, was maximally expressed at 4 and 6 hours, and reduced by 8 hours. Western blot analysis demonstrated a single band with an estimated molecular weight of 10 kD for MIP-1 alpha which was induced after nicotine treatment, suggesting that expression of MIP-1 alpha mRNA could reflect in protein synthesis. In addition. the increase in MIP-1 alpha mRNA expression induced by nicotine was attenuated by co-treatment with the antioxidant N-acetylcysteine (NAC), at doses of 10 and 20 mM, suggesting that the induction of MIP-1 alpha mRNA is mediated via the generation of reactive oxygen species (ROS). To further investigate transcriptional regulation of the MIP-1 alpha gene expression, RAM cells were exposed to nicotine. MIP-1 alpha mRNA levels were significantly increased in nuclear RNA preparations, indicating that transcriptional activation is involved in increased

  8. 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


    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

  9. The ternary complex factor Net/Elk-3 participates in the transcriptional response to hypoxia and regulates HIF-1 alpha.


    Gross, C; Dubois-Pot, H; Wasylyk, B


    The ternary complex factor Net/Elk3 is downregulated in hypoxia and participates in the induction by hypoxia of several genes, including c-fos, vascular endothelial growth factor and egr-1. However, the global role of Net in hypoxia remains to be elucidated. We have identified, in a large-scale analysis of RNA expression using microarrays, more than 370 genes that are regulated by Net in hypoxia. In order to gain insights into the role of Net in hypoxia, we have analysed in parallel the genes regulated by HIF-1alpha, the classical factor involved in the response to hypoxia. We identified about 190 genes that are regulated by HIF-1alpha in hypoxia. Surprisingly, when we compare the genes induced by hypoxia that require either Net or HIF-1alpha, the majority are the same (75%), suggesting that the functions of both factors are closely linked. Interestingly, in hypoxia, Net regulates the expression of several genes known to control HIF-1alpha stability, including PHD2, PHD3 and Siah2, suggesting that Net regulates the stability of HIF-1alpha. We found that inhibition of Net by RNAi leads to decreased HIF-1alpha expression at the protein level in hypoxia. These results indicate that Net participates in the transcriptional response to hypoxia by regulation of HIF-1alpha protein stability. PMID:17704799

  10. 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:


    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.

  11. Novel ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) and 2-methyl-20-epi-1alpha,25-dihydroxyvitamin D(3): transactivation of target genes and modulation of differentiation in human promyelocytic leukemia (HL-60) cells.


    Nakagawa, K; Kurobe, M; Ozono, K; Konno, K; Fujishima, T; Takayama, H; Okano, T


    We evaluated the biological activity of two sets of ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) (2-methyl-1alpha,25(OH)(2)D(3)) and 2-methyl-20-epi-1alpha, 25-dihydroxyvitamin D(3) (2-methyl-20-epi-1alpha,25(OH)(2)D(3)) in terms of the following: transactivation of a rat 25-hydroxyvitamin D(3)-24-hydroxylase gene promoter including two vitamin D response elements (VDREs) and a human osteocalcin gene promoter including a VDRE in transfected human osteosarcoma (MG-63) cells; a vitamin D receptor (VDR)-mediated response using a VDR-GAL4 one-hybrid luciferase reporter system and a retinoid X receptor alpha (RXRalpha)-mediated response using an expressed VDR/RXRalpha-GAL4 modified two-hybrid luciferase reporter system in transfected human epitheloid carcinoma, cervix (HeLa) cells; and modulation of cell surface CD11b antigen expression in human leukemia (HL-60) cells. All the diastereomers of both analogues exhibited unique biological activity profiles depending upon the configurations of the C-1 and C-3 hydroxyl groups, the C-2 methyl group in ring A, and the C-20 methyl group in the side chain. Of the eight possible diastereomers of the 2-methyl analogues, 2alpha-methyl-1alpha,25(OH)(2)D(3) was the most potent and exhibited comparable or even greater biological potency than 1alpha,25(OH)(2)D(3). Of the eight possible diastereomers of the 2-methyl-20-epi analogues, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3) was the most potent and exhibited 100- to 200-fold higher transcriptional potencies than 1alpha,25(OH)(2)D(3) and exceptionally high cell regulatory activities. 2beta-methyl-20-epi-1alpha,25(OH)(2)D(3) was nearly as potent as its 2-epimer, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3), whereas its 20-epimer, 2beta-methyl-1alpha,25(OH)(2)D(3), was almost completely biologically inactive. In these respects, it can be postulated that the double modification of 2-methyl substitution and 20-epimerization to 1alpha,25(OH)(2)D(3) induces remarkable changes

  12. Aβ25-35 Suppresses Mitochondrial Biogenesis in Primary Hippocampal Neurons.


    Dong, Weiguo; Wang, Feng; Guo, Wanqing; Zheng, Xuehua; Chen, Yue; Zhang, Wenguang; Shi, Hong


    Mitochondrial biogenesis is involved in the regulation of mitochondrial content, morphology, and function. Impaired mitochondrial biogenesis has been observed in Alzheimer's disease. Amyloid-β (Aβ) has been shown to cause mitochondrial dysfunction in cultured neurons, but its role in mitochondrial biogenesis in neurons remains poorly defined. AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) are key energy-sensing molecules regulating mitochondrial biogenesis. In addition, peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis, is a target for SIRT1 deacetylase activity. In this study, we investigated the effects of Aβ25-35 on mitochondrial biogenesis in cultured hippocampal neurons and the underlying mechanisms. In primary hippocampal neurons, we found that 24-h incubation with Aβ25-35 suppressed both phosphorylations of AMPK and SIRT1 expression and increased PGC-1α acetylation expression. In addition, Aβ25-35 also resulted in a decrease in mitochondrial DNA copy number, as well as decreases in the expression of mitochondrial biogenesis factors (PGC-1α, NRF 1, NRF 2, and Tfam). Taken together, these data show that Aβ25-35 suppresses mitochondrial biogenesis in hippocampal neurons. Aβ25-35-induced impairment of mitochondrial biogenesis may be associated with the inhibition of the AMPK-SIRT1-PGC-1α pathway. PMID:26055049

  13. [Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].


    Anatskaia, O V; Sidorenko, N V; Matveev, I V; Kropotov, A V; Vinogradov, A E


    Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization. PMID:23074852

  14. Isoflurane attenuates LPS-induced acute lung injury by targeting miR-155-HIF1-alpha.


    Hu, Rong; Zhang, Ying; Yang, Xiaohua; Yan, Jia; Sun, Yu; Chen, Zhifeng; Jiang, Hong


    Isoflurane alleviates the inflammatory response in endotoxin-induced acute lung injury (ALI). In this study, we investigated the protective mechanism of isoflurane postconditioning in lipopolysaccharide (LPS)induced ALI. Exposure to isoflurane decreased miR-155 and upregulated HIF-1 alpha and HO-1 mRNA and protein. The effects of isoflurane on HIF-1 alpha mRNA and protein could be inhibited by overexpression of miR-155. Furthermore, mice overexpressing miR-155 had higher levels of TNF-alpha and IL-1 beta in BALF when exposed to isoflurane after LPS challenge.Conversely, downregulation of miR-155 promoted isoflurane effects on HIF-1 alpha expression. These results suggest that isoflurane posttreatment hr alleviates LPS-induced ALI and cell injury by triggering miR-155-HIF-1 alpha pathway, leading to upregulation of HO-1. PMID:25553444

  15. Retroviral interleukin 1alpha gene transfer in bone marrow stromal cells in a primate model: induction of myelopoiesis stimulation.


    de Revel, Thierry; Becard, Nicolas; Sorg, Tania; Rousseau, Sandrine; Spano, Jean Philippe; Thiebot, Hugues; Methali, Magid; Gras, Gabriel; Le Grand, Roger; Dormont, Dominique


    Effects of interleukin 1-alpha (IL-1alpha), a proinflammatory cytokine with pleiotropic activity, in the myelopoietic setting, is mainly linked to its ability to increase haematopoietic growth factor production by bone marrow stromal cells. In order to minimize systemic effects of IL-1alpha therapy, we proposed a model of retroviral IL-1alpha gene transfer within bone marrow stromal cells in the macaque cynomolgus. Invitro, 10-15% of bone marrow stromal cells was effectively transduced by retroviral vector (murine Moloney leukaemia virus-derived) expressing IL-1alpha/LacZ, or LacZ alone as control marker, as assessed by betaGal staining. IL-1alpha gene expression was upregulated [semiquantitative reverse transcription polymerase chain reaction (RT-PCR)] within the transduced cells and the cell supernatant showed an increased production of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF (enzyme-linked immunosorbent assay) and an increased clonogenic activity (colony-forming cell assay). Ex vivo autologous expanded IL-1alpha/LacZ transduced bone marrow stromal cells were reinfused in two macaques (and two control animals for LacZ alone as controls), without clinical systemic toxicity; LacZ expression by RT-PCR was detected in one animal of each group between d 4 and 9. A slight increase of the peripheral blood leucocyte counts (both polymorphonuclear cells and monocytes) of the two animals transduced with IL-1alpha/LacZ was observed within 10 d, indicating stimulation of myelopoiesis. PMID:12181061

  16. 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.


    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.

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

    SciTech Connect

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


    The authors 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. 32 refs., 8 figs.

  18. United Mitochondrial Disease Foundation


    ... Caregivers! Want to help? Enroll now in the Mitochondrial Disease Community Registry to advance the development of treatments and cures. HOME What is Mitochondrial Disease Types of Mitochondrial Disease Possible Symptoms Getting a ...

  19. What Is Mitochondrial DNA?


    ... 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 ...

  20. Cyclic mechanical stretching and interleukin-1alpha synergistically up-regulate prostacyclin secretion in cultured human uterine myometrial cells.


    Korita, D; Itoh, H; Sagawa, N; Yura, S; Yoshida, M; Kakui, K; Takemura, M; Nuamah, M A; Fujii, S


    Prostacyclin (PGI2), a potent uterine smooth muscle relaxant, is postulated to be a major prostaglandin (PG) secreted from the human myometrium. PGI2 metabolite concentrations in the maternal plasma were reported to be elevated during pregnancy, especially during labor. Recently, we developed cultured human myometrial cells from pregnant women and reported that cyclic mechanical stretching mimicking labor increased PGI2 secretion from these cells by up-regulating PGI2 synthase promoter activities. Since elevation of cervical/vaginal interleukin-1alpha (IL-1alpha) concentrations is also a characteristic feature of delivery, and IL-1alpha is a known stimulator of PG synthesis, we investigated a possible synergistic effect of cyclic mechanical stretching and IL-1alpha on PGI2 production in cultured human myometrial cells. Treatment with IL-1alpha (10 ng/ml) significantly augmented (4- to 60-fold) the secretion of PGI2, prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha) and thromboxane A2 (TXA2) from cultured human myometrial cells obtained from non-pregnant and pregnant women as well as in cultured human umbilical artery and cultured human coronary artery smooth muscle cells (p < 0.05 for all comparisons). However, labor-like cyclic mechanical stretching up-regulated IL-1alpha-augmented PGI2 secretion from myometrial cells obtained from non-pregnant and pregnant women 2.1- to 2.8-fold (p < 0.05 for all comparisons), but not PGE2, PGF2alpha nor TXA2. Moreover, such an augumentation of PGI2 secretion by cyclic mechanical stretching was not observed in cultured human umbilical artery nor in cultured human coronary artery smooth muscle cells. These results suggest that cyclic mechanical stretching by labor, in concert with IL-1alpha stimulation, contributes to the increase in myometrial PGI2 secretion during delivery. PMID:15255281

  1. An investigation of the toxicity of 1alpha-hydroxycholecalciferol to calves.


    Mullen, P A; Bedford, P G; Ingram, P L


    Two calves were treated with 15 micrograms/kg body weight of 1alpha-hydroxycholecalciferol by intramuscular injection on four occasions at seven-day intervals. Anorexia and reduced water consumption persisted for 48 h after each treatment. No clinical signs of iridocyclitis or any other lesions of the eyes were present at any time either macroscopically or microscopically. After the first treatment serum GOT and GD activities increased, serum AP activity fell, serum concentrations of calcium and inorganic phosphate increased, and magnesium concentrations decreased. The reduced serum magnesium concentrations and increased calcium and inorganic phosphate concentrations were maintained for the duration of the experiment, but there was no evidence of a cumulative effect of successive treatments. Blood urea concentrations increased after the third treatment. The gross pathology at post mortem examination was similar to that reported after vitamin D3 supplementation. PMID:542713

  2. Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin.

    PubMed Central

    Hub, E.; Rot, A.


    Based on their ability to induce leukocyte chemotaxis and adhesion to endothelial cells (ECs), chemokines have been implicated in driving inflammatory leukocyte emigration. Recently, it was suggested that chemokines can accomplish their pro-emigratory role more effectively while being bound to the luminal surface of the ECs. Previously, such binding was demonstrated in situ in human skin for the prototype alpha-chemokine interleukin (IL)-8. Here we used an in situ binding assay to investigate the binding characteristics of several beta-chemokines in intact human skin. RANTES, MCP-1, and MCP-3 bound, similar to IL-8, in a specific saturable manner to the ECs of venules and small veins but not arteries or capillaries. RANTES inhibited MCP-1 and MCP-3 binding and vice versa, indicating that the EC binding sites are shared among these beta-chemokines; moreover, IL-8 and RANTES cross-competed for each other's binding, suggesting that the same chemokine binding sites are used by members of alpha- and beta-chemokine subfamilies. Conversely, MIP-1alpha did not bind to the ECs and did not compete for binding of RANTES. Analogous to IL-8, all of the tested beta-chemokines bound to the resident dermal cells. As a novel aspect of chemokine interaction with cells in normal skin, we observed specific, saturable binding of RANTES, MCP-1, and MCP-3 but not MIP-1alpha or IL-8 to the ECs of dermal afferent lymphatic vessels. RANTES, MCP-1, and MCP-3 also cross-competed for each other's binding to lymphatics, suggesting a common binding site with a novel chemokine binding profile. We suggest that the chemokine binding to the ECs of lymphatics may be involved in the process of leukocyte entry into the afferent lymphatic vessels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9502417

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. 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.


    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

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


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


    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. PMID:20236219

  6. In vitro biological activities of a series of 2 beta-substituted analogues of 1 alpha,25-dihydroxyvitamin D3.


    Tsugawa, N; Nakagawa, K; Kurobe, M; Ono, Y; Kubodera, N; Ozono, K; Okano, T


    Biological activities of a series of 2beta-substituted analogues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene regulation of the hydroxyalkyl and hydroxyalkoxy 2beta-substituted analogues were almost comparable to those of 1alpha,25(OH)2D3, while the alkyl and alkenyl analogues were much less active than 1alpha,25(OH)2D3. This study investigated the biological evaluation of a series of 2beta-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2beta-position of 1alpha,25(OH)2D3. PMID:10706413

  7. Intravenous human interleukin-1alpha impairs memory processing in mice: dependence on blood-brain barrier transport into posterior division of the septum.


    Banks, W A; Farr, S A; La Scola, M E; Morley, J E


    Peripherally administered cytokines profoundly affect the central nervous system (CNS). One mechanism by which they could affect the CNS is by crossing the blood-brain barrier (BBB) to interact directly with brain receptors. Human and murine IL-1alpha (hIL-1alpha; mIL-1alpha) are transported across the murine BBB with a high rate of transport into the posterior division of the septum (PDS), but it is unknown whether BBB transport is relevant to their actions. Here, we injected species-specific blocking antibodies into the PDS to determine whether transport across the BBB is required for blood-borne hIL-1alpha to affect memory. Retention was impaired in a dose-dependent manner when hIL-1alpha was injected either by tail vein (i.v.) or into the PDS, with the PDS route being 1000 times more potent. About 70% of the memory impairment induced by i.v. hIL-1alpha was reversed by injecting a blocking antibody (Ab) specific for hIL-1alpha into the PDS. This shows that much of the memory impairment induced by hIL-1alpha depends on its ability to cross the BBB. Ab specific for mIL-1alpha was also effective in reversing memory impairment, showing that hIL-1alpha releases mIL-1alpha from endogenous stores. Whether the mIL-1alpha was released from peripheral stores, which would require it to cross the BBB, or from brain stores is unknown. In conclusion, these results show that exogenous, blood-borne hIL-1alpha affects memory by releasing mIL-1alpha from endogenous stores and by crossing the BBB to act at sites within the PDS. PMID:11602664

  8. N-acetylcysteine inhibits the upregulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    PubMed Central

    Caro, Andres A.; Bell, Matthew; Ejiofor, Shannon; Zurcher, Grant; Petersen, Dennis R.; Ronis, Martin J. J.


    Background Chronic ethanol administration to experimental animals induces hepatic oxidative stress and upregulates mitochondrial biogenesis. The mechanisms by which chronic ethanol upregulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic ethanol feeding. If our hypothesis is correct, co-administration of antioxidants should prevent upregulation of mitochondrial biogenesis genes. Methods Rats were fed an ethanol-containing diet intragastrically by total enteral nutrition for 150 days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7 g/kg/day; control rats were administered isocaloric diets where carbohydrates substituted for ethanol calories. Results Ethanol administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and GSH/GSSG ratio. These effects were inhibited by co-administration of ethanol and NAC. Chronic ethanol increased the expression of mitochondrial biogenesis genes including peroxisome proliferator activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of ethanol and NAC prevented these effects. Chronic ethanol administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC. Conclusions These results suggest that oxidative stress caused by chronic ethanol triggered the upregulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by

  9. 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


    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.

  10. Sequential sampling and analysis of renal hydroxylase activities of cattle given 1 alpha-hydroxyvitamin D3.


    Littledike, E T; Engstrom, G W; Sachs, M


    A new method was developed for sequential sampling of bovine renal cortex. This method results in minimum hemorrhage and adhesions and provides sufficient renal cortex tissue for assay of 25-hydroxyvitamin D 1 alpha-, 24-, and 23-hydroxylase activities. Application of this procedure in calves and pregnant cows treated with 1 alpha-hydroxyvitamin D3 is described. The success of these experiments suggests these techniques could be used to follow enzyme activities that control crucial aspects of vitamin D metabolism in normal peripartum cows and cows with milk fever or other diseases of mineral metabolism. PMID:3722540

  11. Mitochondrial Dynamics and Mitochondrial Dysfunction in Diabetes.


    Wada, Jun; Nakatsuka, Atsuko


    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. PMID:27339203

  12. HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells

    PubMed Central

    Kudová, Jana; Procházková, Jiřina; Vašiček, Ondřej; Perečko, Tomáš; Sedláčková, Miroslava; Pešl, Martin; Pacherník, Jiří


    Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro. PMID:27355368

  13. Roles of adrenomedullin and hypoxia-inducible factor 1 alpha in patients with varicocele.


    Hu, W; Zhou, P-H; Zhang, X-B; Xu, C-G; Wang, W


    This study aimed to assess any changes in the plasma concentrations of adrenomedullin (ADM) and hypoxia-inducible factor 1 alpha (HIF 1a) in patients with varicocele (VC). Plasma concentrations of ADM and HIF 1a were measured in brachial vein (BV) and internal spermatic vein (ISV) of 30 fertile VC subjects and 35 untreated infertile VC patients. The results demonstrated that plasma levels of ADM and HIF 1a were significantly higher in ISV than those in BV in the fertile or infertile group respectively. The values of ADM and HIF 1a in BV or ISV of the infertile group were significantly higher than in BV or ISV of the fertile group respectively. Similar changes in values of reactive oxygen metabolites (ROM) were observed. Plasma HIF 1a concentration positively correlated with ROM levels. Plasma ADM concentration positively correlated with ROM values and HIF 1a levels in the two groups. Moreover, remarkable improvement in clinical sperm parameters was observed 3 months after surgery for the infertile patients. It is concluded that ADM may participate, along with HIF 1a, in mechanisms that aid spermatogenic cells in adapting to hypoxia. These predictors may have potential in infertility development in VC patients. Furthermore, early surgical repair is extremely important for infertile VC patients with poor semen quality. PMID:25335788

  14. 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.


    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

  15. Hypoxia-inducible factor-1alpha: A promising therapeutic target in endometriosis.


    Zhan, Lei; Wang, Wenyan; Zhang, Yu; Song, Enxue; Fan, Yijun; Wei, Bing


    Endometriosis is a common gynecologic disease defined as the presence of ectopic endometrial tissues on the ovaries and pelvic peritoneum, and it is a significant cause of pelvic pain, dysmenorrhea and infertility of women in their reproductive age. However, the etiology of endometriosis remains obscure. In recent years, a growing body of evidence validated that hypoxia developed a close relationship with endometriosis and the expression of hypoxia-inducible factor-1alpha (HIF-1α) was increased significantly in the development of endometriosis. Furthermore, inhibiting the expression of HIF-1α contributed to suppress endometriosis progression, suggesting HIF-1α plays a critical function in endometriosis. Nevertheless, the mechanisms by which HIF-1α associates with endometriosis are still undefined. In this brief review, we had a general understanding of HIF-1α firstly, and then we tried to sum up the collective knowledge of HIF-1α in endometriosis. Finally, we will discuss kinds of novel therapeutic approaches to endometriosis based on the functions of HIF-1α. PMID:26898675

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


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


    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. PMID:22645760

  17. The existence of 25-hydroxyvitamin D sub 3 -1. alpha. -hydroxylase in the liver of carp and bastard halibut

    SciTech Connect

    Takeuchi, Atsuko; Okano, Toshio; Kobayashi, Tadashi )


    We have found that carp and bastard halibut contain 25-hydroxyvitamin D{sub 3}(25-D{sub 3})-1{alpha}-hydroxylase in the liver besides in the kidney by the following in vivo and in vitro experiments. When ({sup 3}H)-25-D{sub 3} was intraperitoneally injected to vitamin D(D)-deficient carp and normal bastard halibut, the profiles of high-performance liquid chromatography (HPLC) of the plasma lipid extract showed the formation of a peak corresponding to ({sup 3}H)-1{alpha},25-dihydroxyvitamin D{sub 3}(1,25-D{sub 3}). When ({sup 3}H)25-D{sub 3} was incubated with liver homogenates of the fish, a peak corresponding to ({sup 3}H)-1,25-D{sub 3} was also observed in the profile of HPLC. The formation of the metabolite was confirmed by the thermal isomerization into the pre-isomer and mass fragmentography. Although the 1{alpha}-hydroxylase was also observed in the kidney, the activity of the enzyme was lower than that in the liver. The results suggest that 25-D{sub 3}-1{alpha}-hydroxylase exists in the liver of carp and bastard halibut and the 25-D{sub 3} formed from D{sub 3} in the liver is immediately metabolized into 1,25-D{sub 3} in the same tissue.

  18. Sphingosine kinase 1: a new modulator of hypoxia inducible factor 1alpha during hypoxia in human cancer cells.


    Ader, Isabelle; Brizuela, Leyre; Bouquerel, Pierre; Malavaud, Bernard; Cuvillier, Olivier


    Here, we provide the first evidence that sphingosine kinase 1 (SphK1), an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids, modulates the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1-dependent stabilization of HIF-1alpha levels is mediated by the Akt/glycogen synthase kinase-3beta signaling pathway that prevents its von Hippel-Lindau protein-mediated degradation by the proteasome. The pharmacologic and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1alpha and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung), suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia, giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences. PMID:18922940

  19. Whole-body in-vivo neutron activation analysis in assessing treatment of renal osteodystrophy with 1-alpha-hydroxycholecalciferol.


    Naik, R B; Gosling, P; Price, C P; Robinson, B H; Dabek, J T; Heath, D A; James, H M; Kanis, J A; Smith, R


    Four selected adults with different patterns of osteodystrophy receiving regular dialysis were treated with 1-alpha-hydroxycholecalciferol (1-alpha-OHD3) 0-5-2 mug/day for 10 to 12 months. In two patients, one with osteitis fibrosa and the other with osteomalacia, significant biochemical, radiological, and histological improvements occurred, and total body calcium measured by in-vivo neutron activation analysis increased. In two patients, in whom there were no increases of whole-body calcium, neither biochemical improvement nor healing of bone lesions occurred during the study; in one of these patients the effect of 1-alpha-OHD3 on bone resorption may have contributed to loss of body calcium and deterioration of bone disease. 1-alpha-OHD3 may therefore be a valuable adjunct in the treatment of only some patients with renal osteodystrophy. Whole-body in-vivo neutron activation seems to provide a sensitive and non-invasive index of early response to treatment. PMID:1276820

  20. Hypoxia-induced compensatory effect as related to Shh and HIF-1alpha in ischemia embryo rat heart.


    Hwang, Jin-Ming; Weng, Yi-Jiun; Lin, James A; Bau, Da-Tian; Ko, Fu-Yang; Tsai, Fuu-Jen; Tsai, Chang-Hai; Wu, Chieh-Hsi; Lin, Pei-Cheng; Huang, Chih-Yang; Kuo, Wei-Wen


    Chronic cardiac ischemia/hypoxia induces coronary collateral formation and cardiomyocyte proliferation. Hypoxia can induce cellular adaptive responses, such as synthesis of VEGF for angiogenesis and IGF-2 for proliferation. Both reduce apoptotic effects to minimize injury or damage. To investigate the mechanism of neoangiogenesis and proliferation of fetal heart under umbilical cord compression situation, we used H9c2 cardiomyoblast cell culture, and in vivo embryonic hearts as our study models. Results showed hypoxia induced not only the increase of IGF-2 and VEGF expression but also the activation of their upstream regulatory genes, HIF-1alpha and Shh. The relationship between HIF-1alpha and Shh was further studied by using cyclopamine and 2-ME2, inhibitor of Shh and HIF-1alpha signaling, respectively, in the cardiomyoblast cell culture under hypoxia. We found that the two inhibitors not only blocked their own signal pathway, but also inhibited each other. The observations revealed when fetal heart under hypoxia that HIF-1alpha and Shh pathways maybe involve in cell proliferation and neoangiogenesis to minimize injury or damage, whereas the complex cross-talk between the two pathways remains unknown. PMID:18228117

  1. Hepatocyte nuclear factor-1alpha is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo.


    Bosse, Tjalling; van Wering, Herbert M; Gielen, Marieke; Dowling, Lauren N; Fialkovich, John J; Piaseckyj, Christina M; Gonzalez, Frank J; Akiyama, Taro E; Montgomery, Robert K; Grand, Richard J; Krasinski, Stephen D


    Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling. PMID:16223943

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

    SciTech Connect

    Dehne, Nathalie; Bruene, Bernhard


    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.

  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


    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. HIF-1alpha and HIF-2alpha Are Differentially Activated in Distinct Cell Populations in Retinal Ischaemia

    PubMed Central

    Mowat, Freya M.; Luhmann, Ulrich F. O.; Smith, Alexander J.; Lange, Clemens; Duran, Yanai; Harten, Sarah; Shukla, Deepa; Maxwell, Patrick H.; Ali, Robin R.; Bainbridge, James W. B.


    Background Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. Methodology/Principal Findings We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. Conclusions/Significance Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in

  5. Kaempferol is an estrogen-related receptor alpha and gamma inverse agonist.


    Wang, Junjian; Fang, Fang; Huang, Zhiyan; Wang, Yanfei; Wong, Chiwai


    Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRalpha and ERRgamma). We demonstrated that kaempferol binds to ERRalpha and ERRgamma and blocks their interaction with coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors. PMID:19171140

  6. Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

    PubMed Central

    Gardner, Lauren H.; Mathews, Juanita; Yamazaki, Yuki; Allsopp, Richard C.


    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. PMID:27148974

  7. Altered Hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies

    PubMed Central

    Wikenheiser, Jamie; Wolfram, Julie A.; Gargesha, Madhusudhana; Yang, Ke; Karunamuni, Ganga; Wilson, David L.; Semenza, Gregg L.; Agani, Faton; Fisher, Steven A.; Ward, Nicole; Watanabe, Michiko


    The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1α), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a “template” of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1α protein expression and its downstream target genes without high mortality. We also altered HIF-1α gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1α (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression. PMID:19777592

  8. Hypoxia-inducible factor 1 alpha in high-risk breast cancer: an independent prognostic parameter?

    PubMed Central

    Gruber, Günther; Greiner, Richard H; Hlushchuk, Ruslan; Aebersold, Daniel M; Altermatt, Hans J; Berclaz, Gilles; Djonov, Valentin


    Background Hypoxia-inducible factor 1 alpha (hif-1α) furnishes tumor cells with the means of adapting to stress parameters like tumor hypoxia and promotes critical steps in tumor progression and aggressiveness. We investigated the role of hif-1α expression in patients with node-positive breast cancer. Methods Tumor samples from 77 patients were available for immunohistochemistry. The impact of hif-1α immunoreactivity on survival endpoints was determined by univariate and multivariate analyses, and correlations to clinicopathological characteristics were determined by cross-tabulations. Results hif-1α was expressed in 56% (n = 43/77) of the patients. Its expression correlated with progesterone receptor negativity (P = 0.002). The Kaplan–Meier curves revealed significantly shorter distant metastasis-free survival (DMFS) (P = 0.04, log-rank) and disease-free survival (DFS) (P = 0.04, log-rank) in patients with increased hif-1α expression. The difference in overall survival (OS) did not attain statistical significance (5-year OS, 66% without hif-1α expression and 55% with hif-1α expression; P = 0.21). The multivariate analysis failed to reveal an independent prognostic value for hif-1α expression in the whole patient group. The only significant parameter for all endpoints was the T stage (T3/T4 versus T1/T2: DMFS, relative risk = 3.16, P = 0.01; DFS, relative risk = 2.57, P = 0.03; OS, relative risk = 3.03, P = 0.03). Restricting the univariate and multivariate analyses to T1/T2 tumors, hif-1α expression was a significant parameter for DFS and DMFS. Conclusions hif-1α is expressed in the majority of patients with node-positive breast cancer. It can serve as a prognostic marker for an unfavorable outcome in those with T1/T2 tumors and positive axillary lymph nodes. PMID:15084243

  9. 1alpha,25-dihydroxyvitamin D3 rapidly inhibits fibroblast-induced collagen gel contraction.


    Greiling, D; Thieroff-Ekerdt, R


    1alpha,25-Dihydroxyvitamin D3 (1,25-D3) inhibits the proliferation of fibroblasts in vitro in monolayer culture. We investigated the effect of 1,25-D3 on normal murine and human fibroblasts cultured in collagen type I gels, which more closely resembles the in vivo situation in the dermis. In this culture system 1,25-D3 had no effect on fibroblast proliferation; however, the fibroblast-induced collagen gel contraction was inhibited in a time- and concentration-dependent manner in the nanomolar concentration range. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were inactive. 1,25-D3 had no effect in fibroblasts lacking a functional vitamin D receptor. Pretreatment of fibroblasts in monolayer culture for 5 min was sufficient to trigger the inhibition of collagen gel contraction. Nifedipine increased collagen gel contraction and counteracted the effect of 1,25-D3. The inhibition of collagen gel contraction by 1,25-D3 is supposed to be mediated by the vitamin D receptor because a functional vitamin D receptor is required, and vitamin D metabolites with low affinity to the vitamin D receptor were inactive. Brief pretreatment of fibroblasts was sufficient to trigger the inhibitory effect of 1,25-D3, suggesting a nongenomic effect. A genomic mode of action could not be ruled out, however, because the inhibition was first measured after 24 h. The antagonism of the calcium channel antagonist nifedipine probably represents the sum of two opposite effects rather than supporting evidence for a nongenomic mode of action of 1,25-D3. In conclusion, 1,25-D3 has a specific and rapidly triggered inhibitory effect on fibroblast-induced collagen gel contraction. PMID:8752663

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

    PubMed Central

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


    AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The PI3K/AKT pathway and HIF-1α are both involved in the process of glycolysis in esophageal cancer cells. PMID:27239113

  11. Nuclear respiratory factor 1 controls myocyte enhancer factor 2A transcription to provide a mechanism for coordinate expression of respiratory chain subunits.


    Ramachandran, Bindu; Yu, Gengsheng; Gulick, Tod


    Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. We used genome comparisons to conclude that the promoter regions of COX6A(H) and COX7A(H) lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. We show that MEF2A mRNA is induced with forced expression of NRF1 and that the MEF2A 5'-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, we used an isoform-specific antibody in ChIP to confirm MEF2A binding to the COX6A(H) promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 --> MEF2A --> COX(H) transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1alpha in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. Our findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4. PMID:18222924

  12. Mitochondrial disease and epilepsy.


    Rahman, Shamima


    Mitochondrial respiratory chain disorders are relatively common inborn errors of energy metabolism, with a combined prevalence of one in 5000. These disorders typically affect tissues with high energy requirements, and cerebral involvement occurs frequently in childhood, often manifesting in seizures. Mitochondrial diseases are genetically heterogeneous; to date, mutations have been reported in all 37 mitochondrially encoded genes and more than 80 nuclear genes. The major genetic causes of mitochondrial epilepsy are mitochondrial DNA mutations (including those typically associated with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [MELAS] and myoclonic epilepsy with ragged red fibres [MERRF] syndromes); mutations in POLG (classically associated with Alpers syndrome but also presenting as the mitochondrial recessive ataxia syndrome [MIRAS], spinocerebellar ataxia with epilepsy [SCAE], and myoclonus, epilepsy, myopathy, sensory ataxia [MEMSA] syndromes in older individuals) and other disorders of mitochondrial DNA maintenance; complex I deficiency; disorders of coenzyme Q(10) biosynthesis; and disorders of mitochondrial translation such as RARS2 mutations. It is not clear why some genetic defects, but not others, are particularly associated with seizures. Epilepsy may be the presenting feature of mitochondrial disease but is often part of a multisystem clinical presentation. Mitochondrial epilepsy may be very difficult to manage, and is often a poor prognostic feature. At present there are no curative treatments for mitochondrial disease. Individuals with mitochondrial epilepsy are frequently prescribed multiple anticonvulsants, and the role of vitamins and other nutritional supplements and the ketogenic diet remain unproven. PMID:22283595

  13. The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

    SciTech Connect

    Serasinghe, Madhavika N.; Yoon, Yisang


    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.

  14. Hypoxia-inducible factor-1 {alpha} expression predicts superior survival in patients with diffuse large B-cell lymphoma treated with R-CHOP.


    Evens, Andrew M; Sehn, Laurie H; Farinha, Pedro; Nelson, Beverly P; Raji, Adekunle; Lu, Yi; Brakman, Adam; Parimi, Vamsi; Winter, Jane N; Schumacker, Paul T; Gascoyne, Randy D; Gordon, Leo I


    PURPOSE Hypoxia-inducible factor (HIF) controls the expression of genes in response to hypoxia, as well as a wide range of other cellular processes. We previously showed constitutive stabilization of HIF-1alpha in the majority of patients with diffuse large B-cell lymphoma (DLBCL). To our knowledge, the prognostic significance of HIF in lymphoma has never been investigated. PATIENTS AND METHODS We studied the immunohistochemical protein expression of HIF-1alpha on tissue microarrays from 153 patients with DLBCL treated in sequential cohorts with cyclophosphamide, doxorubicin, oncovin, and prednisone (CHOP) or rituximab-CHOP (R-CHOP) from 1999 to 2002. Results were correlated with patient outcome. Results Median follow-up for all patients was 80 months. Among all patients, HIF-1alpha was expressed in 62% of germinal center and 59% of non-germinal center patients. With HIF-1alpha analyzed as a dependent variable, there were no survival differences in CHOP-treated patients. In the R-CHOP group, however, HIF-1alpha protein expression correlated with significantly improved progression-free survival (PFS) and overall survival (OS). Five-year PFS for HIF-1alpha-positive patients was 71% v 43% for HIF-1alpha-negative patients (P = .0187), whereas 5-year OS was 75% and 54%, respectively (P = .025). In multivariate analysis with International Prognostic Index criteria, HIF-1alpha remained a significant predictor for PFS (P = .026) and OS (P = .043). Compared with other biomarkers, HIF-1alpha correlated only with BCL6 (P = .004). In terms of gene expression, we found several common gene associations of HIF-1alpha and the stromal-1 signature with genes predominantly involved in regulation of the extracellular matrix (eg, BGN, COL1A2, COL5A1, and PLOD2). CONCLUSION The expression of HIF-1alpha protein is an important independent favorable prognostic factor for survival in patients with DLBCL treated with R-CHOP. PMID:20048181

  15. Nitrate-containing beetroot enhances myocyte metabolism and mitochondrial content

    PubMed Central

    Vaughan, Roger A.; Gannon, Nicholas P.; Carriker, Colin R.


    Beetroot (甜菜 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. PMID:26870674

  16. Nitrate-containing beetroot enhances myocyte metabolism and mitochondrial content.


    Vaughan, Roger A; Gannon, Nicholas P; Carriker, Colin R


    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. PMID:26870674

  17. Steroid signalling in human ovarian surface epithelial cells: the response to interleukin-1alpha determined by microarray analysis.


    Rae, M T; Niven, D; Ross, A; Forster, T; Lathe, R; Critchley, H O D; Ghazal, P; Hillier, S G


    The human ovarian surface epithelium (HOSE) is a common site of gynaecological disease including endometriosis and ovarian cancer, probably due to serial injury-repair events associated with successive ovulations. To comprehend the importance of steroid signalling in the regulation of the HOSE, we used a custom microarray to catalogue the expression of over 250 genes involved in the synthesis and reception of steroid hormones, sterols and retinoids. The array included a subset of non-steroidogenic genes commonly involved in pro-/anti-inflammatory signalling. HOSE cells donated by five patients undergoing surgery for non-malignant gynaecological conditions were cultured for 48 h in the presence and absence of 500 pg/ml interleukin-1alpha (IL-1alpha). Total RNA was reverse-transcribed into biotin-labelled cDNA, which was hybridised to the array and visualised by gold-particle resonance light scattering and charge-coupled device (CCD) camera detection. Results for selected genes were verified by quantitative reverse-transcription PCR. In five out of five cases, untreated HOSE cells expressed genes encoding enzymes required for de novo biosynthesis of cholesterol from acetate and subsequent formation of C21-pregnane and C19-androstane steroids. Consistent with the inability of HOSE cells to synthesise glucocorticoids, oestrogens or 5alpha-reduced androgens de novo, CYP21, CYP19 and 5alpha-reductase were not detected. The only steroidogenic gene significantly up-regulated by IL-1alpha was 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1). Other cytokine-induced genes were IL-6, IL-8, nuclear factor kappaB (NFkappaB) inhibitor alpha, metallothionein-IIA and lysyl oxidase: inflammation-associated genes that respond to glucocorticoids. The only steroidogenic gene significantly suppressed by IL-1alpha was 3betaHSD1. Other genes suppressed by IL-1alpha were aldehyde dehydrogenase (ALDH) 1, ALDH 10, gonadotrophin hormone-releasing hormone receptor, peroxisome

  18. 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


    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

  19. Isolation and characterization of three cassava elongation factor 1 alpha (MeEF1A) promoters.


    Suhandono, Sony; Apriyanto, Ardha; Ihsani, Nisa


    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, MeEF1A5 and MeEF1A6 were successfully isolated [corrected]. 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

  20. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.


    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude


    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  1. HIF-1alpha Expression Profile in Intratumoral and Peritumoral Inflammatory Cells as a Prognostic Marker for Squamous Cell Carcinoma of the Oral Cavity

    PubMed Central

    Mendes, Suzanny Oliveira; dos Santos, Marcelo; Peterle, Gabriela Tonini; Maia, Lucas de Lima; Stur, Elaine; Agostini, Lidiane Pignaton; de Carvalho, Marcos Brasilino; Tajara, Eloiza Helena; Louro, Iúri Drumond; Trivilin, Leonardo Oliveira; da Silva-Conforti, Adriana Madeira Álvares


    The HIF-1 transcriptional complex is responsible for controlling transcription of over 100 genes involved in cell hypoxia response. HIF-1alpha subunit is stabilized in hypoxia conditions, creating the HIF-1 nuclear transcription factor. In inflammatory cells, high HIF-1alpha expression induces lymphocytic immunosuppression, decreasing tumoral antigen recognition, which promotes tumor growth. The present work investigated the relationship between HIF-1alpha expression in lymphocytes populating the intratumoral and peritumoral region of 56 patients with oral cancer. Our data indicates a prognostic value for this expression. High HIF-1alpha expression in peritumoral inflammatory cells is significantly related to worse patient outcome, whereas high expression in the intratumoral lymphoid cells correlates with a better prognosis. A risk profile indicating the chance of disease relapse and death was designed based on HIF-1alpha expression in tumoral inflammatory cells, defining low, intermediate and high risks. This risk profile was able to determine that high HIF-1alpha expression in peritumoral cells correlates with worse prognosis, independently of intratumoral expression. Low HIF-1alpha in tumor margins and high expression in the tumor was considered a low risk profile, showing no cases of disease relapse and disease related death. Intermediate risk was associated with low expression in tumor and tumor margins. Our results suggest that HIF-1alpha expression in tumor and peritumoral inflammatory cells may play an important role as prognostic tumor marker. PMID:24416312

  2. Imatinib resistance associated with BCR-ABL upregulation is dependent on HIF-1alpha-induced metabolic reprograming.


    Zhao, F; Mancuso, A; Bui, T V; Tong, X; Gruber, J J; Swider, C R; Sanchez, P V; Lum, J J; Sayed, N; Melo, J V; Perl, A E; Carroll, M; Tuttle, S W; Thompson, C B


    As chronic myeloid leukemia (CML) progresses from the chronic phase to blast crisis, the levels of BCR-ABL increase. In addition, blast-transformed leukemic cells display enhanced resistance to imatinib in the absence of BCR-ABL-resistance mutations. In this study, we show that when BCR-ABL-transformed cell lines were selected for imatinib resistance in vitro, the cells that grew out displayed a higher BCR-ABL expression comparable to the increase seen in accelerated forms of the disease. This enhanced expression of BCR-ABL was associated with an increased rate of glycolysis but with a decreased rate of proliferation. The higher level of BCR-ABL expression in the selected cells correlated with a nonhypoxic induction of hypoxia-inducible factor-1alpha (HIF-1alpha) that was required for cells to tolerate enhanced BCR-ABL signaling. HIF-1alpha induction resulted in an enhanced rate of glycolysis but with reduced glucose flux through both the tricarboxylic acid cycle and the oxidative arm of the pentose phosphate pathway (PPP). The reduction in oxidative PPP-mediated ribose synthesis was compensated by the HIF-1alpha-dependent activation of the nonoxidative PPP enzyme, transketolase, in imatinib-resistant CML cells. In both primary cultures of cells from patients exhibiting blast transformation and in vivo xenograft tumors, use of oxythiamine, which can inhibit both the pyruvate dehydrogenase complex and transketolase, resulted in enhanced imatinib sensitivity of tumor cells. Together, these results suggest that oxythiamine can enhance imatinib efficacy in patients who present an accelerated form of the disease. PMID:20228846

  3. Studies on the 1alpha, 25-dihydroxycholecalciferol-like activity in a calcinogenic plant. Cestrum diurnum, in the chick.


    Wasserman, R H; Corradino, R A; Krook, L; Hughes, M R; Haussler, M R


    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. PMID:1255265

  4. Expression of 1alpha-HYD and 24-HYD in bovine kidney mediated by vitamin D3 supplementation.


    Rezende, L R; Delgado, E F; Júnior, A R L; Gasparin, G; Jorge, E C; Mourão, G B; Coutinho, L L


    In order to better understand vitamin D3 in cattle metabolism, we quantified 1alpha-HYD and 24-HYD gene expression. In the kidneys of 35 male Nellore cattle, these were divided into a control group and two treatment groups (2 x 10(6) international units of vitamin D3 administered for 2 or 8 consecutive days pre-slaughter). Vitamin D3 supplementation resulted in a significant increase in 1alpha-HYD gene expression; however, significantly increased 24-HYD gene expression was only detected in cattle that had 8 days of supplementation. The finding of upregulation of 24-HYD due to vitamin D supplementation is in line with the expected rise in 24,25-di-hydroxy-vitamin D3 synthesis observed when plasma vitamin D3 concentrations are high, stimulating excretion by the organism. On the other hand, upregulation of 1alpha-HYD was unexpected, since vitamin D3 supplementation has been reported to impact these two genes in opposite directions. We conclude that vitamin D3 metabolism in these animals is more complex than previously reported. PMID:24391007

  5. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    PubMed Central

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran


    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed. PMID:20307565

  6. 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.

  7. Hypoxia inducible factor-1 alpha as a therapeutic target in multiple myeloma

    PubMed Central

    Terragna, Carolina; Martello, Marina; Dico, Angela F.; Solaini, Giancarlo; Baracca, Alessandra; Sgarbi, Gianluca; Pasquinelli, Gianandrea; Valente, Sabrina; Zamagni, Elena; Tacchetti, Paola; Martinelli, Giovanni; Cavo, Michele


    The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment. PMID:24732040

  8. [Mitochondrial disease and mitochondrial DNA depletion syndromes].


    Huang, Chin-Chang; Hsu, Chang-Huang


    Mitochondria is an intracellular double membrane-bound structure and it can provide energy for intracellular metabolism. The metabolism includes Krebs cycle, beta-oxidation and lipid synthesis. The density of mitochondria is different in various tissues dependent upon the demands of oxidative phosphorylation. Mitochondrial diseases can occur by defects either in mitochondrial DNA or nuclear DNA. Human mitochondrial DNA (mtDNA) encoding for 22 tRNAs, 2 rRNAs and 13 mRNAs that are translated in the mitochondria. Mitochondrial genetic diseases are most resulted from defects in the mtDNA which may be point mutations, deletions, or mitochondrial DNA depletion. These patterns of inheritance in mitochondrial diseases include sporadic, maternally inherited, or of Mendelian inheritance. Mitochondrial DNA depletion is caused by defects in the nuclear genes that are responsible for maintenance of integrity of mtDNA or deoxyribonucelotide pools and mtDNA biogenesis. The mtDNA depletion syndrome (MDS) includes the following categories: progressive external ophthalmoplegia (PEO), predominant myopathy, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), sensory-ataxic neuropathy, dysarthria, and ophthalmoplegia (SANDO) and hepato-encephalopathy. The most common tissues or organs involved in MDS and related disorders include the brain, liver and muscles. These involved genes are divided into two groups including 1) DNA polymerase gamma (POLG, POLG2) and Twinkle genes whose products function directly at the mtDNA replication fork, and 2) adenine nucleotide translocator 1, thymidine phosphorylase, thymidine kinase 2, deoxyguanosine kinase, ADP-forming succinyl-CoA synthetase ligase, MPV17 whose products supply the mitochondria with deoxyribonucleotide triphosphate pools needed for mtDNA replication, and possible mutation in the RRM2B gene. The development has provided new information about the importance of the biosynthetic pathway of the nucleotides for mtDNA replication

  9. 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:


    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.

  10. Aerobic Interval Training Attenuates Mitochondrial Dysfunction in Rats Post-Myocardial Infarction: Roles of Mitochondrial Network Dynamics

    PubMed Central

    Jiang, Hong-Ke; Wang, You-Hua; Sun, Lei; He, Xi; Zhao, Mei; Feng, Zhi-Hui; Yu, Xiao-Jiang; Zang, Wei-Jin


    Aerobic interval training (AIT) can favorably affect cardiovascular diseases. However, the effects of AIT on post-myocardial infarction (MI)—associated mitochondrial dysfunctions remain unclear. In this study, we investigated the protective effects of AIT on myocardial mitochondria in post-MI rats by focusing on mitochondrial dynamics (fusion and fission). Mitochondrial respiratory functions (as measured by the respiratory control ratio (RCR) and the ratio of ADP to oxygen consumption (P/O)); complex activities; dynamic proteins (mitofusin (mfn) 1/2, type 1 optic atrophy (OPA1) and dynamin-related protein1 (DRP1)); nuclear peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α); and the oxidative signaling of extracellular signal-regulated kinase (ERK) 1/2, c-Jun NH2-terminal protein kinase (JNK) and P53 were observed. Post-MI rats exhibited mitochondrial dysfunction and adverse mitochondrial network dynamics (reduced fusion and increased fission), which was associated with activated ERK1/2-JNK-P53 signaling and decreased nuclear PGC-1α. After AIT, MI-associated mitochondrial dysfunction was improved (elevated RCR and P/O and enhanced complex I, III and IV activities); in addition, increased fusion (mfn2 and OPA1), decreased fission (DRP1), elevated nuclear PGC-1α and inactivation of the ERK1/2-JNK-P53 signaling were observed. These data demonstrate that AIT may restore the post-MI mitochondrial function by inhibiting dynamics pathological remodeling, which may be associated with inactivation of ERK1/2-JNK-P53 signaling and increase in nuclear PGC-1α expression. PMID:24675698

  11. 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. )


    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.

  12. Suppression of hypoxia-inducible factor-1alpha and its downstream genes reduces acute hyperglycemia-enhanced hemorrhagic transformation in a rat model of cerebral ischemia.


    Chen, Chunhua; Ostrowski, Robert P; Zhou, Changman; Tang, Jiping; Zhang, John H


    We evaluated a role of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream genes in acute hyperglycemia-induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats weighing 280-300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF-1alpha inhibitors, 2-methoxyestradiol (2ME2) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF-1alpha inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF-1alpha was increased in ischemic brain tissues after MCAO and reduced by HIF-1alpha inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase-2 and -9 (MMP-2/MMP-9) in the ipsilateral hemisphere. Both 2ME2 and YC-1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF-1alpha and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia. PMID:20155812

  13. 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


    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.

  14. 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


    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.

  15. Mitochondrial phospholipids: role in mitochondrial function.


    Mejia, Edgard M; Hatch, Grant M


    Mitochondria are essential components of eukaryotic cells and are involved in a diverse set of cellular processes that include ATP production, cellular signalling, apoptosis and cell growth. These organelles are thought to have originated from a symbiotic relationship between prokaryotic cells in an effort to provide a bioenergetic jump and thus, the greater complexity observed in eukaryotes (Lane and Martin 2010). Mitochondrial processes are required not only for the maintenance of cellular homeostasis, but also allow cell to cell and tissue to tissue communication (Nunnari and Suomalainen 2012). Mitochondrial phospholipids are important components of this system. Phospholipids make up the characteristic outer and inner membranes that give mitochondria their shape. In addition, these membranes house sterols, sphingolipids and a wide variety of proteins. It is the phospholipids that also give rise to other characteristic mitochondrial structures such as cristae (formed from the invaginations of the inner mitochondrial membrane), the matrix (area within cristae) and the intermembrane space (IMS) which separates the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). Phospholipids are the building blocks that make up these structures. However, the phospholipid composition of the OMM and IMM is unique in each membrane. Mitochondria are able to synthesize some of the phospholipids it requires, but the majority of cellular lipid biosynthesis takes place in the endoplasmic reticulum (ER) in conjunction with the Golgi apparatus (Fagone and Jackowski 2009). In this review, we will focus on the role that mitochondrial phospholipids play in specific cellular functions and discuss their biosynthesis, metabolism and transport as well as the differences between the OMM and IMM phospholipid composition. Finally, we will focus on the human diseases that result from disturbances to mitochondrial phospholipids and the current research being performed to help

  16. Mitochondrial DNA, mitochondrial dysfunction, and cardiac manifestations.


    Lee, Sung Ryul; Kim, Nari; Noh, Yeonhee; Xu, Zhelong; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin


    Mitochondria, the powerhouses of cells, have their own DNA (mtDNA). They regulate the transport of metabolites and ions, which determine cell physiology, survival, and death. Mitochondrial dysfunction, including impaired oxidative phosphorylation, preferentially affects heart function via imbalance of energy supply and demand. Recently, mitochondrial mutations and associated mitochondrial dysfunction were suggested as a causal factor of cardiac manifestations. Oxidative stress largely influences mtDNA stability due to oxidative modifications of mtDNA. Furthermore, the continuous replicative state of mtDNA and presence of minimal nucleoid structure render mitochondria vulnerable to oxidative damage and subsequent mutations, which impair mitochondrial functions. However, the occurrence of mtDNA heteroplasmy in the same mitochondrion or cell and presence of nuclear DNA-encoded mtDNA repair systems raise questions regarding whether oxidative stress-mediated mtDNA mutations are the major driving force in accumulation of mtDNA mutations. Here, we address the possible causes of mitochondrial DNA mutations and their involvement in cardiac manifestations. Current strategies for treatment related to mitochondrial mutations and/or dysfunction in cardiac manifestations are briefly discussed. PMID:27100514

  17. 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.


    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.

  18. 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.


    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

  19. Noscapine inhibits hypoxia-mediated HIF-1alpha expression andangiogenesis in vitro: a novel function for an old drug.


    Newcomb, Elizabeth W; Lukyanov, Yevgeniy; Schnee, Tona; Ali, M Aktar; Lan, Li; Zagzag, David


    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

  20. Modulation of the bovine innate immune response by production of 1alpha,25-dihydroxyvitamin D(3) in bovine monocytes.


    Nelson, C D; Reinhardt, T A; Thacker, T C; Beitz, D C; Lippolis, J D


    In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] from 25-hydroxyvitamin D(3) [25(OH)D(3)] by 1alpha-hydroxylase (1alpha-OHase). Based on human studies, it was hypothesized that bovine monocytes could produce 1,25(OH)(2)D(3) upon activation and 1,25(OH)(2)D(3) would regulate expression of vitamin D-responsive genes in monocytes. First, the effects of 1,25(OH)(2)D(3) on bovine monocytes isolated from peripheral blood were tested. Treatment of nonstimulated monocytes with 1,25(OH)(2)D(3) increased expression of the gene for the vitamin D 24-hydroxylase (24-OHase) enzyme by 51+/-13 fold, but 1,25(OH)(2)D(3) induction of 24-OHase expression was blocked by lipopolysaccharide (LPS) stimulation. In addition, 1,25(OH)(2)D(3) increased the gene expression of inducible nitric oxide synthase and the chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) in LPS-stimulated monocytes 69+/-13 and 40+/-12 fold, respectively. Next, the ability of bovine monocytes to express 1alpha-OHase and produce 1,25(OH)(2)D(3) was tested. Activation of monocytes with LPS, tripalmitoylated lipopeptide (Pam3CSK4), or peptidoglycan caused 43+/-9, 17+/-3, and 19+/-3 fold increases in 1alpha-OHase gene expression, respectively. Addition of 25(OH)D(3) to LPS-stimulated monocytes enhanced expression of inducible nitric oxide synthase and RANTES and nitric oxide production in a dose-dependent manner, giving evidence that activated monocytes convert 25(OH)D(3) to 1,25(OH)(2)D(3). In conclusion, bovine monocytes produce 1,25(OH)(2)D(3) in response to toll-like receptor signaling, and 1,25(OH)(2)D(3) production in monocytes increased the expression of genes involved in the innate immune system. Vitamin D status of cattle might be important for optimal innate immune function because 1,25(OH)(2)D(3) production in activated monocytes and subsequent upregulation of inducible nitric oxide synthase and RANTES expression

  1. Mitochondrial dysfunction during sepsis.


    Azevedo, Luciano Cesar Pontes


    Sepsis and multiple organ failure remain leading causes of death in intensive care patients. Recent advances in our understanding of the pathophysiology of these syndromes include a likely prominent role for mitochondria. Patient studies have shown that the degree of mitochondrial dysfunction is related to the eventual outcome. Associated mechanisms include damage to mitochondria or inhibition of the electron transport chain enzymes by nitric oxide and other reactive oxygen species (the effects of which are amplified by co-existing tissue hypoxia), hormonal influences that decrease mitochondrial activity, and downregulation of mitochondrial protein expression. Notably, despite these findings, there is minimal cell death seen in most affected organs, and these organs generally regain reasonably normal function should the patient survive. It is thus plausible that multiple organ failure following sepsis may actually represent an adaptive state whereby the organs temporarily 'shut down' their normal metabolic functions in order to protect themselves from an overwhelming and prolonged insult. A decrease in energy supply due to mitochondrial inhibition or injury may trigger this hibernation/estivation-like state. Likewise, organ recovery may depend on restoration of normal mitochondrial respiration. Data from animal studies show histological recovery of mitochondria after a septic insult that precedes clinical improvement. Stimulation of mitochondrial biogenesis could offer a new therapeutic approach for patients in multi-organ failure. This review will cover basic aspects of mitochondrial function, mechanisms of mitochondrial dysfunction in sepsis, and approaches to prevent, mitigate or speed recovery from mitochondrial injury. PMID:20509844

  2. Mitochondrial threshold effects.

    PubMed Central

    Rossignol, Rodrigue; Faustin, Benjamin; Rocher, Christophe; Malgat, Monique; Mazat, Jean-Pierre; Letellier, Thierry


    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

  3. MYC and Mitochondrial Biogenesis

    PubMed Central

    Morrish, Fionnuala; Hockenbery, David


    Mitochondria, the powerhouses of the cell, face two imperatives concerning biogenesis. The first is the requirement for dividing cells to replicate their mitochondrial content by growth of existing mitochondria. The second is the dynamic regulation of mitochondrial content in response to organismal and cellular cues (e.g., exercise, caloric restriction, energy status, temperature). MYC provides the clearest example of a programmed expansion of mitochondrial content linked to the cell cycle. As an oncogene, MYC also presents intriguing questions about the role of its mitochondrial targets in cancer-related phenotypes, such as the Warburg effect and MYC-dependent apoptosis. PMID:24789872

  4. Mitochondrial and Metabolic Gene Expression in the Aged Rat Heart.


    Barton, Gregory P; Sepe, Joseph J; McKiernan, Susan H; Aiken, Judd M; Diffee, Gary M


    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

  5. 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.


    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

  6. Treatment of hypophosphataemic vitamin D-resistant rickets with massive doses of 1 alpha-hydroxy-vitamin D3 during childhood.

    PubMed Central

    Seino, Y; Shimotsuji, T; Ishii, T; Ishida, M; Ikehara, C; Yamaoka, K; Yabuuchi, H; Dokoh, S


    Plasma levels of 1,25 dihydroxy-vitamin D (1,25-(OH)2-D) were low in 3 children with hypophosphataemic vitamin D-resistant rickets (HVDRR) during childhood, but increased after very large doses (0.5 to 2 micrograms/kg per day) of 1 alpha-hydroxy-vitamin D (1 alpha-OH-D3). This treatment has two advantages. Firstly, hypercalcaemia is easily controlled by reducing the dose of 1 alpha-OH-D3 because of its short half-life. Secondly, the administration of 1 alpha-OH-D3 to patients with HVDRR can enhance the tubular reabsorption of phosphate, and this seems desirable in treating HVDRR. PMID:6246841

  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


    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. Correlation of Hypoxia-Inducible Factor 1{alpha} with Angiogenesis in Liver Tumors After Transcatheter Arterial Embolization in an Animal Model

    SciTech Connect

    Liang Bin; Zheng Chuansheng Feng, Gan-Sheng; Wu Hanping; Wang Yong; Zhao Hui; Qian Jun; Liang Huimin


    This study sought to determine the expression of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) and its relation to angiogenesis in liver tumors after transcatheter arterial embolization (TAE) in an animal model. A total of 20 New Zealand White rabbits were implanted with VX2 tumor in liver. TAE-treated group animals (n = 10) received TAE with polyvinyl alcohol particles. Control group animals (n = 10) received sham embolization with distilled water. Six hours or 3 days after TAE, animals were humanely killed, and tumor samples were collected. Immunohistochemical staining was performed to evaluate HIF-1{alpha} and vascular endothelial growth factor (VEGF) protein expression and microvessel density (MVD). Real-time polymerase chain reaction was performed to examine VEGF mRNA levels. The levels of HIF-1{alpha} protein were significantly higher in TAE-treated tumors than those in the control tumors (P = 0.001). HIF-1{alpha} protein was expressed in viable tumor cells that were located predominantly at the periphery of necrotic tumor regions. The levels of VEGF protein and mRNA, and mean MVD were significantly increased in TAE-treated tumors compared with the control tumors (P = 0.001, 0.000, and 0.001, respectively). HIF-1{alpha} protein level was significantly correlated with VEGF mRNA (r = 0.612, P = 0.004) and protein (r = 0.554, P = 0.011), and MVD (r = 0.683, P = 0.001). We conclude that HIF-1{alpha} is overexpressed in VX2 tumors treated with TAE as a result of intratumoral hypoxia generated by the procedure and involved in activation of the TAE-associated tumor angiogenesis. HIF-1{alpha} might represent a promising therapeutic target for antiangiogenesis in combination with TAE against liver tumors.

  9. Stem cell homing and angiomyogenesis in transplanted hearts are enhanced by combined intramyocardial SDF-1alpha delivery and endogenous cytokine signaling.


    Zhao, Tiemin; Zhang, Dongsheng; Millard, Ronald W; Ashraf, Muhammad; Wang, Yigang


    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. PMID:19181961

  10. Two splice variants of the hypoxia-inducible factor HIF-1alpha as potential dimerization partners of ARNT2 in neurons.


    Drutel, G; Kathmann, M; Héron, A; Gros, C; Macé, S; Schwartz, J C; Arrang, J M


    The hypoxia-inducible factor (HIF-1alpha), a basic helix-loop-helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF-1alpha and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF-1alpha is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single-minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF-1alpha in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF-1alpha/ARNT1 complex, does not recognize the HIF-1-binding site of the hypoxia-induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF-1alpha and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF-1alpha, ARNT1 and ARNT2, downregulation of either HIF-1alpha or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation. PMID:11029639

  11. The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression.


    Axelos, M; Bardet, C; Liboz, T; Le Van Thai, A; Curie, C; Lescure, B


    The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes. PMID:2615757

  12. Expressions of endothelin-1, fibronectin, and interleukin-1alpha of human umbilical vein endothelial cells under prolonged culture.


    Kiyonaga, H; Doi, Y; Karasaki, Y; Arashidani, K; Itoh, H; Fujimoto, S


    We examined human umbilical vein endothelial cells (HUVECs) under prolonged culture by electron microscopy and by light and electron immunocytochemistry including double immunolabeling. Based on the cell area of HUVECs through multiple passages, we divided the cells into first, second, and third stages, which exhibited distinct morphological and immunocytochemical characteristics. During the first stage, HUVECs were polygonal in shape and had already formed the monolayer confluence. During the second stage, they were characterized by an increased number of Weibel-Palade (WP) bodies, which were actively segregated from Golgi cisterns. Endothelin (ET)-1 and von Willebrand factor, an endothelial cell marker, were occasionally colocalized in WP bodies. The increase in WP bodies correlated with high ET-1 concentration in the cultured medium, suggesting that these inclusions are involved in storage and release of ET-1 in a manner indicating a regulatory pathway. During the third stage, fibronectin and interleukin (IL)-1alpha were expressed in HUVECs as well as in multinucleated giant cells, which originated from HUVECs, but WP bodies decreased in number in association with a decrease in ET-1 immunoreactivity and concentration. The foregoing changes in immunoreactivities for ET-1, fibronectin, and IL-1alpha affecting HUVECs under prolonged culture may reflect a senescent process of these cells. PMID:11479772

  13. 1 alpha,25-dihydroxyvitamin D3 analogs featuring aromatic and heteroaromatic rings: design, synthesis, and preliminary biological testing.


    Posner, G H; Li, Z; White, M C; Vinader, V; Takeuchi, K; Guggino, S E; Dolan, P; Kensler, T W


    Aromatic compounds 2a-c, analogs of 1 alpha, 25-dihydroxyvitamin (calcitriol, 1), and heteroaromatic compounds 4a-c and 5a-c, analogs of 19-nor-1 alpha, 25-dihydroxyvitamin D3 (3), were designed to simulate the topology of their biologically potent parent compounds while avoiding previtamin D equilibrium. Convergent and facile total syntheses of the analogs (+)-2b, (+)-2c, (-)-4b, and (-)-5b were achieved via carbonyl addition of regiospecifically formed organolithium nucleophiles to the enantiomerically pure C,D-ring ketone (+)-17, characteristic of natural calcitriol (1). Likewise, hybrid analogs 20a-c were prepared to determine whether incorporation of a known potentiating side chain would lead to increased biological activity. Preliminary in vitro biological testing showed that aromatic analogs (+)-2b, (+)-2c, and 20a-c as well as heteroaromatic analogs (-)-4b and (-)-5b have very low affinities for the calf thymus vitamin D receptor but considerable antiproliferative activities in murine keratinocytes at micromolar concentration. No biological advantage was observed in this keratinocyte assay for the doubly modified hybrid analogs 20a-c over the singly modified parent (+)-2b. Analog (+)-2b, but surprisingly not the corresponding analog 20b differing from (+)-2b only in the side chain, showed considerable activity in nongenomic opening of calcium channels in rat osteosarcoma cells. PMID:7473581

  14. Fish oil diet modulates epididymal and inguinal adipocyte metabolism in mice.


    Bargut, Thereza Cristina Lonzetti; Souza-Mello, Vanessa; Mandarim-de-Lacerda, Carlos Alberto; Aguila, Marcia Barbosa


    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. PMID:26876019

  15. 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.


    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.

  16. The human mitochondrial transcriptome

    PubMed Central

    Mercer, Tim R.; Neph, Shane; Dinger, Marcel E.; Crawford, Joanna; Smith, Martin A.; Shearwood, Anne-Marie J.; Haugen, Eric; Bracken, Cameron P.; Rackham, Oliver; Stamatoyannopoulos, John A.; Filipovska, Aleksandra; Mattick, John S.


    Summary The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs and rRNAs. Here we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA, and provides a resource for future studies of mitochondrial function (accessed at PMID:21854988

  17. Twin Mitochondrial Sequence Analysis.


    Bouhlal, Yosr; Martinez, Selena; Gong, Henry; Dumas, Kevin; Shieh, Joseph T C


    When applying genome-wide sequencing technologies to disease investigation, it is increasingly important to resolve sequence variation in regions of the genome that may have homologous sequences. The human mitochondrial genome challenges interpretation given the potential for heteroplasmy, somatic variation, and homologous nuclear mitochondrial sequences (numts). Identical twins share the same mitochondrial DNA (mtDNA) from early life, but whether the mitochondrial sequence remains similar is unclear. We compared an adult monozygotic twin pair using high throughput-sequencing and evaluated variants with primer extension and mitochondrial pre-enrichment. Thirty-seven variants were shared between the twin individuals, and the variants were verified on the original genomic DNA. These studies support highly identical genetic sequence in this case. Certain low-level variant calls were of high quality and homology to the mitochondrial DNA, and they were further evaluated. When we assessed calls in pre-enriched mitochondrial DNA templates, we found that these may represent numts, which can be differentiated from mtDNA variation. We conclude that twin identity extends to mitochondrial DNA, and it is critical to differentiate between numts and mtDNA in genome sequencing, particularly since significant heteroplasmy could influence genome interpretation. Further studies on mtDNA and numts will aid in understanding how variation occurs and persists. PMID:24040623

  18. Mitochondrial syndromes with leukoencephalopathies.


    Wong, Lee-Jun C


    White matter involvement has recently been recognized as a common feature in patients with multisystem mitochondrial disorders that may be caused by molecular defects in either the mitochondrial genome or the nuclear genes. It was first realized in classical mitochondrial syndromes associated with mitochondrial DNA (mtDNA) mutations, such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leigh's disease, and Kearns-Sayre's syndrome. Deficiencies in respiratory chain complexes I, II, IV, and V often cause Leigh's disease; most of them are due to nuclear defects that may lead to severe early-onset leukoencephalopathies. Defects in a group of nuclear genes involved in the maintenance of mtDNA integrity may also affect the white matter; for example, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) caused by thymidine phosphorylase deficiency, Navajo neurohepatopathy (NNH) due to MPV17 mutations, and Alpers syndrome due to defects in DNA polymerase gamma (POLG). More recently, leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) has been reported to be caused by autosomal recessive mutations in a mitochondrial aspartyl-tRNA synthetase, DARS2 gene. A patient with leukoencephalopathy and neurologic complications in addition to a multisystem involvement warrants a complete evaluation for mitochondrial disorders. A definite diagnosis may be achieved by molecular analysis of candidate genes based on the biochemical, clinical, and imaging results. PMID:22422207

  19. Myoclonus in mitochondrial disorders.


    Mancuso, Michelangelo; Orsucci, Daniele; Angelini, Corrado; Bertini, Enrico; Catteruccia, Michela; Pegoraro, Elena; Carelli, Valerio; Valentino, Maria L; Comi, Giacomo P; Minetti, Carlo; Bruno, Claudio; Moggio, Maurizio; Ienco, Elena Caldarazzo; Mongini, Tiziana; Vercelli, Liliana; Primiano, Guido; Servidei, Serenella; Tonin, Paola; Scarpelli, Mauro; Toscano, Antonio; Musumeci, Olimpia; Moroni, Isabella; Uziel, Graziella; Santorelli, Filippo M; Nesti, Claudia; Filosto, Massimiliano; Lamperti, Costanza; Zeviani, Massimo; Siciliano, Gabriele


    Myoclonus is a possible manifestation of mitochondrial disorders, and its presence is considered, in association with epilepsy and the ragged red fibers, pivotal for the syndromic diagnosis of MERRF (myoclonic epilepsy with ragged red fibers). However, its prevalence in mitochondrial diseases is not known. The aims of this study are the evaluation of the prevalence of myoclonus in a big cohort of mitochondrial patients and the clinical characterization of these subjects. Based on the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases," we reviewed the clinical and molecular data of mitochondrial patients with myoclonus among their clinical features. Myoclonus is a rather uncommon clinical feature of mitochondrial diseases (3.6% of 1,086 patients registered in our database). It is not strictly linked to a specific genotype or phenotype, and only 1 of 3 patients with MERRF harbors the 8344A>G mutation (frequently labeled as "the MERRF mutation"). Finally, myoclonus is not inextricably linked to epilepsy in MERRF patients, but more to cerebellar ataxia. In a myoclonic patient, evidences of mitochondrial dysfunction must be investigated, even though myoclonus is not a common sign of mitochondriopathy. Clinical, histological, and biochemical data may predict the finding of a mitochondrial or nuclear DNA mutation. Finally, this study reinforces the notion that myoclonus is not inextricably linked to epilepsy in MERRF patients, and therefore the term "myoclonic epilepsy" seems inadequate and potentially misleading. PMID:24510442

  20. Mitochondrial Therapeutics for Cardioprotection

    PubMed Central

    Carreira, Raquel S.; Lee, Pamela; Gottlieb, Roberta A.


    Mitochondria represent approximately one-third of the mass of the heart and play a critical role in maintaining cellular function—however, they are also a potent source of free radicals and pro-apoptotic factors. As such, maintaining mitochondrial homeostasis is essential to cell survival. As the dominant source of ATP, continuous quality control is mandatory to ensure their ongoing optimal function. Mitochondrial quality control is accomplished by the dynamic interplay of fusion, fission, autophagy, and mitochondrial biogenesis. This review examines these processes in the heart and considers their role in the context of ischemia-reperfusion injury. Interventions that modulate mitochondrial turnover, including pharmacologic agents, exercise, and caloric restriction are discussed as a means to improve mitochondrial quality control, ameliorate cardiovascular dysfunction, and enhance longevity. PMID:21718247

  1. Negative regulation of mitochondrial transcription by mitochondrial topoisomerase I

    PubMed Central

    Sobek, Stefan; Dalla Rosa, Ilaria; Pommier, Yves; Bornholz, Beatrice; Kalfalah, Faiza; Zhang, Hongliang; Wiesner, Rudolf J.; von Kleist-Retzow, Jürgen-Christoph; Hillebrand, Frank; Schaal, Heiner; Mielke, Christian; Christensen, Morten O.; Boege, Fritz


    Mitochondrial topoisomerase I is a genetically distinct mitochondria-dedicated enzyme with a crucial but so far unknown role in the homeostasis of mitochondrial DNA metabolism. Here, we present data suggesting a negative regulatory function in mitochondrial transcription or transcript stability. Deficiency or depletion of mitochondrial topoisomerase I increased mitochondrial transcripts, whereas overexpression lowered mitochondrial transcripts, depleted respiratory complexes I, III and IV, decreased cell respiration and raised superoxide levels. Acute depletion of mitochondrial topoisomerase I triggered neither a nuclear mito-biogenic stress response nor compensatory topoisomerase IIβ upregulation, suggesting the concomitant increase in mitochondrial transcripts was due to release of a local inhibitory effect. Mitochondrial topoisomerase I was co-immunoprecipitated with mitochondrial RNA polymerase. It selectively accumulated and rapidly exchanged at a subset of nucleoids distinguished by the presence of newly synthesized RNA and/or mitochondrial RNA polymerase. The inactive Y559F-mutant behaved similarly without affecting mitochondrial transcripts. In conclusion, mitochondrial topoisomerase I dampens mitochondrial transcription and thereby alters respiratory capacity. The mechanism involves selective association of the active enzyme with transcriptionally active nucleoids and a direct interaction with mitochondrial RNA polymerase. The inhibitory role of topoisomerase I in mitochondrial transcription is strikingly different from the stimulatory role of topoisomerase I in nuclear transcription. PMID:23982517

  2. Effects of Long-Term Rice Bran Extract Supplementation on Survival, Cognition and Brain Mitochondrial Function in Aged NMRI Mice.


    Hagl, Stephanie; Asseburg, Heike; Heinrich, Martina; Sus, Nadine; Blumrich, Eva-Maria; Dringen, Ralf; Frank, Jan; Eckert, Gunter P


    Aging represents a major risk factor for the development of neurodegenerative diseases like Alzheimer's disease (AD). As mitochondrial dysfunction plays an important role in brain aging and occurs early in the development of AD, the prevention of mitochondrial dysfunction might help to slow brain aging and the development of neurodegenerative diseases. Rice bran extract (RBE) contains high concentrations of vitamin E congeners and γ-oryzanol. We have previously shown that RBE increased mitochondrial function and protected from mitochondrial dysfunction in vitro and in short-term in vivo feeding studies. To mimic the use of RBE as food additive, we have now investigated the effects of a long-term (6 months) feeding of RBE on survival, behavior and brain mitochondrial function in aged NMRI mice. RBE administration significantly increased survival and performance of aged NMRI mice in the passive avoidance and Y-maze test. Brain mitochondrial dysfunction found in aged mice was ameliorated after RBE administration. Furthermore, data from mRNA and protein expression studies revealed an up-regulation of mitochondrial proteins in RBE-fed mice, suggesting an increase in mitochondrial content which is mediated by a peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)-dependent mechanism. Our findings suggest that a long-term treatment with a nutraceutical containing RBE could be useful for slowing down brain aging and thereby delaying or even preventing AD. PMID:27350374

  3. Coordination of metabolic plasticity in skeletal muscle.


    Hood, David A; Irrcher, Isabella; Ljubicic, Vladimir; Joseph, Anna-Maria


    Skeletal muscle is a highly malleable tissue, capable of pronounced metabolic and morphological adaptations in response to contractile activity (i.e. exercise). Each bout of contractile activity results in a coordinated alteration in the expression of a variety of nuclear DNA and mitochondrial DNA (mtDNA) gene products, leading to phenotypic adaptations. This results in an increase in muscle mitochondrial volume and changes in organelle composition, referred to as mitochondrial biogenesis. The functional consequence of this biogenesis is an improved resistance to fatigue. Signals initiated by the exercise bout involve changes in intracellular Ca2+ as well as alterations in energy status (i.e. ATP/ADP ratio) and the consequent activation of downstream kinases such as AMP kinase and Ca2+-calmodulin-activated kinases. These kinases activate transcription factors that bind DNA to affect the transcription of genes, the most evident manifestation of which occurs during the post-exercise recovery period when energy metabolism is directed toward anabolism, rather than contractile activity. An important protein that is affected by exercise is the transcriptional coactivator PGC-1alpha, which cooperates with multiple transcription factors to induce the expression of nuclear genes encoding mitochondrial proteins. Once translated in the cytosol, these mitochondrially destined proteins are imported into the mitochondrial outer membrane, inner membrane or matrix space via specific import machinery transport components. Contractile activity affects the expression of the import machinery, as well as the kinetics of import, thus facilitating the entry of newly synthesized proteins into the expanding organelle. An important set of proteins that are imported are the mtDNA transcription factors, which influence the expression and replication of mtDNA. While mtDNA contributes only 13 proteins to the synthesis of the organelle, these proteins are vital for the proper assembly of multi

  4. Kinetic Stability May Determine the Interaction Dynamics of the Bifunctional Protein DCoH1, the Dimerization Cofactor of the Transcription Factor HNF-1[alpha

    SciTech Connect

    Rho, H.; Jones, C.N.; Rose, R.B.


    The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1{alpha} (hepatocyte nuclear factor-1{alpha}), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1{alpha} complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1{alpha} through simple mixing. To further investigate regulation of DCoH/HNF-1{alpha} complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1{alpha}. The 1.8 {angstrom} resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1{alpha} complex formation are discussed.

  5. IL-1 alpha, but not IL-1 beta, is required for contact-allergen-specific T cell activation during the sensitization phase in contact hypersensitivity.


    Nakae, S; Naruse-Nakajima, C; Sudo, K; Horai, R; Asano, M; Iwakura, Y


    Contact hypersensitivity (CHS) is a T cell-mediated cellular immune response caused by epicutaneous exposure to contact allergens. In this reaction, after the first epicutaneous allergen sensitization, Langerhans cells (LC) catch allergens and migrate from the skin to draining lymph nodes (LN) and activate naive T cells. Although IL-1 is suggested to be involved in these processes, the mechanisms have not been elucidated completely. In this report, to elucidate roles of IL-1alpha and IL-1beta in CHS, we analyzed ear swelling in 2,4,6-trinitrochlorobenzene (TNCB)-induced CHS using gene-targeted mice. We found that ear swelling was suppressed in IL-1alpha-deficient (IL-1alpha(-/-)) mice but not in IL-1beta(-/-) mice. LC migration from the skin into LN was delayed in both IL-1alpha(-/-) and IL-1beta(-/-) mice, suggesting that this defect was not the direct cause for the reduced CHS in these mice. However, we found that the proliferative response of trinitrophenyl (TNP)-specific T cells after sensitization with TNCB was specifically reduced in IL-1alpha(-/-) mice. Furthermore, adoptive transfer of TNP-conjugated IL-1-deficient epidermal cells (EC) into wild-type mice indicated that only IL-1alpha, but not IL-1beta, produced by antigen-presenting cells in EC could prime allergen-specific T cells. These observations indicate that IL-1alpha, but not IL-1beta, plays a crucial role in TNCB-induced CHS by sensitizing TNP-specific T cells. PMID:11717188

  6. 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


    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.

  7. Salidroside Stimulates Mitochondrial Biogenesis and Protects against H2O2-Induced Endothelial Dysfunction

    PubMed Central

    Xing, Shasha; Yang, Xiaoyan; Li, Wenjing; Bian, Fang; Wu, Dan; Chi, Jiangyang; Xu, Gao; Zhang, Yonghui; Jin, Si


    Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙−) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways. PMID:24868319

  8. Identification and biological activities of a new antiangiogenic small molecule that suppresses mitochondrial reactive oxygen species

    SciTech Connect

    Kim, Ki Hyun; Park, Ju Yeol; Jung, Hye Jin; Kwon, Ho Jeong


    Research highlights: {yields} YCG063 was screened as a new angiogenesis inhibitor which suppresses mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library. {yields} The compound inhibited in vitro and in vivo angiogenesis in a dose-dependent manner. {yields} This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions. -- Abstract: Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1{alpha} and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

  9. Treatment of Mitochondrial Disorders

    PubMed Central

    Avula, Sreenivas; Parikh, Sumit; Demarest, Scott; Kurz, Jonathan; Gropman, Andrea


    Opinion statement While numerous treatments for mitochondrial disorders have been suggested, relatively few have undergone controlled clinical trials. Treatment of these disorders is challenging, as only symptomatic therapy is available. In this review we will focus on newer drugs and treatment trials in mitochondrial diseases, with a special focus on medications to avoid in treating epilepsy and ICU patient with mitochondrial disease, which has not been included in such a review. Readers are also referred to the opinion statement in A Modern Approach to the Treatment of Mitochondrial Disease published in Current Treatment Options in Neurology 2009. Many of the supplements used for treatment were reviewed in the previous abstract, and dosing guidelines were provided. The focus of this review is on items not previously covered in depth, and our discussion includes more recently studied compounds as well as any relevant updates on older compounds. We review a variety of vitamins and xenobiotics, including dichloroacetate (DCA), arginine, coenzyme Q10, idebenone, EPI-743, and exercise training. Treatment of epilepsy, which is a common feature in many mitochondrial phenotypes, warrants special consideration due to the added toxicity of certain medications, and we provide a discussion of these unique treatment challenges. Interesting, however, with only a few exceptions, the treatment strategies for epilepsy in mitochondrial cytopathies are the same as for epilepsy without mitochondrial dysfunction. We also discuss intensive care management, building upon similar reviews, adding new dimensions, and demonstrating the complexity of overall care of these patients. PMID:24700433

  10. Sporadic cutaneous angiosarcomas generally lack hypoxia-inducible factor 1alpha: a histologic and immunohistochemical study of 45 cases.


    Abedalthagafi, Malak; Rushing, Elisabeth J; Auerbach, Aaron; Desouki, Mohamed M; Marwaha, Jason; Wang, Zengfeng; Fanburg-Smith, Julie C


    Cutaneous angiosarcoma (AS) is a rare malignant neoplasm of dermis composed of infiltrating cells of endothelial phenotype with overall poor prognosis. Although autocrine stimulation by vascular endothelial growth factor secretion may play a role in the pathogenesis of angiosarcoma, its mechanism has not been fully established. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to hypoxia.. The stability of HIF can regulate key proteins in angiogenesis and the alpha-subunit has been found in epithelial tumors, only 1 case of human retroperitoneal angiosarcoma, and rare vascular proliferations and tumors in knockout mice. We wanted to observe the utility of HIF-1alpha as a marker or explanatory factor in AS. Cases coded as "angiosarcoma" of dermis were culled and re-reviewed for inclusion as AS, based on patient folder, slides, and obtained immunohistochemistry including CD31 and smooth muscle actin (SMA). Hypoxia-inducible factor-1alpha was performed on a subset of cases, with additional available material. Forty-five cases met the criteria for AS; there were 17% females and 83% males, with a mean age at presentation of 67 years (range, 27-88 years). Tumors presented most commonly in the skin of the scalp followed by the left lower leg, face, nose, lower arm, neck, thigh, eyelid, ear, and temple. Associated basal cell carcinoma was noted in 1 patient; no others had other neoplasms or unrelated surgeries. There was no history of other primary, lymphedema, radiation, breast-associated, or thorotrast-induced angiosarcoma. The tumors ranged in size from 0.4 up to 9.5 cm, with a mean size of 2.4 cm. Histopathologically, most tumors were vasoformative, with either solid architecture (n = 35) or papillary endothelial hyperplasia-like foci (n = 7). All cases demonstrated infiltrative growth pattern, cytologic atypia, and mitotic activity, including atypical forms. Surface ulceration was present in 44% and

  11. Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1alpha, interleukin-1beta and tumor necrosis factor alpha.


    Yeşilada, E; Ustün, O; Sezik, E; Takaishi, Y; Ono, Y; Honda, G


    In this study, in vitro inhibitory effects of 55 extracts or fractions obtained from 10 plant species on interleukin-1 (IL-1alpha, IL-1beta) and tumor necrosis factor (TNF-alpha) biosynthesis were studied. The following plant materials from Turkish folk medicine for the treatment of various diseases which are thought to be inflammatory in nature e.g. rheumatism, fever, infections, edemas or related inflammatory diseases were selected as the subject of this study: Cistus laurifolius leaves, Clematis flammna flowering herbs, Crataegus orientalis roots, Daphne oleoides ssp. oleoides whole plant, Ecbalium elaterium roots, Rosa canina roots, Rubus discolor roots, Rubus hirtus roots, Sambucus ebulus flowers and leaves, Sambucus nigra flowers and leaves. All plants showed inhibitory activity against at least one of these models in various percentages depending upon the concentration, thus supporting the folkloric utilization. Daphne oleoides was found to be the most active plant against the test models. PMID:9324006

  12. Acridinium ester labelled cytokines: receptor binding studies with human interleukin-1 alpha, interleukin-1 beta and interferon-gamma.


    Joss, U R; Towbin, H


    As a consequence of environmental protection and legal restrictions, increasing efforts are made to avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6-dimethyl-4-(N-succinimidyloxy-carbonyl)phenyl 10-methylacridinium-9-carboxylate methosulphate (DMAE-NHS). When exposed to hydrogen peroxide in a basic solution, the DMAE-moiety decays with emission of a short-lasting chemiluminescent flash. With the goal of replacing the radioactive label in protein ligands with a DMAE label, and of increasing the efficiency by using microtitre plate technology for DMAE detection, we compared the receptor binding properties of iodinated interleukin-1 alpha (125I-IL-1 alpha), interleukin-1 beta (125I-IL-1 beta) and interferon-gamma (125I-IFN-gamma) with the corresponding DMAE-labelled ligands. The luminescence signal was assessed in a single-tube luminometer and in the prototype of a chemiluminescent microtitre plate reader. Derivatization of the three proteins with DMAE-N-hydroxy-succinimide resulted in photon yields of up to 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss of receptor binding affinity was observed, which might have been expected as a consequence of the chemical modification of the proteins. The use of DMAE labelling of proteins has the following advantages as compared to iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE-labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence of higher specific activity of the DMAE label.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8154300

  13. Mitochondrial diseases and epilepsy.


    Bindoff, Laurence A; Engelsen, Bernt A


    The mitochondrial respiratory chain is the final common pathway for energy production. Defects affecting this pathway can give rise to disease that presents at any age and affects any tissue. However, irrespective of genetic defect, epilepsy is common and there is a significant risk of status epilepticus. This review summarizes our current understanding of the epilepsy that occurs in mitochondrial disease, focusing on three of the most common disorders: mitochondrial myopathy encephalopathy, lactic acidosis and stroke-like episodes (MELAS), myoclonus epilepsy and ragged-red fibers (MERRF), and polymerase gamma (POLG) related disease. In addition, we review the pathogenesis and possible treatment of these disorders. PMID:22946726

  14. The effects of HIF-1alpha on gene expression profiles of NCI-H446 human small cell lung cancer cells

    PubMed Central


    Background Gene targeted therapy refers to any therapy focused on one of the many biological features of the tumor. Such features are mediated by specific genes that are involved in tumor metastasis, recurrence, poor response to chemotherapy and others. Hypoxia is an important pathognomonic feature of many malignant tumors including SCLC (small cell lung cancer). HIF-1alpha, which is induced by hypoxia, is the most important regulatory factor of many specific genes that can influence the biological features of tumors. Methods In this study, we tried to elucidate the changes in gene expression profiles of SCLC NCI-H446 cells mediated by HIF-1alpha. According to different treatments of cells, three experimental pairwise comparisons were designed: hypoxia group vs. control group, Ad5-HIF-1alpha group vs. Ad5 group, and Ad5-siHIF-1 alpha group Vs Ad5 group. Results Results from the analysis of gene expression profiles indicated that there were 65 genes upregulated and 28 genes downregulated more than two-fold in all three experimental pairwise comparisons. These genes were involved in transport, signal-transduction, cell adhesion/motility, growth factor/cytokines, transcription, inflammatory response, metabolic process, in addition to others. SOCS1, IGFBP5, IL-6 and STAT3 were also upregulated at protein level. SOCS1 could significantly induce apoptosis and suppress growth of NCI-H446 cells but HIF-1alpha could induce growth and suppress apoptosis. Conclusions Through this research, we are trying to find novel functional genes that are mediated by HIF-1alpha and provide the theoretical basis for new therapeutic targets. HIF-1 alpha maybe upregulate the expression of SOCS1 through mediation of STAT3 and IL-6. In addition, SOCS1 could significantly induce apoptosis and suppress growth of NCI-H446 cells. This was contrary to HIF-1alpha and it indicated that there might be an antagonism effect between HIF-1alpha and SOCS1 on regulating growth and apoptosis of NCI-H446

  15. Mitochondrial biogenesis: pharmacological approaches.


    Valero, Teresa


    Organelle biogenesis is concomitant to organelle inheritance during cell division. It is necessary that organelles double their size and divide to give rise to two identical daughter cells. Mitochondrial biogenesis occurs by growth and division of pre-existing organelles and is temporally coordinated with cell cycle events [1]. However, mitochondrial biogenesis is not only produced in association with cell division. It can be produced in response to an oxidative stimulus, to an increase in the energy requirements of the cells, to exercise training, to electrical stimulation, to hormones, during development, in certain mitochondrial diseases, etc. [2]. Mitochondrial biogenesis is therefore defined as the process via which cells increase their individual mitochondrial mass [3]. Recent discoveries have raised attention to mitochondrial biogenesis as a potential target to treat diseases which up to date do not have an efficient cure. Mitochondria, as the major ROS producer and the major antioxidant producer exert a crucial role within the cell mediating processes such as apoptosis, detoxification, Ca2+ buffering, etc. This pivotal role makes mitochondria a potential target to treat a great variety of diseases. Mitochondrial biogenesis can be pharmacologically manipulated. This issue tries to cover a number of approaches to treat several diseases through triggering mitochondrial biogenesis. It contains recent discoveries in this novel field, focusing on advanced mitochondrial therapies to chronic and degenerative diseases, mitochondrial diseases, lifespan extension, mitohormesis, intracellular signaling, new pharmacological targets and natural therapies. It contributes to the field by covering and gathering the scarcely reported pharmacological approaches in the novel and promising field of mitochondrial biogenesis. There are several diseases that have a mitochondrial origin such as chronic progressive external ophthalmoplegia (CPEO) and the Kearns- Sayre syndrome (KSS

  16. Abnormal regulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity by calcium and calcitonin in renal cortex from hypophosphatemic (Hyp) mice.


    Fukase, M; Avioli, L V; Birge, S J; Chase, L R


    25-Hydroxyvitamin D3-1 alpha-hydroxylase activity was assayed in primary serum-free monolayer tissue culture of renal cortical cells from hypophosphatemic (Hyp) mice and normal litter mates. Morphological and growth characteristics of cells from the two genotypes were indistinguishable. Basal enzyme activity was not significantly different in either type of cell over a wide range of substrate concentration. The enzyme from both genotypes was stimulated by PTH and suppressed by increased phosphate concentration in the culture medium. Whereas 1 alpha-hydroxylase activity in cells from normal mice was increased in low calcium medium and suppressed in high calcium medium, the enzyme in cells from Hyp mice was not altered by similar changes in the medium calcium concentration. Salmon calcitonin caused a significant increase in 1 alpha-hydroxylase in cells from normal mice, but did not stimulate enzyme activity in cells from Hyp mice. These studies indicate that control of 1 alpha-hydroxylase activity is abnormal in renal cortical cells from Hyp mice. Impaired control of this enzyme could result in the inappropriately low circulating concentrations of 1,25-dihydroxyvitamin D3 that have been observed in humans with hypophosphatemic rickets and in the relatively low activity of 1 alpha-hydroxylase in renal cortical homogenates of Hyp mice compared to that in normal mice on a low phosphate diet. PMID:6705736

  17. [Hydroxysafflor yellow A up-regulates HIF-1alpha via inhibition of VHL and p53 in Eahy 926 cell line exposed to hypoxia].


    Lian, Ze-Qin; Zhao, Da-Long; Zhu, Hai-Bo


    In present study, we investigated the mechanism of regulating HIF-1alpha expression by hydroxysafflor yellow A (HSYA) in Eahy 926 cell line under 1% O2 hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 micromol x L(-1)) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1alpha, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1alpha, VHL and p53 were assayed by Western blotting method. HSYA at 100 micromol x L(-1) increased Eahy 926 cells proliferation rate under hypoxia. HIF-1alpha mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1alpha expression partially via its inhibition of VHL and p53 expression. PMID:18717335

  18. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    SciTech Connect

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong


    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

  19. Inherited mitochondrial neuropathies.


    Finsterer, Josef


    Mitochondrial disorders (MIDs) occasionally manifest as polyneuropathy either as the dominant feature or as one of many other manifestations (inherited mitochondrial neuropathy). MIDs in which polyneuropathy is the dominant feature, include NARP syndrome due to the transition m.8993T>, CMT2A due to MFN2 mutations, CMT2K and CMT4A due to GDAP1 mutations, and axonal/demyelinating neuropathy with external ophthalmoplegia due to POLG1 mutations. MIDs in which polyneuropathy is an inconstant feature among others is the MELAS syndrome, MERRF syndrome, LHON, Mendelian PEO, KSS, Leigh syndrome, MNGIE, SANDO; MIRAS, MEMSA, AHS, MDS (hepato-cerebral form), IOSCA, and ADOA syndrome. In the majority of the cases polyneuropathy presents in a multiplex neuropathy distribution. Nerve conduction studies may reveal either axonal or demyelinated or mixed types of neuropathies. If a hereditary neuropathy is due to mitochondrial dysfunction, the management of these patients is at variance from non-mitochondrial hereditary neuropathies. Patients with mitochondrial hereditary neuropathy need to be carefully investigated for clinical or subclinical involvement of other organs or systems. Supportive treatment with co-factors, antioxidants, alternative energy sources, or lactate lowering agents can be tried. Involvement of other organs may require specific treatment. Mitochondrial neuropathies should be included in the differential diagnosis of hereditary neuropathies. PMID:21402391

  20. Mitochondrial Ryanodine Receptors and Other Mitochondrial Ca2+ Permeable Channels

    PubMed Central

    Ryu, Shin-Young; Beutner, Gisela; Dirksen, Robert T.; Kinnally, Kathleen W.; Sheu, Shey-Shing


    Ca2+ channels that underlie mitochondrial Ca2+ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca2+ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca2+ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca2+ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca2+ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca2+ channels. PMID:20096690

  1. Abnormal parathyroid hormone stimulation of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in the hypophosphatemic mouse. Evidence for a generalized defect of vitamin D metabolism.

    PubMed Central

    Nesbitt, T; Drezner, M K; Lobaugh, B


    Abnormal regulation of vitamin D metabolism is a feature of X-linked hypophosphatemic rickets in man and of the murine homologue of the disease in the hypophosphatemic (Hyp)-mouse. We previously reported that mutant mice have abnormally low renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity for the prevailing degree of hypophosphatemia. To further characterize this defect, we examined whether Hyp-mouse renal 1 alpha-hydroxylase activity responds normally to other stimulatory and inhibitory controls of enzyme function. We studied stimulation by parathyroid hormone (PTH) using: (a) a calcium-deficient (0.02% Ca) diet to raise endogenous PTH; or (b) 24-h continuous infusion of 0.25 IU/h bovine PTH via osmotic minipump. In both cases enzyme activity of identically treated normal mice increased to greater levels than those attained by Hyp-mice. The relative inability of PTH to stimulate 1 alpha-hydroxylase activity is not a function of the hypophosphatemia in the Hyp-mouse since PTH-infused, phosphate-depleted normal mice sustained a level of enzyme activity greater than that of normal and Hyp-mice. In further studies we investigated inhibition of enzyme activity by using: (a) a calcium-loaded (1.2% Ca) diet to suppress endogenous PTH; or (b) 24-h continuous infusion of 0.2 ng/h 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 1 alpha-hydroxylase activity of normal and Hyp-mice was significantly reduced to similar absolute levels following maintenance on the calcium-loaded diet. Further, infusion of 1,25(OH)2D3 caused a comparable reduction of 1 alpha-hydroxylase activity in normal, Hyp-, and phosphate-depleted normal mice. These observations indicate that the inhibitory control of 1 alpha-hydroxylase by reduced levels of PTH or increased 1,25(OH)2D3 concentrations is intact in the mutants. However, the inability of PTH and hypophosphatemia to stimulate enzyme activity in a manner analogous to that in normal and phosphate-depleted mice indicates

  2. Overexpression of Intrinsic Hypoxia Markers HIF1{alpha} and CA-IX Predict for Local Recurrence in Stage T1-T2 Glottic Laryngeal Carcinoma Treated With Radiotherapy

    SciTech Connect

    Schrijvers, M.L.; Laan, B.F.A.M. van der; Bock, G.H. de; Pattje, W.J.; Mastik, M.F.; Menkema, L.; Langendijk, J.A.; Kluin, P.M.; Schuuring, E.; Wal, J.E. van der


    Purpose: To examine the prognostic value of three endogenous hypoxia markers (hypoxia inducible factor 1 {alpha} subunit [HIF1{alpha}], carbonic anhydrase IX [CA-IX], and glucose transporter type 1 [GLUT-1]) on the clinical outcome in patients with early-stage glottic carcinoma primarily treated with radiotherapy (RT) and to determine the predictive hypoxic profile to choose the optimal treatment of early-stage laryngeal carcinoma. Methods and Materials: Immunohistochemistry for HIF1{alpha}, CA-IX, and GLUT-1 was performed on formalin-fixed, paraffin-embedded, pretreatment tissue samples of 91 glottic squamous cell carcinoma specimens. The patient group consisted only of those with early-stage (T1-T2) glottic carcinoma, and all patients were treated with RT only. Relative tumor staining was scored on the tissue samples. Receiver operating curve analysis was performed to determine the optimal cutoff value for each tumor marker. Cox regression analyses for the variables HIF1{alpha}, CA-IX, GLUT-1, gender, age, hemoglobin level, T category, N category, tobacco use, and alcohol use were performed with local control and overall survival as endpoints. Results: HIF1{alpha} overexpression in early-stage glottic carcinoma correlated significantly with worse local control (hazard ratio [HR], 3.05; p = 0.021) and overall survival (HR, 2.92; p = 0.016). CA-IX overexpression correlated significantly with worse local control (HR, 2.93; p = 0.020). GLUT-1 overexpression did not show any correlation with the clinical outcome parameters. Tumors with a nonhypoxic profile (defined as low HIF1{alpha} and low CA-IX expression) had significantly better local control (HR, 6.32; p 0.013). Conclusion: The results of our study have shown that early-stage glottic laryngeal carcinomas with low HIF1{alpha} and CA-IX expression are highly curable with RT. For this group, RT is a good treatment option. For tumors with HIF1{alpha} or CA-IX overexpression, hypoxic modification before RT or primary

  3. Protecting Neural Structures and Cognitive Function During Prolonged Space Flight by Targeting the Brain Derived Neurotrophic Factor Molecular Network

    NASA Technical Reports Server (NTRS)

    Schmidt, M. A.; Goodwin, T. J.


    Brain derived neurotrophic factor (BDNF) is the main activity-dependent neurotrophin in the human nervous system. BDNF is implicated in production of new neurons from dentate gyrus stem cells (hippocampal neurogenesis), synapse formation, sprouting of new axons, growth of new axons, sprouting of new dendrites, and neuron survival. Alterations in the amount or activity of BDNF can produce significant detrimental changes to cortical function and synaptic transmission in the human brain. This can result in glial and neuronal dysfunction, which may contribute to a range of clinical conditions, spanning a number of learning, behavioral, and neurological disorders. There is an extensive body of work surrounding the BDNF molecular network, including BDNF gene polymorphisms, methylated BDNF gene promoters, multiple gene transcripts, varied BDNF functional proteins, and different BDNF receptors (whose activation differentially drive the neuron to neurogenesis or apoptosis). BDNF is also closely linked to mitochondrial biogenesis through PGC-1alpha, which can influence brain and muscle metabolic efficiency. BDNF AS A HUMAN SPACE FLIGHT COUNTERMEASURE TARGET Earth-based studies reveal that BDNF is negatively impacted by many of the conditions encountered in the space environment, including oxidative stress, radiation, psychological stressors, sleep deprivation, and many others. A growing body of work suggests that the BDNF network is responsive to a range of diet, nutrition, exercise, drug, and other types of influences. This section explores the BDNF network in the context of 1) protecting the brain and nervous system in the space environment, 2) optimizing neurobehavioral performance in space, and 3) reducing the residual effects of space flight on the nervous system on return to Earth

  4. How mitochondrial dynamism orchestrates mitophagy

    PubMed Central

    Shirihai, Orian; Song, Moshi; Dorn, Gerald W


    Mitochondria are highly dynamic, except in adult cardiomyocytes. Yet, the fission and fusion-promoting proteins that mediate mitochondrial dynamism are highly expressed in, and essential to the normal functioning of, hearts. Here, we review accumulating evidence supporting important roles for mitochondrial fission and fusion in cardiac mitochondrial quality control, focusing on the PINK1-Parkin mitophagy pathway.Based in part on recent findings from in vivo mouse models in which mitofusin-mediated mitochondrial fusion or Drp1-mediated mitochondrial fission were conditionally interrupted in cardiac myocytes, we propose several new concepts that may provide insight into the cardiac mitochondrial dynamism-mitophagy interactome. PMID:25999423

  5. Molecular Genetics of Mitochondrial Biogenesis in Maize.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mitochondrial genome encodes proteins essential for mitochondrial respiration and ATP synthesis. Nuclear gene products, however, are required for the expression of mitochondrial genes and the elaboration of functional mitochondrial protein complexes. We are exploiting a unique collection of maiz...

  6. The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy

    PubMed Central

    Vincent, Amy E.; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M.; McFarland, Robert; Gorman, Grainne S.; Taylor, Robert W.; Turnbull, Doug M.; Picard, Martin


    Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553

  7. The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.


    Vincent, Amy E; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M; McFarland, Robert; Gorman, Grainne S; Taylor, Robert W; Turnbull, Doug M; Picard, Martin


    Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553

  8. A Polymorphism in Hepatocyte Nuclear Factor 1 Alpha, rs7310409, Is Associated with Left Main Coronary Artery Disease

    PubMed Central

    Liu, Rui; Liu, Hanning; Gu, Haiyong; Teng, Xiao; Nie, Yu; Zhou, Zhou; Zhao, Yan; Hu, Shengshou; Zheng, Zhe


    Coronary artery disease is the leading cause of mortality and morbidity in the world. Left main coronary artery disease (LMCAD) is a particularly severe phenotypic form of CAD and has a genetic basis. We hypothesized that some inflammation- and hyperhomocysteinemia-related gene polymorphisms may contribute to LMCAD susceptibility in a Chinese population. We studied the association between polymorphisms in the genes hepatocyte nuclear factor 1 alpha (HNF1A; rs7310409, G/A), C-reactive protein (rs1800947 and rs3093059 T/C), methylenetetrahydrofolate reductase (rs1801133, C/T), and methylenetetrahydrofolate dehydrogenase (rs1076991, A/G) in 402 LMCAD and 804 more peripheral CAD patients in a Chinese population. Genotyping was performed using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method. When the HNF1A rs7310409 GG homozygote genotype was used as the reference group, both the individual, GA and AA, and combined GA/AA genotypes were associated with an increased risk of LMCAD. This single nucleotide polymorphism (rs7310409) is strongly associated with plasma CRP levels. In conclusion, the present study provides evidence that the HNF1A rs7310409 G/A functional polymorphism may contribute to the risk of LMCAD. PMID:25202455

  9. Tissue-specific metallothionein gene expression in liver and intestine by dexamethasone, interleukin-1. alpha. and elevated zinc status

    SciTech Connect

    Hempe, J.M.; Carlson, J.M.; Cousins, R.J. )


    Intestinal metallothionein has been implicated in the regulation of zinc absorption. Glucocorticoids and cytokines mediate hepatic metallothionein gene expression but the effects of these hormones in the small intestine are unclear. In this experiment, rats were injected ip with dexamethasone (DEX), recombinant human interleukin-1{alpha} (ILK-1), or ZnSO{sub 4}. Data collected 0. 3, 6,9, or 12 hour post-injection showed tissue specific regulation of metallothionein gene expression. Liver metallothionein mRNA (determined by hybridization analysis) were increased by DEX, IL-1 and ZnSO{sub 4}. In contrast, the intestine was completely refractory to IL-1. DEX did not affect intestinal metallothionein but did enhance mucosal accumulation of {sup 65}Zn by ligated duodenal loops. Absorption of {sup 65}Zn was not affected by IL-1 or DEX but was inversely related to elevated intestinal metallothionein protein induced in response to ZnSO. Plasma zinc was depressed by DEX and IL-1 and elevated in rats injected with ZnSO{sub 4} but was not related to {sup 54}Zn absorption. Tissue-specific induction of metallothionein may constitute a mechanism for independently regulating both tissue zinc distribution and zinc absorption.

  10. Expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor in human alcoholic brain.


    Lewohl, J M; Crane, D I; Dodd, P R


    The expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the alpha class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patient's age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because alpha 1 expression was raised in both groups of alcoholics of controls. The two groups of alcoholics could be differentiated on the basis of regional variations in alpha 1 expression. In frontal cortex, alpha 1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, alpha 1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform. PMID:9098573