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Sample records for differentially expressed mitochondrial

  1. The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation

    PubMed Central

    Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A.

    2015-01-01

    The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2–3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2–3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. PMID:26182383

  2. Differential expression and glycative damage affect specific mitochondrial proteins with aging in rat liver.

    PubMed

    Bakala, Hilaire; Ladouce, Romain; Baraibar, Martin A; Friguet, Bertrand

    2013-12-01

    Aging is accompanied by the gradual deterioration of cell functions. Particularly, mitochondrial dysfunction, associated with an accumulation of damaged proteins, is of key importance due to the central role of these organelles in cellular metabolism. However, the detailed molecular mechanisms involved in such impairment have not been completely elucidated. In the present study, proteomic analyses looking at both changes at the expression level as well as to glycative modifications of the mitochondrial proteome were performed. Two-dimensional difference gel electrophoresis analysis revealed 16 differentially expressed proteins with aging. Thirteen exhibited a decreased expression and are crucial enzymes related to OXPHOS chain complex I/V components, TCA cycle or fatty acid β-oxidation reaction. On the other hand, 2 enzymes involved in fatty acid β-oxidation cycle were increased in aged mitochondria. Immunodetection and further identification of glycated proteins disclosed a set of advanced glycation end product-modified proteins, including 6 enzymes involved in the fatty acid β-oxidation process, and 2 enzymes of the TCA/urea cycles. A crucial antioxidant enzyme, catalase, was among the most strongly glycated proteins. In addition, several AGE-damaged enzymes (aldehyde dehydrogenase 2, medium chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA dehydrogenase) exhibited a decreased activity with age. Taken together, these data suggest that liver mitochondria in old rats suffer from a decline in their capacity for energy production, due to (i) decreased expression of OXPHOS complex I/V components and (ii) glycative damage to key fatty acid β-oxidation and TCA/urea cycle enzymes. PMID:23906978

  3. Differential Expression of Adenine Nucleotide Converting Enzymes in Mitochondrial Intermembrane Space: A Potential Role of Adenylate Kinase Isozyme 2 in Neutrophil Differentiation

    PubMed Central

    Tanimura, Ayako; Horiguchi, Taigo; Miyoshi, Keiko; Hagita, Hiroko; Noma, Takafumi

    2014-01-01

    Adenine nucleotide dynamics in the mitochondrial intermembrane space (IMS) play a key role in oxidative phosphorylation. In a previous study, Drosophila adenylate kinase isozyme 2 (Dak2) knockout was reported to cause developmental lethality at the larval stage in Drosophila melanogaster. In addition, two other studies reported that AK2 is a responsible gene for reticular dysgenesis (RD), a human disease that is characterized by severe combined immunodeficiency and deafness. Therefore, mitochondrial AK2 may play an important role in hematopoietic differentiation and ontogenesis. Three additional adenine nucleotide metabolizing enzymes, including mitochondrial creatine kinases (CKMT1 and CKMT2) and nucleoside diphosphate kinase isoform D (NDPK-D), have been found in IMS. Although these kinases generate ADP for ATP synthesis, their involvement in RD remains unclear and still an open question. In this study, mRNA and protein expressions of these mitochondrial kinases were firstly examined in mouse ES cells, day 8 embryos, and 7-week-old adult mice. It was found that their expressions are spatiotemporally regulated, and Ak2 is exclusively expressed in bone marrow, which is a major hematopoietic tissue in adults. In subsequent experiments, we identified increased expression of both AK2 and CKMT1 during macrophage differentiation and exclusive production of AK2 during neutrophil differentiation using HL-60 cells as an in vitro model of hematopoietic differentiation. Furthermore, AK2 knockdown specifically inhibited neutrophil differentiation without affecting macrophage differentiation. These data suggest that AK2 is indispensable for neutrophil differentiation and indicate a possible causative link between AK2 deficiency and neutropenia in RD. PMID:24587121

  4. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    PubMed

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

    2016-03-14

    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

  5. Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei.

    PubMed

    Vertommen, Didier; Van Roy, Joris; Szikora, Jean-Pierre; Rider, Mark H; Michels, Paul A M; Opperdoes, Fred R

    2008-04-01

    Label-free semi-quantitative differential three-dimensional liquid chromatography coupled to mass spectrometry (3D-LC-MS/MS) was used to compare the glycosomal and mitochondrial proteomes of the bloodstream- and insect-form of Trypanosoma brucei. The abundance of glycosomal marker proteins identified in the two life-cycle stages corresponded well with the relative importance of biochemical pathways present in the glycosomes of the two stages and the peptide spectral count ratios of selected enzymes were in good agreement with published data about their enzymatic specific activities. This approach proved extremely useful for the generation of large scale proteomics data for the comparison of different life-cycle stages. Several proteins involved in oxidative stress protection, sugar-nucleotide synthesis, purine salvage, nucleotide-monophosphate formation and purine-nucleotide cycle were identified as glycosomal proteins. PMID:18242729

  6. Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function.

    PubMed

    Waldman, Maayan; Bellner, Lars; Vanella, Luca; Schragenheim, Joseph; Sodhi, Komal; Singh, Shailendra P; Lin, Daohong; Lakhkar, Anand; Li, Jiangwei; Hochhauser, Edith; Arad, Michael; Darzynkiewicz, Zbigniew; Kappas, Atallah; Abraham, Nader G

    2016-07-15

    Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhanced peroxisome proliferation-activated receptor γ coactivator α (PGC-1α) expression, and increased mitochondrial function in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase, P < 0.05, in PGC-1α and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase in heme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1α as cells deficient in PGC-1α (PGC-1α knockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treated with EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1α-dependent manner, P < 0.05. The increase in PGC-1α was associated with an increase in β-catenin, P < 0.05, adiponectin expression, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS (MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specific transcript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/paracrine manner to increase PGC-1α is required for activation of HO-1 expression. This is the first study to dissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1α signaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1 expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargement and terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in the canonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation. PMID:27224420

  7. Differential expression of citA gene encoding the mitochondrial citrate synthase of Aspergillus nidulans in response to developmental status and carbon sources.

    PubMed

    Min, In Sook; Bang, Ji Young; Seo, Soon Won; Lee, Cheong Ho; Maeng, Pil Jae

    2010-04-01

    As an extension of our previous studies on the mitochondrial citrate synthase of Aspergillus nidulans and cloning of its coding gene (citA), we analyzed differential expression of citA in response to the progress of development and change of carbon source. The cDNA consisted of 1,700 nucleotides and was predicted to encode a 474-amino acid protein. By comparing the cDNA sequence with the corresponding genomic sequence, we confirmed that citA gene contains 7 introns and that its transcription starts at position -26 (26-nucleotide upstream from the initiation codon). Four putative CreA binding motifs and three putative stress-response elements (STREs) were found within the 1.45-kb citA promoter region. The mode of citA expression was examined by both Northern blot and confocal microscopy using green fluorescent protein (sGFP) as a vital reporter. During vegetative growth and asexual development, the expression of citA was ubiquitous throughout the whole fungal body including mycelia and conidiophores. During sexual development, the expression of citA was quite strong in cleistothecial shells, but significantly weak in the content of cleistothecia including ascospores. Acetate showed a strong inductive effect on citA expression, which is subjected to carbon catabolite repression (CCR) caused by glucose. The recombinant fusion protein CitA(40)::sGFP (sGFP containing the 40-amino acid N-terminal segment of CitA) was localized into mitochondria, which supports that a mitochondrial targeting signal is included within the 40-amino acid N-terminal segment of CitA. PMID:20437151

  8. Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation

    PubMed Central

    Schoenfeld, Robert; Wong, Alice; Silva, Jillian; Li, Ming; Itoh, Aki; Horiuchi, Makoto; Itoh, Takayuki; Pleasure, David; Cortopassi, Gino

    2011-01-01

    Demyelination occurs in multiple inherited mitochondrial diseases. We studied which genes were induced as a consequence of differentiation in rodent and human oligodendroglia. Cholesterol, myelin and mitochondrial genes were significantly increased with oligodendroglial differentiation. Mitochondrial DNA content per cell and acetyl CoA-related transcripts increased significantly; thus, the large buildup of cholesterol necessary for myelination appears to require mitochondrial production of acetyl-CoA. Oligodendroglia were treated with low doses of the mitochondrial inhibitor rotenone to test the dependence of differentiation on mitochondrial function. Undifferentiated cells were resistant to rotenone, whereas differentiating cells were much more sensitive. Very low doses of rotenone that did not affect viability or ATP synthesis still inhibited differentiation, as measured by reduced levels of the myelin transcripts 2′,3′-Cyclic Nucleotide-3′-Phosphodiesterase and Myelin Basic Protein. Thus, mitochondrial transcripts and mtDNA are amplified during oligodendroglial differentiation, and differentiating oligodendroglia are especially sensitive to mitochondrial inhibition, suggesting mechanisms for demyelination observed in mitochondrial disease. PMID:20005986

  9. Differential expression of cardiac muscle mitochondrial matrix proteins in broilers from ascites-resistant and susceptible lines.

    PubMed

    Cisar, C R; Balog, J M; Anthony, N B; Donoghue, A M

    2005-05-01

    Ascites is a metabolic disorder of modern broilers that is distinguished by cardiopulmonary insufficiency in the face of intense oxygen demands of rapidly growing tissues. Broilers with ascites exhibit sustained elevation of pulmonary arterial pressure and right ventricular hypertrophy, the end result of which is heart failure. It has been shown that mitochondrial function is impaired in broilers with ascites. In the current study, mitochondrial matrix protein levels were compared between ascites-resistant line broilers and ascites-susceptible line broilers with and without ascites using two-dimensional (2-D) gel electrophoresis. One hundred seventy-two protein spots were detected on the gels, and 9 of the spots were present at different levels in the 4 groups of broilers. These 9 protein spots were selected for identification by mass spectrometry. Two of the spots were found to contain single mitochondrial matrix proteins. Both mitochondrial matrix proteins, the dihydrolipoamide succinyltransferase component of the 2-oxoglutarate dehydrogenase complex and the alpha-subunit of mitochondrial trifunctional enzyme, were present at higher levels in ascites-resistant line broilers with ascites in the present study. The elevated levels of 2 key proteins in aerobic metabolism in ascites-resistant line broilers with ascites observed in the present study suggests that the mitochondria of broilers with this disease may respond inappropriately to hypoxia. PMID:15913181

  10. Mitochondrial respiratory uncoupling promotes keratinocyte differentiation and blocks skin carcinogenesis

    PubMed Central

    Lago, CU; Nowinski, SM; Rundhaug, JE; Pfeiffer, ME; Kiguchi, K; Hirasaka, K; Yang, X; Abramson, EM; Bratton, SB; Rho, O; Colavitti, R; Kenaston, MA; Nikawa, T; Trempus, C; DiGiovanni, J; Fischer, SM; Mills, EM

    2013-01-01

    Decreased mitochondrial oxidative metabolism is a hallmark bioenergetic characteristic of malignancy that may have an adaptive role in carcinogenesis. By stimulating proton leak, mitochondrial uncoupling proteins (UCP1-3) increase mitochondrial respiration and may thereby oppose cancer development. To test this idea, we generated a mouse model that expresses an epidermal-targeted keratin-5-UCP3 (K5-UCP3) transgene and exhibits significantly increased cutaneous mitochondrial respiration compared with wild type (FVB/N). Remarkably, we observed that mitochondrial uncoupling drove keratinocyte/epidermal differentiation both in vitro and in vivo. This increase in epidermal differentiation corresponded to the loss of markers of the quiescent bulge stem cell population, and an increase in epidermal turnover measured using a bromodeoxyuridine (BrdU)-based transit assay. Interestingly, these changes in K5-UCP3 skin were associated with a nearly complete resistance to chemically-mediated multistage skin carcinogenesis. These data suggest that targeting mitochondrial respiration is a promising novel avenue for cancer prevention and treatment. PMID:22266853

  11. Mitochondrial pyruvate dehydrogenase phosphatase 1 regulates the early differentiation of cardiomyocytes from mouse embryonic stem cells.

    PubMed

    Heo, Hye Jin; Kim, Hyoung Kyu; Youm, Jae Boum; Cho, Sung Woo; Song, In-Sung; Lee, Sun Young; Ko, Tae Hee; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin

    2016-01-01

    Mitochondria are crucial for maintaining the properties of embryonic stem cells (ESCs) and for regulating their subsequent differentiation into diverse cell lineages, including cardiomyocytes. However, mitochondrial regulators that manage the rate of differentiation or cell fate have been rarely identified. This study aimed to determine the potential mitochondrial factor that controls the differentiation of ESCs into cardiac myocytes. We induced cardiomyocyte differentiation from mouse ESCs (mESCs) and performed microarray assays to assess messenger RNA (mRNA) expression changes at differentiation day 8 (D8) compared with undifferentiated mESCs (D0). Among the differentially expressed genes, Pdp1 expression was significantly decreased (27-fold) on D8 compared to D0, which was accompanied by suppressed mitochondrial indices, including ATP levels, membrane potential, ROS and mitochondrial Ca(2+). Notably, Pdp1 overexpression significantly enhanced the mitochondrial indices and pyruvate dehydrogenase activity and reduced the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate compared to a mock control. In confirmation of this, a knockdown of the Pdp1 gene promoted the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate. In conclusion, our results suggest that mitochondrial PDP1 is a potential regulator that controls cardiac differentiation at an early differentiation stage in ESCs. PMID:27538372

  12. Mitochondrial pyruvate dehydrogenase phosphatase 1 regulates the early differentiation of cardiomyocytes from mouse embryonic stem cells

    PubMed Central

    Heo, Hye Jin; Kim, Hyoung Kyu; Youm, Jae Boum; Cho, Sung Woo; Song, In-Sung; Lee, Sun Young; Ko, Tae Hee; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin

    2016-01-01

    Mitochondria are crucial for maintaining the properties of embryonic stem cells (ESCs) and for regulating their subsequent differentiation into diverse cell lineages, including cardiomyocytes. However, mitochondrial regulators that manage the rate of differentiation or cell fate have been rarely identified. This study aimed to determine the potential mitochondrial factor that controls the differentiation of ESCs into cardiac myocytes. We induced cardiomyocyte differentiation from mouse ESCs (mESCs) and performed microarray assays to assess messenger RNA (mRNA) expression changes at differentiation day 8 (D8) compared with undifferentiated mESCs (D0). Among the differentially expressed genes, Pdp1 expression was significantly decreased (27-fold) on D8 compared to D0, which was accompanied by suppressed mitochondrial indices, including ATP levels, membrane potential, ROS and mitochondrial Ca2+. Notably, Pdp1 overexpression significantly enhanced the mitochondrial indices and pyruvate dehydrogenase activity and reduced the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate compared to a mock control. In confirmation of this, a knockdown of the Pdp1 gene promoted the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate. In conclusion, our results suggest that mitochondrial PDP1 is a potential regulator that controls cardiac differentiation at an early differentiation stage in ESCs. PMID:27538372

  13. Differential Expression of PGC-1α and Metabolic Sensors Suggest Age-Dependent Induction of Mitochondrial Biogenesis in Friedreich Ataxia Fibroblasts

    PubMed Central

    García-Giménez, José Luis; Gimeno, Amparo; Gonzalez-Cabo, Pilar; Dasí, Francisco; Bolinches-Amorós, Arantxa; Mollá, Belén; Palau, Francesc; Pallardó, Federico V.

    2011-01-01

    Background Friedreich's ataxia (FRDA) is a mitochondrial rare disease, which molecular origin is associated with defect in the expression of frataxin. The pathological consequences are degeneration of nervous system structures and cardiomyopathy with necrosis and fibrosis, among others. Principal Findings Using FRDA fibroblasts we have characterized the oxidative stress status and mitochondrial biogenesis. We observed deficiency of MnSOD, increased ROS levels and low levels of ATP. Expression of PGC-1α and mtTFA was increased and the active form of the upstream signals p38 MAPK and AMPK in fibroblasts from two patients. Interestingly, the expression of energetic factors correlated with the natural history of disease of the patients, the age when skin biopsy was performed and the size of the GAA expanded alleles. Furthermore, idebenone inhibit mitochondriogenic responses in FRDA cells. Conclusions The induction of mitochondrial biogenesis in FRDA may be a consequence of the mitochondrial impairment associated with disease evolution. The increase of ROS and the involvement of the oxidative phosphorylation may be an early event in the cell pathophysiology of frataxin deficiency, whereas increase of mitochondriogenic response might be a later phenomenon associated to the individual age and natural history of the disease, being more evident as the patient age increases and disease evolves. This is a possible explanation of heart disease in FRDA. PMID:21687738

  14. Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals.

    PubMed

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Estraño, Carlos E

    2014-01-01

    Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components. PMID:25159305

  15. Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

    PubMed Central

    Cline, Susan D.

    2012-01-01

    How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

  16. Levetiracetam Differentially Alters CD95 Expression of Neuronal Cells and the Mitochondrial Membrane Potential of Immune and Neuronal Cells in vitro.

    PubMed

    Rogers, Susannah K; Shapiro, Lee A; Tobin, Richard P; Tow, Benjamin; Zuzek, Aleksej; Mukherjee, Sanjib; Newell-Rogers, M Karen

    2014-01-01

    Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s) of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side-effects. The current study examined the effects of levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if levetiracetam alters the expression of immune receptor-ligand pairs. The results show that levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action. PMID:24600432

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

    PubMed

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

    2016-01-01

    Aging is associated with a decline in cardiac function. Exercise intervention has been suggested as a way to improve this decrement. Age-related decline in cardiac function is associated with decreases in fatty acid oxidation, mitochondrial function, and AMP-activated protein kinase (AMPK) activity. The molecular mechanisms involved with age-related changes in mitochondrial function and substrate metabolism are poorly understood. We determined gene expression differences in hearts of Young (6 mo), Old (33 mo), and old exercise trained (Old + EXE) (34 mo) FBN rats, using Qiagen PCR arrays for Glucose, Fatty acid, and Mitochondrial metabolism. Old rats demonstrated decreased (p < 0.05) expression for key genes in fatty acid oxidation, mitochondrial function, and AMPK signaling. There were no differences in the expression of genes involved in glucose metabolism with age. These gene expression changes occurred prior to altered protein translation as we found no differences in the protein content of peroxisome proliferator activated receptor gamma, coactivators 1 alpha (PGC-1α), peroxisome proliferator activated receptor alpha (PPARα), and AMPKα2 between young and old hearts. Four months of exercise training did not attenuate the decline in the gene expression in aged hearts. Despite this lack of change in gene expression, exercise-trained rats demonstrated increased exercise capacity compared to their sedentary counterparts. Taken together, our results show that differential expression of genes associated with fatty acid metabolism, AMPK signaling and mitochondrial function decrease in the aging heart which may play a role in age-related declines in fatty acid oxidation, AMPK activity, and mitochondrial function in the heart. PMID:27601998

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

    PubMed Central

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

    2016-01-01

    Aging is associated with a decline in cardiac function. Exercise intervention has been suggested as a way to improve this decrement. Age-related decline in cardiac function is associated with decreases in fatty acid oxidation, mitochondrial function, and AMP-activated protein kinase (AMPK) activity. The molecular mechanisms involved with age-related changes in mitochondrial function and substrate metabolism are poorly understood. We determined gene expression differences in hearts of Young (6 mo), Old (33 mo), and old exercise trained (Old + EXE) (34 mo) FBN rats, using Qiagen PCR arrays for Glucose, Fatty acid, and Mitochondrial metabolism. Old rats demonstrated decreased (p < 0.05) expression for key genes in fatty acid oxidation, mitochondrial function, and AMPK signaling. There were no differences in the expression of genes involved in glucose metabolism with age. These gene expression changes occurred prior to altered protein translation as we found no differences in the protein content of peroxisome proliferator activated receptor gamma, coactivators 1 alpha (PGC-1α), peroxisome proliferator activated receptor alpha (PPARα), and AMPKα2 between young and old hearts. Four months of exercise training did not attenuate the decline in the gene expression in aged hearts. Despite this lack of change in gene expression, exercise-trained rats demonstrated increased exercise capacity compared to their sedentary counterparts. Taken together, our results show that differential expression of genes associated with fatty acid metabolism, AMPK signaling and mitochondrial function decrease in the aging heart which may play a role in age-related declines in fatty acid oxidation, AMPK activity, and mitochondrial function in the heart. PMID:27601998

  19. Changes, and the Relevance Thereof, in Mitochondrial Morphology during Differentiation into Endothelial Cells

    PubMed Central

    Shin, Ji Won; Park, So Hee; Kang, Yun Gyeong; Wu, Yanru; Choi, Hyun Ju

    2016-01-01

    The roles of mitochondria in various physiological functions of vascular endothelial cells have been investigated extensively. Morphological studies in relation to physiological functions have been performed. However, there have been few reports of morphological investigations related to stem cell differentiation. This was the first morphological study of mitochondria in relation to endothelial differentiation and focused on quantitative analysis of changes in mitochondrial morphology, number, area, and length during differentiation of human mesenchymal stem cells (hMSCs) into endothelial-like cells. To induce differentiation, we engaged vascular endothelial growth factors and flow-induced shear stress. Cells were classified according to the expression of von Willebrand factor as hMSCs, differentiating cells, and almost fully differentiated cells. Based on imaging analysis, we investigated changes in mitochondrial number, area, and length. In addition, mitochondrial networks were quantified on a single-mitochondrion basis by introducing a branch form factor. The data indicated that the mitochondrial number, area per cell, and length were decreased with differentiation. The mitochondrial morphology became simpler with progression of differentiation. These findings could be explained in view of energy level during differentiation; a higher level of energy is needed during differentiation, with larger numbers of mitochondria with branches. Application of this method to differentiation into other lineages will explain the energy levels required to control stem cell differentiation. PMID:27517609

  20. Changes, and the Relevance Thereof, in Mitochondrial Morphology during Differentiation into Endothelial Cells.

    PubMed

    Shin, Ji Won; Park, So Hee; Kang, Yun Gyeong; Wu, Yanru; Choi, Hyun Ju; Shin, Jung-Woog

    2016-01-01

    The roles of mitochondria in various physiological functions of vascular endothelial cells have been investigated extensively. Morphological studies in relation to physiological functions have been performed. However, there have been few reports of morphological investigations related to stem cell differentiation. This was the first morphological study of mitochondria in relation to endothelial differentiation and focused on quantitative analysis of changes in mitochondrial morphology, number, area, and length during differentiation of human mesenchymal stem cells (hMSCs) into endothelial-like cells. To induce differentiation, we engaged vascular endothelial growth factors and flow-induced shear stress. Cells were classified according to the expression of von Willebrand factor as hMSCs, differentiating cells, and almost fully differentiated cells. Based on imaging analysis, we investigated changes in mitochondrial number, area, and length. In addition, mitochondrial networks were quantified on a single-mitochondrion basis by introducing a branch form factor. The data indicated that the mitochondrial number, area per cell, and length were decreased with differentiation. The mitochondrial morphology became simpler with progression of differentiation. These findings could be explained in view of energy level during differentiation; a higher level of energy is needed during differentiation, with larger numbers of mitochondria with branches. Application of this method to differentiation into other lineages will explain the energy levels required to control stem cell differentiation. PMID:27517609

  1. SIRT3, a Mitochondrial NAD+-Dependent Deacetylase, Is Involved in the Regulation of Myoblast Differentiation

    PubMed Central

    Abdel Khalek, Waed; Cortade, Fabienne; Ollendorff, Vincent; Lapasset, Laure; Tintignac, Lionel

    2014-01-01

    Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD+-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity. PMID:25489948

  2. Maintenance and Expression of Mammalian Mitochondrial DNA.

    PubMed

    Gustafsson, Claes M; Falkenberg, Maria; Larsson, Nils-Göran

    2016-06-01

    Mammalian mitochondrial DNA (mtDNA) encodes 13 proteins that are essential for the function of the oxidative phosphorylation system, which is composed of four respiratory-chain complexes and adenosine triphosphate (ATP) synthase. Remarkably, the maintenance and expression of mtDNA depend on the mitochondrial import of hundreds of nuclear-encoded proteins that control genome maintenance, replication, transcription, RNA maturation, and mitochondrial translation. The importance of this complex regulatory system is underscored by the identification of numerous mutations of nuclear genes that impair mtDNA maintenance and expression at different levels, causing human mitochondrial diseases with pleiotropic clinical manifestations. The basic scientific understanding of the mechanisms controlling mtDNA function has progressed considerably during the past few years, thanks to advances in biochemistry, genetics, and structural biology. The challenges for the future will be to understand how mtDNA maintenance and expression are regulated and to what extent direct intramitochondrial cross talk between different processes, such as transcription and translation, is important. PMID:27023847

  3. Murine Mesenchymal Stem Cell Commitment to Differentiation Is Regulated by Mitochondrial Dynamics.

    PubMed

    Forni, Maria Fernanda; Peloggia, Julia; Trudeau, Kyle; Shirihai, Orian; Kowaltowski, Alicia J

    2016-03-01

    Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105(+) CD90(+) CD73(+) CD29(+) CD34(-) mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells. PMID:26638184

  4. Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

    PubMed

    Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

    2016-04-01

    Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms

  5. Extraocular mitochondrial myopathies and their differential diagnoses.

    PubMed

    Schoser, Benedikt G H; Pongratz, Dieter

    2006-06-01

    The diagnosis of mitochondrial myopathy depends upon a constellation of findings, family history, type of muscle involvement, specific laboratory abnormalities, and the results of histological, pathobiochemical and genetic analysis. In the present paper, the authors describe the diagnostic approach to mitochondrial myopathies manifesting as extraocular muscle disease. The most common ocular manifestation of mitochondrial myopathy is progressive external ophthalmoplegia (PEO). To exclude myasthenia gravis, ocular myositis, thyroid associated orbitopathy, oculopharyngeal muscular dystrophy, and congenital fibrosis of the extraocular muscles in patients with an early onset or long-lasting very slowly progressive ptosis and external ophthalmoplegia, almost without any diplopia, and normal to mildly elevated serum creatine kinase and lactate, electromyography, nerve conduction studies and MRI of the orbits should be performed. A PEO phenotype forces one to look comprehensively for other multisystemic mitochondrial features (e.g., exercise induced weakness, encephalopathy, polyneuropathy, diabetes, heart disease). Thereafter, and presently even in familiar PEO, a diagnostic muscle biopsy should be taken. Histological and ultrastructural hallmarks are mitochondrial proliferations and structural abnormalities, lipid storage, ragged-red fibers, or cytochrome-C negative myofibers. In addition, Southern blotting may reveal the common deletion, or molecular analysis may verify specific mutations of distinct mitochondrial or nuclear genes. PMID:16760117

  6. Mitochondrial dysfunction impairs tumor suppressor p53 expression/function.

    PubMed

    Compton, Shannon; Kim, Chul; Griner, Nicholas B; Potluri, Prasanth; Scheffler, Immo E; Sen, Sabyasachi; Jerry, D Joseph; Schneider, Sallie; Yadava, Nagendra

    2011-06-10

    Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy. PMID:21502317

  7. Differential expression of cysteine desulfurases in soybean

    PubMed Central

    2011-01-01

    Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression. PMID:22099069

  8. Inventory of the Human Mitochondrial Gene Expression Machinery with Links to Disease

    PubMed Central

    Shutt, Timothy E.; Shadel, Gerald S.

    2010-01-01

    Mammalian mitochondrial DNA encodes thirty-seven essential genes required for ATP production via oxidative phosphorylation, instability or misregulation of which is associated with human diseases and aging. Other than the mtDNA-encoded RNA species (thirteen mRNAs, 12S and 16S rRNAs, and twenty-two tRNAs), the many remaining factors needed for mitochondrial gene expression (i.e. transcription, RNA processing/modification and translation), including a dedicated set of mitochondrial ribosomal proteins, are products of nuclear genes that are imported into the mitochondrial matrix. Herein, we inventory the human mitochondrial gene expression machinery, and while doing so highlight specific associations of these regulatory factors with human disease. Major new breakthroughs have been made recently in this burgeoning area that set the stage for exciting future studies on the key outstanding issue of how mitochondrial gene expression is regulated differentially in vivo. This should promote a greater understanding of why mtDNA mutations and dysfunction cause the complex and tissue-specific pathology characteristic of mitochondrial disease states and how mitochondrial dysfunction contributes to more common human pathology and aging. PMID:20544879

  9. Mitochondrial aerobic respiration is activated during hair follicle stem cell differentiation, and its dysfunction retards hair regeneration

    PubMed Central

    Tang, Yan; Luo, Binping; Deng, Zhili; Wang, Ben; Liu, Fangfen; Li, Jinmao; Shi, Wei; Xie, Hongfu; Hu, Xingwang

    2016-01-01

    Background. Emerging research revealed the essential role of mitochondria in regulating stem/progenitor cell differentiation of neural progenitor cells, mesenchymal stem cells and other stem cells through reactive oxygen species (ROS), Notch or other signaling pathway. Inhibition of mitochondrial protein synthesis results in hair loss upon injury. However, alteration of mitochondrial morphology and metabolic function during hair follicle stem cells (HFSCs) differentiation and how they affect hair regeneration has not been elaborated upon. Methods. We compared the difference in mitochondrial morphology and activity between telogen bulge cells and anagen matrix cells. Expression levels of mitochondrial ROS and superoxide dismutase 2 (SOD2) were measured to evaluate redox balance. In addition, the level of pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase (PDH) were estimated to present the change in energetic metabolism during differentiation. To explore the effect of the mitochondrial metabolism on regulating hair regeneration, hair growth was observed after application of a mitochondrial respiratory inhibitor upon hair plucking. Results. During HFSCs differentiation, mitochondria became elongated with more abundant organized cristae and showed higher activity in differentiated cells. SOD2 was enhanced for redox balance with relatively stable ROS levels in differentiated cells. PDK increased in HFSCs while differentiated cells showed enhanced PDH, indicating that respiration switched from glycolysis to oxidative phosphorylation during differentiation. Inhibiting mitochondrial respiration in differentiated hair follicle cells upon hair plucking repressed hair regeneration in vivo. Conclusions. Upon HFSCs differentiation, mitochondria are elongated with more abundant cristae and show higher activity, accompanying with activated aerobic respiration in differentiated cells for higher energy supply. Also, dysfunction of mitochondrial respiration delays hair

  10. Mitochondrial aerobic respiration is activated during hair follicle stem cell differentiation, and its dysfunction retards hair regeneration.

    PubMed

    Tang, Yan; Luo, Binping; Deng, Zhili; Wang, Ben; Liu, Fangfen; Li, Jinmao; Shi, Wei; Xie, Hongfu; Hu, Xingwang; Li, Ji

    2016-01-01

    Background. Emerging research revealed the essential role of mitochondria in regulating stem/progenitor cell differentiation of neural progenitor cells, mesenchymal stem cells and other stem cells through reactive oxygen species (ROS), Notch or other signaling pathway. Inhibition of mitochondrial protein synthesis results in hair loss upon injury. However, alteration of mitochondrial morphology and metabolic function during hair follicle stem cells (HFSCs) differentiation and how they affect hair regeneration has not been elaborated upon. Methods. We compared the difference in mitochondrial morphology and activity between telogen bulge cells and anagen matrix cells. Expression levels of mitochondrial ROS and superoxide dismutase 2 (SOD2) were measured to evaluate redox balance. In addition, the level of pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase (PDH) were estimated to present the change in energetic metabolism during differentiation. To explore the effect of the mitochondrial metabolism on regulating hair regeneration, hair growth was observed after application of a mitochondrial respiratory inhibitor upon hair plucking. Results. During HFSCs differentiation, mitochondria became elongated with more abundant organized cristae and showed higher activity in differentiated cells. SOD2 was enhanced for redox balance with relatively stable ROS levels in differentiated cells. PDK increased in HFSCs while differentiated cells showed enhanced PDH, indicating that respiration switched from glycolysis to oxidative phosphorylation during differentiation. Inhibiting mitochondrial respiration in differentiated hair follicle cells upon hair plucking repressed hair regeneration in vivo. Conclusions. Upon HFSCs differentiation, mitochondria are elongated with more abundant cristae and show higher activity, accompanying with activated aerobic respiration in differentiated cells for higher energy supply. Also, dysfunction of mitochondrial respiration delays hair

  11. Defining a Model for Mitochondrial Function in mESC Differentiation

    EPA Science Inventory

    Defining a Model for Mitochondrial Function in mESC DifferentiationDefining a Model for Mitochondrial Function in mESC Differentiation Differentiating embryonic stem cells (ESCs) undergo mitochondrial maturation leading to a switch from a system dependent upon glycolysis to a re...

  12. The Differential DRP1 Phosphorylation and Mitochondrial Dynamics in the Regional Specific Astroglial Death Induced by Status Epilepticus

    PubMed Central

    Ko, Ah-Reum; Hyun, Hye-Won; Min, Su-Ji; Kim, Ji-Eun

    2016-01-01

    The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE). Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic) astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1 (an inhibitor of mitochondrial fission) effectively attenuated astroglial apoptosis, but WY14643 (an enhancer of mitochondrial fission) aggravated it. In addition, Mdivi-1 accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein 1 (DRP1; a mitochondrial fission protein) phosphorylation, not optic atrophy 1 (OPA1; a mitochondrial fusion protein) expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE. PMID:27242436

  13. Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics

    PubMed Central

    Hahn, Wendy S.; Kuzmicic, Jovan; Burrill, Joel S.; Donoghue, Margaret A.; Foncea, Rocio; Jensen, Michael D.; Lavandero, Sergio; Arriaga, Edgar A.

    2014-01-01

    Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics. Macrophage infiltration of adipose tissue and the chronic low-grade production of inflammatory cytokines have been mechanistically linked to the development of insulin resistance, the forerunner of type 2 diabetes mellitus. In this study, we evaluated the chronic effects of TNFα, IL-6, and IL-1β on adipocyte mitochondrial metabolism and morphology using the 3T3-L1 model cell system. TNFα treatment of cultured adipocytes led to significant changes in mitochondrial bioenergetics, including increased proton leak, decreased ΔΨm, increased basal respiration, and decreased ATP turnover. In contrast, although IL-6 and IL-1β decreased maximal respiratory capacity, they had no effect on ΔΨm and varied effects on ATP turnover, proton leak, or basal respiration. Only TNFα treatment of 3T3-L1 cells led to an increase in oxidative stress (as measured by superoxide anion production and protein carbonylation) and C16 ceramide synthesis. Treatment of 3T3-L1 adipocytes with cytokines led to decreased mRNA expression of key transcription factors and control proteins implicated in mitochondrial biogenesis, including PGC-1α and eNOS as well as deceased expression of COX IV and Cyt C. Whereas each cytokine led to effects on expression of mitochondrial markers, TNFα exclusively led to mitochondrial fragmentation and decreased the total level of OPA1 while increasing OPA1 cleavage, without expression of levels of mitofusin 2, DRP-1, or mitofilin being affected. In summary, these results indicate that inflammatory cytokines have unique and specialized effects on adipocyte metabolism, but each leads to decreased mitochondrial function and a reprogramming of fat cell biology. PMID:24595304

  14. Effect of mitochondrial fission inhibition on C2C12 differentiation.

    PubMed

    Bloemberg, Darin; Quadrilatero, Joe

    2016-06-01

    The differentiation of skeletal muscle is commonly examined in cell culture using the C2C12 line of mouse skeletal myoblasts. This process shares many similarities with that which occurs during embryonic development, such as the transient activation of caspases. Here, we examined the effect of inhibiting mitochondrial fission, using mdivi-1, on the ability of C2C12 cells to terminally differentiate. This was performed using immunofluorescent identification of cell morphology and myosin expression, as well as immunoblotting for markers of muscle differentiation. Furthermore, the effect of mdivi-1 administration on activation of caspase-2 and -3 was assessed using spectrofluorometric measurement of specific enzyme activity. PMID:27054170

  15. Mitochondrial Hspa9/Mortalin regulates erythroid differentiation via iron-sulfur cluster assembly.

    PubMed

    Shan, Yuxi; Cortopassi, Gino

    2016-01-01

    Mitochondrial iron-sulfur cluster (ISC) biogenesis provides iron-sulfur cofactors to several mitochondrial proteins, but the extent to which ISC biogenesis regulates hematopoiesis has been unclear. The blood disease Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis, and the disease overlaps with the gene Hspa9/Mortalin in multiple ways: the HSPA9 locus maps to 5q31.2 that is frequently deleted in human MDS; mutant Hspa9 causes zebrafish MDS; and Hspa9 knockdown mice have decreased hematopoiesis. We show here that HSPA9 functions in mitochondrial ISC biogenesis, and is required for erythroid differentiation. HSPA9 interacts with and stabilizes the mitochondrial ISC biogenesis proteins frataxin, Nfs1, ISCU, and Nfu. MDS-causing mutations in HSPA9 protein change its interactions with ISC biogenesis proteins. Depletion of HSPA9 decreases aconitase activity, which requires an ISC at its active site, but not that of the non-ISC requiring malate dehydrogenase, and increases IRP1 binding activity. In erythroid cell lines, Hspa9 depletion inhibited erythroid differentiation, post-transcriptionally regulating the expression of Alas2 and FeCH, as expected through known ISC control of the IRE response elements in these genes. By contrast, the Alas2 open reading frame rescued the Hspa9-dependent defect in erythroid differentiation, but not when uncoupled from its 5'-IRE sequence. Thus, Hspa9 depletion causes a mitochondrial ISC deficit, altering IRP1-IRE binding and FeCH stability, which consequently inhibits Alas2 translation, heme synthesis, and erythroid differentiation, i.e.: Hspa9->ISC->IRP/IRE->Alas2->heme synthesis->erythroid differentiation. Thus Hspa9 regulates erythroid differentiation through ISC cluster assembly, providing a pathophysiological mechanism for an MDS subtype characterized by HSPA9 haploinsufficiency, and suggests hemin and other pharmacological stimulators of ISC synthesis as potential routes to therapy. PMID:26702583

  16. A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism

    PubMed Central

    Mei, Yu-qin; Pan, Zong-fu; Chen, Wen-teng; Xu, Min-hua; Zhu, Dan-yan; Yu, Yong-ping; Lou, Yi-jia

    2016-01-01

    Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

  17. A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism.

    PubMed

    Mei, Yu-Qin; Pan, Zong-Fu; Chen, Wen-Teng; Xu, Min-Hua; Zhu, Dan-Yan; Yu, Yong-Ping; Lou, Yi-Jia

    2016-01-01

    Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

  18. Differential regulation of γ-glutamyltransferase and glutamate cysteine ligase expression after mitochondrial uncoupling: γ-glutamyltransferase is regulated in an Nrf2- and NFκB-independent manner.

    PubMed

    Ravuri, C; Svineng, G; Huseby, N-E

    2013-05-01

    The enzymes γ-glutamyltransferase (GGT) and glutamate cysteine ligase (GCL) have important roles in glutathione (GSH) homeostasis, and both are frequently upregulated after acute oxidative stress. Mitochondria are major producers of ROS, and incubating the colorectal adenocarcinoma cell line HT-29 cells with mitochondrial uncouplers significantly increased endogenous ROS as well as mRNA for both GGT and GCLC (the catalytic subunit of GCL). However, no elevation in GGT protein or activity was detected, in contrast to the increased levels of GCLC protein found. The uncouplers initiated endoplasmic reticulum (ER) stress, as demonstrated by highly increased levels of CHOP and GRP78 mRNA. Using inhibitors of proteasomes and ER-associated degradation (ERAD) together with a mitochondrial uncoupler, increased GGT protein and activity levels were obtained indicating that GGT may be a substrate for ERAD. Uncoupling increased the mRNA levels of the two redox-regulated transcription factors Nrf2 and NFκB. Using siRNA to suppress Nrf2 and NFκB expression, downregulation of GCLC expression both at the basal level and after mitochondrial uncoupling was achieved. In contrast, the expression level of GGT was not affected by this treatment. These data strongly indicate a discrepancy between the regulation of GCLC and of GGT following the oxidative stress situation due to mitochondrial uncoupling. Both the enzymes are considered to be part of the cellular antioxidant system; however, the role of GGT as a consistent oxidative response parameter needs to be reevaluated. PMID:23448276

  19. Differential Gene Expression in Glaucoma

    PubMed Central

    Jakobs, Tatjana C.

    2014-01-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell–matrix interactions and adhesion, the cell cycle, and the endothelin system. PMID:24985133

  20. Expression of a family of noncoding mitochondrial RNAs distinguishes normal from cancer cells.

    PubMed

    Burzio, Verónica A; Villota, Claudio; Villegas, Jaime; Landerer, Eduardo; Boccardo, Enrique; Villa, Luisa L; Martínez, Ronny; Lopez, Constanza; Gaete, Fancy; Toro, Viviana; Rodriguez, Ximena; Burzio, Luis O

    2009-06-01

    We reported the presence in human cells of a noncoding mitochondrial RNA that contains an inverted repeat (IR) of 815 nucleotides (nt) covalently linked to the 5' end of the mitochondrial 16S RNA (16S mtrRNA). The transcript contains a stem-loop structure and is expressed in human proliferating cells but not in resting cells. Here, we demonstrate that, in addition to this transcript, normal human proliferating cells in culture express 2 antisense mitochondrial transcripts. These transcripts also contain stem-loop structures but strikingly they are down-regulated in tumor cell lines and tumor cells present in 17 different tumor types. The differential expression of these transcripts distinguishes normal from tumor cells and might contribute a unique vision on cancer biology and diagnostics. PMID:19470459

  1. Expression of a family of noncoding mitochondrial RNAs distinguishes normal from cancer cells

    PubMed Central

    Burzio, Verónica A.; Villota, Claudio; Villegas, Jaime; Landerer, Eduardo; Boccardo, Enrique; Villa, Luisa L.; Martínez, Ronny; Lopez, Constanza; Gaete, Fancy; Toro, Viviana; Rodriguez, Ximena; Burzio, Luis O.

    2009-01-01

    We reported the presence in human cells of a noncoding mitochondrial RNA that contains an inverted repeat (IR) of 815 nucleotides (nt) covalently linked to the 5′ end of the mitochondrial 16S RNA (16S mtrRNA). The transcript contains a stem-loop structure and is expressed in human proliferating cells but not in resting cells. Here, we demonstrate that, in addition to this transcript, normal human proliferating cells in culture express 2 antisense mitochondrial transcripts. These transcripts also contain stem-loop structures but strikingly they are down-regulated in tumor cell lines and tumor cells present in 17 different tumor types. The differential expression of these transcripts distinguishes normal from tumor cells and might contribute a unique vision on cancer biology and diagnostics. PMID:19470459

  2. Mitochondrial Cyclic AMP Response Element-binding Protein (CREB) Mediates Mitochondrial Gene Expression and Neuronal Survival*S

    PubMed Central

    Lee, Junghee; Kim, Chun-Hyung; Simon, David K.; Aminova, Lyaylya R.; Andreyev, Alexander Y.; Kushnareva, Yulia E.; Murphy, Anne N.; Lonze, Bonnie E.; Kim, Kwang-Soo; Ginty, David D.; Ferrante, Robert J.; Ryu, Hoon; Ratan, Rajiv R.

    2008-01-01

    Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders. PMID:16207717

  3. Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells

    PubMed Central

    Henzi, Thomas; Schwaller, Beat

    2015-01-01

    Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in “PV-ergic” cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by

  4. Mitochondrial Gene Expression Is Responsive to Starvation Stress and Developmental Transition in Trypanosoma cruzi

    PubMed Central

    Shaw, Aubie K.; Kalem, Murat C.

    2016-01-01

    ABSTRACT Trypanosoma cruzi parasites causing Chagas disease are passed between mammals by the triatomine bug vector. Within the insect, T. cruzi epimastigote-stage cells replicate and progress through the increasingly nutrient-restricted digestive tract, differentiating into infectious, nonreplicative metacyclic trypomastigotes. Thus, we evaluated how nutrient perturbations or metacyclogenesis affects mitochondrial gene expression in different insect life cycle stages. We compared mitochondrial RNA abundances in cultures containing fed, replicating epimastigotes, differentiating cultures containing both starved epimastigotes and metacyclic trypomastigotes and epimastigote starvation cultures. We observed increases in mitochondrial rRNAs and some mRNAs in differentiating cultures. These increases predominated only for the edited CYb mRNA in cultures enriched for metacyclic trypomastigotes. For the other transcripts, abundance increases were linked to starvation and were strongest in culture fractions with a high population of starved epimastigotes. We show that loss of both glucose and amino acids results in rapid increases in RNA abundances that are quickly reduced when these nutrients are returned. Furthermore, the individual RNAs exhibit distinct temporal abundance patterns, suggestive of multiple mechanisms regulating individual transcript abundance. Finally, increases in mitochondrial respiratory complex subunit mRNA abundances were not matched by increases in abundances of nucleus-encoded subunit mRNAs, nor were there statistically significant increases in protein levels of three nucleus-encoded subunits tested. These results show that, similarly to that in T. brucei, the mitochondrial genome in T. cruzi has the potential to alter gene expression in response to environmental or developmental stimuli but for an as-yet-unknown purpose. IMPORTANCE Chagas disease is caused by insect-transmitted Trypanosoma cruzi. Halting T. cruzi’s life cycle in one of its

  5. Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells

    PubMed Central

    Váraljai, Renáta; Islam, Abul B.M.M.K.; Beshiri, Michael L.; Rehman, Jalees; Lopez-Bigas, Nuria; Benevolenskaya, Elizaveta V.

    2015-01-01

    The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype. PMID:26314709

  6. Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells.

    PubMed

    Váraljai, Renáta; Islam, Abul B M M K; Beshiri, Michael L; Rehman, Jalees; Lopez-Bigas, Nuria; Benevolenskaya, Elizaveta V

    2015-09-01

    The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype. PMID:26314709

  7. Identification of a novel putative mitochondrial protein FAM210B associated with erythroid differentiation.

    PubMed

    Kondo, Aiko; Fujiwara, Tohru; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Nakamura, Yukio; Sawada, Kenichi; Harigae, Hideo

    2016-04-01

    The transcription factor GATA-1 plays an essential role in erythroid differentiation. To identify novel GATA-1 target genes, we analyzed a merged ChIP-seq and expression profiling dataset. We identified FAM210B as a putative novel GATA-1 target gene. Study results demonstrated that GATA-1 directly regulates FAM210B expression, presumably by binding to an intronic enhancer region. Both human and murine FAM210B are abundantly expressed in the later stages of erythroblast development. Moreover, the deduced amino acid sequence predicted that FAM210B is a membrane protein, and Western blot analysis demonstrated its mitochondrial localization. Loss-of-function analysis in erythroid cells suggested that FAM210B may be involved in erythroid differentiation. The identification and characterization of FAM210B provides new insights in the study of erythropoiesis and hereditary anemias. PMID:26968549

  8. Limitations of allotopic expression of mitochondrial genes in mammalian cells.

    PubMed Central

    Oca-Cossio, Jose; Kenyon, Lesley; Hao, Huiling; Moraes, Carlos T

    2003-01-01

    The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria. PMID:14573482

  9. Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

    PubMed

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Cardoso, Ana L; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

    2015-03-01

    Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases. PMID:25634573

  10. Could mitochondrial dysfunction be a differentiating marker between chronic fatigue syndrome and fibromyalgia?

    PubMed

    Castro-Marrero, Jesús; Cordero, Mario D; Sáez-Francas, Naia; Jimenez-Gutierrez, Conxita; Aguilar-Montilla, Francisco J; Aliste, Luisa; Alegre-Martin, José

    2013-11-20

    Chronic fatigue syndrome (CFS) and fibromyalgia (FM) are complex and serious illnesses that affect approximately 2.5% and 5% of the general population worldwide, respectively. The etiology is unknown; however, recent studies suggest that mitochondrial dysfunction has been involved in the pathophysiology of both conditions. We have investigated the possible association between mitochondrial biogenesis and oxidative stress in patients with CFS and FM. We studied 23 CFS patients, 20 FM patients, and 15 healthy controls. Peripheral blood mononuclear cell showed decreased levels of Coenzyme Q10 from CFS patients (p<0.001 compared with controls) and from FM subjects (p<0.001 compared with controls) and ATP levels for CFS patients (p<0.001 compared with controls) and for FM subjects (p<0.001 compared with controls). On the contrary, CFS/FM patients had significantly increased levels of lipid peroxidation, respectively (p<0.001 for both CFS and FM patients with regard to controls) that were indicative of oxidative stress-induced damage. Mitochondrial citrate synthase activity was significantly lower in FM patients (p<0.001) and, however, in CFS, it resulted in similar levels than controls. Mitochondrial DNA content (mtDNA/gDNA ratio) was normal in CFS and reduced in FM patients versus healthy controls, respectively (p<0.001). Expression levels of peroxisome proliferator-activated receptor gamma-coactivator 1-alpha and transcription factor A, mitochondrial by immunoblotting were significantly lower in FM patients (p<0.001) and were normal in CFS subjects compared with healthy controls. These data lead to the hypothesis that mitochondrial dysfunction-dependent events could be a marker of differentiation between CFS and FM, indicating the mitochondria as a new potential therapeutic target for these conditions. PMID:23600892

  11. A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

    PubMed

    Wilson, William C; Hornig-Do, Hue-Tran; Bruni, Francesco; Chang, Jeong Ho; Jourdain, Alexis A; Martinou, Jean-Claude; Falkenberg, Maria; Spåhr, Henrik; Larsson, Nils-Göran; Lewis, Richard J; Hewitt, Lorraine; Baslé, Arnaud; Cross, Harold E; Tong, Liang; Lebel, Robert R; Crosby, Andrew H; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N

    2014-12-01

    The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes. PMID:25008111

  12. Suppression of cytoplasmic male sterility by nuclear genes alters expression of a novel mitochondrial gene region.

    PubMed Central

    Singh, M; Brown, G G

    1991-01-01

    To identify regions of the mitochondrial genome that potentially could specify the "Polima" (pol) cytoplasmic male sterility (CMS) of Brassica napus, transcripts of 14 mitochondrial genes from nap (male fertile), pol (male sterile), and nuclear fertility-restored pol cytoplasm plants were analyzed. Transcriptional differences among these plants were detected only with the ATPase subunit 6 (atp6) gene. Structural analysis of the atp6 gene regions of pol and nap mitochondrial DNAs showed that rearrangements in the pol mitochondrial genome occurring upstream of atp6 have generated a chimeric 224-codon open reading frame, designated orf224, that is cotranscribed with atp6. In CMS plants, most transcripts of this region are dicistronic, comprising both orf224 and atp6 sequences. Nuclear restorer genes at either of two distinct loci appear to specifically alter this transcript pattern such that monocistronic atp6 transcripts predominate. The differences in expression of this region appear to result, in part, from differential processing of a tRNA-like element comprising a tRNA pseudogene present immediately upstream of atp6 in both the sterile and fertile mitochondrial DNAs. Possible mechanisms by which expression of the orf224/atp6 locus and the Polima CMS trait may be specifically related are considered. PMID:1840901

  13. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  14. Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells

    PubMed Central

    Vega-Naredo, I; Loureiro, R; Mesquita, K A; Barbosa, I A; Tavares, L C; Branco, A F; Erickson, J R; Holy, J; Perkins, E L; Carvalho, R A; Oliveira, P J

    2014-01-01

    The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies. PMID:24832466

  15. Mitochondrial quality, dynamics and functional capacity in Parkinson’s disease cybrid cell lines selected for Lewy body expression

    PubMed Central

    2013-01-01

    Background Lewy bodies (LB) are a neuropathological hallmark of Parkinson’s disease (PD) and other synucleinopathies. The role their formation plays in disease pathogenesis is not well understood, in part because studies of LB have been limited to examination of post-mortem tissue. LB formation may be detrimental to neuronal survival or merely an adaptive response to other ongoing pathological processes. In a human cytoplasmic hybrid (cybrid) neural cell model that expresses mitochondrial DNA from PD patients, we observed spontaneous formation of intracellular protein aggregates (“cybrid LB” or CLB) that replicate morphological and biochemical properties of native, cortical LB. We studied mitochondrial morphology, bioenergetics and biogenesis signaling by creating stable sub-clones of three PD cybrid cell lines derived from cells expressing CLB. Results Cloning based on CLB expression had a differential effect on mitochondrial morphology, movement and oxygen utilization in each of three sub-cloned lines, but no long-term change in CLB expression. In one line (PD63CLB), mitochondrial function declined compared to the original PD cybrid line (PD63Orig) due to low levels of mtDNA in nucleoids. In another cell line (PD61Orig), the reverse was true, and cellular and mitochondrial function improved after sub-cloning for CLB expression (PD61CLB). In the third cell line (PD67Orig), there was no change in function after selection for CLB expression (PD67CLB). Conclusions Expression of mitochondrial DNA derived from PD patients in cybrid cell lines induced the spontaneous formation of CLB. The creation of three sub-cloned cybrid lines from cells expressing CLB resulted in differential phenotypic changes in mitochondrial and cellular function. These changes were driven by the expression of patient derived mitochondrial DNA in nucleoids, rather than by the presence of CLB. Our studies suggest that mitochondrial DNA plays an important role in cellular and mitochondrial

  16. Mitochondrial encephalomyopathy: variable clinical expression within a single kindred.

    PubMed Central

    Crimmins, D; Morris, J G; Walker, G L; Sue, C M; Byrne, E; Stevens, S; Jean-Francis, B; Yiannikas, C; Pamphlett, R

    1993-01-01

    The clinical manifestations of mitochondrial encephalomyopathy are described in four generations of a single kindred. The age of onset of major neurological disturbance varied from 3-70 years. In some patients, deafness was the only manifestation; in others, recurrent bouts of status epilepticus associated with focal neurological deficits and headache, caused severe disability or death. Examples of all three adult forms of mitochondrial encephalomyopathy: MELAS, MERFF and Kearns Sayre syndrome, were represented within the kindred. Associated features included deafness, short stature, non-insulin-dependent diabetes mellitus, migraine, peptic ulceration and severe constipation. The nt 3243 A-G MELAS mutation was detected in two members of the kindred. This study highlights the diversity of clinical expression of a mitochondrial mutation within a single kindred. Images PMID:8350109

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

    PubMed

    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

    2010-07-01

    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

  18. Canstatin inhibits isoproterenol-induced apoptosis through preserving mitochondrial morphology in differentiated H9c2 cardiomyoblasts.

    PubMed

    Okada, Muneyoshi; Morioka, Suiri; Kanazawa, Hiroki; Yamawaki, Hideyuki

    2016-08-01

    Canstatin, a non-collagenous fragment, is cleaved from type IV collagen α2 chain, an essential component of basement membrane surrounding cardiomyocytes. Although canstatin is known as an endogenous anti-angiogenic factor, its effects on cardiomyocytes have not been clarified. This study examined the effects of canstatin on isoproterenol-induced apoptosis in differentiated H9c2 cardiomyoblasts. Retinoic acid was used to differentiate H9c2 myoblast to cardiomyocyte-like phenotype. Cell viability was determined by a cell counting assay. Western blotting was performed to detect expression of cleaved casepase-3 and phosphorylation of dynamin related protein (Drp)1 at Ser637 which regulates mitochondrial fission. Mito Sox Red staining was performed to examine a mitochondria-dependent production of reactive oxygen species (ROS). Mitochondrial morphology was detected by Mito Tracker Red staining. Isoproterenol (100 μM, 48 h) significantly decreased cell viability and increased cleaved caspase-3 expression, which were inhibited by canstatin (10-250 ng/ml) in a concentration-dependent manner. Canstatin suppressed the isoproterenol-induced mitochondrial fission but not ROS. Canstatin also inhibited the isoproterenol-induced dephosphorylation of Drp1 at Ser637. In conclusion, canstatin inhibits isoproterenol-induced apoptosis through the inhibition of mitochondrial fission via the suppression of dephosphorylation of Drp1 at Ser637 in differentiated H9c2 cardiomyoblasts. PMID:27315818

  19. Effect of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.

    PubMed

    Chen, Yu-Mei; Li, Xing; Song, Gui-Xian; Liu, Ming; Fan, Yi; Wu, Li-Jie; Li, Hua; Zhang, Qi-Jun; Liu, Yao-Qiu; Qian, Ling-Mei

    2016-02-01

    To explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8(CCK-8) proliferation assays and cell cycle analysis demonstrated that LYRM1 gene silencing significantly inhibited P19 cell proliferation. Flow cytometry and measurement of their caspase-3 activities revealed that knockdown of LYRM1 increased P19 cell apoptosis. Observation of morphological changes using an inverted microscope and expression analysis of specific differentiation marker genes using quantitative real-time PCR and Western blotting revealed that knockdown of LYRM1 significantly inhibited the differentiation of P19 cells into cardiomyocytes. Furthermore, real-time quantitative PCR applied to detect mitochondrial DNA (mtDNA) copy number implied that there was no significant difference in the LYRM1 knockdown group compared with the control group. Cellular ATP production investigated by luciferase-based luminescence assay was dramatically decreased in differentiated cells transfected with LYRM1 RNAi. Fluorescence microscopy and flow cytometery were used to detect the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) showed that the level of ROS was dramatically increased and MMP was obviously decreased in differentiated cells transfected with LYRM1 RNAi. Collectively, knockdown of LYRM1 promoted apoptosis and suppressed proliferation and differentiation in P19 cells. In addition, knockdown of LYRM1 induced mitochondrial impairment in P19 cells during differentiation, which was reflected by decreased ATP synthesis, lower MMP and increased ROS levels. PMID:26759027

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

    SciTech Connect

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

    2010-07-02

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

  1. Effects of moderate global maternal nutrient reduction on fetal baboon renal mitochondrial gene expression at 0.9 gestation.

    PubMed

    Pereira, Susana P; Oliveira, Paulo J; Tavares, Ludgero C; Moreno, António J; Cox, Laura A; Nathanielsz, Peter W; Nijland, Mark J

    2015-06-01

    Early life malnutrition results in structural alterations in the kidney, predisposing offspring to later life renal dysfunction. Kidneys of adults who were growth restricted at birth have substantial variations in nephron endowment. Animal models have indicated renal structural and functional consequences in offspring exposed to suboptimal intrauterine nutrition. Mitochondrial bioenergetics play a key role in renal energy metabolism, growth, and function. We hypothesized that moderate maternal nutrient reduction (MNR) would adversely impact fetal renal mitochondrial expression in a well-established nonhuman primate model that produces intrauterine growth reduction at term. Female baboons were fed normal chow diet or 70% of control diet (MNR). Fetal kidneys were harvested at cesarean section at 0.9 gestation (165 days gestation). Human Mitochondrial Energy Metabolism and Human Mitochondria Pathway PCR Arrays were used to analyze mitochondrially relevant mRNA expression. In situ protein content was detected by immunohistochemistry. Despite the smaller overall size, the fetal kidney weight-to-body weight ratio was not affected. We demonstrated fetal sex-specific differential mRNA expression encoding mitochondrial metabolite transport and dynamics proteins. MNR-related differential gene expression was more evident in female fetuses, with 16 transcripts significantly altered, including 14 downregulated and 2 upregulated transcripts. MNR impacted 10 transcripts in male fetuses, with 7 downregulated and 3 upregulated transcripts. The alteration in mRNA levels was accompanied by a decrease in mitochondrial protein cytochrome c oxidase subunit VIc. In conclusion, transcripts encoding fetal renal mitochondrial energy metabolism proteins are nutrition sensitive in a sex-dependent manner. We speculate that these differences lead to decreased mitochondrial fitness that contributes to renal dysfunction in later life. PMID:25761880

  2. The Impact of Mitochondrial Complex Inhibition on mESC Differentiation

    EPA Science Inventory

    The Impact of Mitochondrial Complex Inhibition on mESC Differentiation JE Royland, SH Warren, S Jeffay, MR Hoopes, HP Nichols, ES Hunter U.S. Environmental Protection Agency, Integrated Systems Toxicology Division, Research Triangle Park, NC The importance of mitochondrial funct...

  3. Mitochondrial DNA copy number and replication in reprogramming and differentiation.

    PubMed

    St John, Justin C

    2016-04-01

    Until recently, it was thought that the role of the mitochondrial genome was confined to encoding key proteins that generate ATP through the process of oxidative phosphorylation in the electron transfer chain. However, with increasing new evidence, it is apparent that the mitochondrial genome has a major role to play in a number of diseases and phenotypes. For example, mitochondrial variants and copy number have been implicated in the processes of fertilisation outcome and development and the onset of tumorigenesis. On the other hand, mitochondrial DNA (mtDNA) haplotypes have been implicated in a variety of diseases and most likely account for the adaptation that our ancestors achieved in order that they were fit for their environments. The mechanisms, which enable the mitochondrial genome to either protect or promote the disease phenotype, require further elucidation. However, there appears to be significant 'crosstalk' between the chromosomal and mitochondrial genomes that enable this to take place. One such mechanism is the regulation of DNA methylation by mitochondrial DNA, which is often perturbed in reprogrammed cells that have undergone dedifferentiation and affects mitochondrial DNA copy number. Furthermore, it appears that the mitochondrial genome interacts with the chromosomal genome to regulate the transcription of key genes at certain stages during development. Additionally, the mitochondrial genome can accumulate a series of mtDNA variants, which can lead to diseases such as cancer. It is likely that a combination of certain mitochondrial variants and aberrant patterns of mtDNA copy number could indeed account for many diseases that have previously been unaccounted for. This review focuses on the role that the mitochondrial genome plays especially during early stages of development and in cancer. PMID:26827792

  4. Effects of TCDD on the Expression of Nuclear Encoded Mitochondrial Genes

    PubMed Central

    Forgacs, Agnes L.; Burgoon, Lyle D.; Lynn, Scott G.; LaPres, John J.; Zacharewski, Timothy

    2014-01-01

    Generation of mitochondrial reactive oxygen species (ROS) can be perturbed following exposure to environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Reports indicate that the aryl hydrocarbon receptor (AhR) mediates TCDD-induced sustained hepatic oxidative stress by decreasing hepatic ATP levels and through hyperpolarization of the inner mitochondrial membrane. To further elucidate the effects of TCDD on the mitochondria, high-throughput quantitative real-time PCR (HTP-QRTPCR) was used to evaluate the expression of 90 genes encoding mitochondrial proteins involved in electron transport, oxidative phosphorylation, uncoupling, and associated chaperones. HTP-QRTPCR analysis of time course (30 μg/kg TCDD at 2, 4, 8, 12, 18, 24, 72, and 168 hrs) liver samples obtained from orally gavaged immature, ovariectomized C57BL/6 mice identified 54 differentially expressed genes (|fold change|>1.5 and P-value <0.1). Of these, 8 exhibited a dose response (0.03 to 300 μg/kg TCDD) at 4, 24 or 72 hrs. Dose responsive genes encoded proteins associated with electron transport chain (ETC) complex I (NADH dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) and could be generally categorized as having proton gradient, ATP synthesis, and chaperone activities. In contrast, transcript levels of ETC complex II, succinate dehydrogenase, remained unchanged. Putative dioxin response elements were computationally found in the promoter regions of the 8 dose-responsive genes. This high-throughput approach suggests that TCDD alters the expression of genes associated with mitochondrial function which may contribute to TCDD-elicited mitochondrial toxicity. PMID:20399798

  5. Effects of TCDD on the expression of nuclear encoded mitochondrial genes

    SciTech Connect

    Forgacs, Agnes L.; Burgoon, Lyle D.; Lynn, Scott G.; LaPres, John J.; Zacharewski, Timothy

    2010-07-15

    Generation of mitochondrial reactive oxygen species (ROS) can be perturbed following exposure to environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Reports indicate that the aryl hydrocarbon receptor (AhR) mediates TCDD-induced sustained hepatic oxidative stress by decreasing hepatic ATP levels and through hyperpolarization of the inner mitochondrial membrane. To further elucidate the effects of TCDD on the mitochondria, high-throughput quantitative real-time PCR (HTP-QRTPCR) was used to evaluate the expression of 90 nuclear genes encoding mitochondrial proteins involved in electron transport, oxidative phosphorylation, uncoupling, and associated chaperones. HTP-QRTPCR analysis of time course (30 {mu}g/kg TCDD at 2, 4, 8, 12, 18, 24, 72, and 168 h) liver samples obtained from orally gavaged immature, ovariectomized C57BL/6 mice identified 54 differentially expressed genes (|fold change| > 1.5 and P-value < 0.1). Of these, 8 exhibited a sigmoidal or exponential dose-response profile (0.03 to 300 {mu}g/kg TCDD) at 4, 24 or 72 h. Dose-responsive genes encoded proteins associated with electron transport chain (ETC) complexes I (NADH dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) and could be generally categorized as having proton gradient, ATP synthesis, and chaperone activities. In contrast, transcript levels of ETC complex II, succinate dehydrogenase, remained unchanged. Putative dioxin response elements were computationally found in the promoter regions of all 8 dose-responsive genes. This high-throughput approach suggests that TCDD alters the expression of genes associated with mitochondrial function which may contribute to TCDD-elicited mitochondrial toxicity.

  6. Mitochondrial-related gene expression changes are sensitive to agonal-pH state: implications for brain disorders

    PubMed Central

    Vawter, MP; Tomita, H; Meng, F; Bolstad, B; Li, J; Evans, S; Choudary, P; Atz, M; Shao, L; Neal, C; Walsh, DM; Burmeister, M; Speed, T; Myers, R; Jones, EG; Watson, SJ; Akil, H; Bunney, WE

    2010-01-01

    Mitochondrial defects in gene expression have been implicated in the pathophysiology of bipolar disorder and schizophrenia. We have now contrasted control brains with low pH versus high pH and showed that 28% of genes in mitochondrial-related pathways meet criteria for differential expression. A majority of genes in the mitochondrial, chaperone and proteasome pathways of nuclear DNA-encoded gene expression were decreased with decreased brain pH, whereas a majority of genes in the apoptotic and reactive oxygen stress pathways showed an increased gene expression with a decreased brain pH. There was a significant increase in mitochondrial DNA copy number and mitochondrial DNA gene expression with increased agonal duration. To minimize effects of agonal-pH state on mood disorder comparisons, two classic approaches were used, removing all subjects with low pH and agonal factors from analysis, or grouping low and high pH as a separate variable. Three groups of potential candidate genes emerged that may be mood disorder related: (a) genes that showed no sensitivity to pH but were differentially expressed in bipolar disorder or major depressive disorder; (b) genes that were altered by agonal-pH in one direction but altered in mood disorder in the opposite direction to agonal-pH and (c) genes with agonal-pH sensitivity that displayed the same direction of changes in mood disorder. Genes from these categories such as NR4A1 and HSPA2 were confirmed with Q-PCR. The interpretation of postmortem brain studies involving broad mitochondrial gene expression and related pathway alterations must be monitored against the strong effect of agonal-pH state. Genes with the least sensitivity to agonal-pH could present a starting point for candidate gene search in neuropsychiatric disorders. PMID:16636682

  7. Mitochondrial-related gene expression changes are sensitive to agonal-pH state: implications for brain disorders.

    PubMed

    Vawter, M P; Tomita, H; Meng, F; Bolstad, B; Li, J; Evans, S; Choudary, P; Atz, M; Shao, L; Neal, C; Walsh, D M; Burmeister, M; Speed, T; Myers, R; Jones, E G; Watson, S J; Akil, H; Bunney, W E

    2006-07-01

    Mitochondrial defects in gene expression have been implicated in the pathophysiology of bipolar disorder and schizophrenia. We have now contrasted control brains with low pH versus high pH and showed that 28% of genes in mitochondrial-related pathways meet criteria for differential expression. A majority of genes in the mitochondrial, chaperone and proteasome pathways of nuclear DNA-encoded gene expression were decreased with decreased brain pH, whereas a majority of genes in the apoptotic and reactive oxygen stress pathways showed an increased gene expression with a decreased brain pH. There was a significant increase in mitochondrial DNA copy number and mitochondrial DNA gene expression with increased agonal duration. To minimize effects of agonal-pH state on mood disorder comparisons, two classic approaches were used, removing all subjects with low pH and agonal factors from analysis, or grouping low and high pH as a separate variable. Three groups of potential candidate genes emerged that may be mood disorder related: (a) genes that showed no sensitivity to pH but were differentially expressed in bipolar disorder or major depressive disorder; (b) genes that were altered by agonal-pH in one direction but altered in mood disorder in the opposite direction to agonal-pH and (c) genes with agonal-pH sensitivity that displayed the same direction of changes in mood disorder. Genes from these categories such as NR4A1 and HSPA2 were confirmed with Q-PCR. The interpretation of postmortem brain studies involving broad mitochondrial gene expression and related pathway alterations must be monitored against the strong effect of agonal-pH state. Genes with the least sensitivity to agonal-pH could present a starting point for candidate gene search in neuropsychiatric disorders. PMID:16636682

  8. Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Regulate Cortical Neural Progenitor Differentiation

    PubMed Central

    Hou, Yan; Mattson, Mark P.; Cheng, Aiwu

    2013-01-01

    In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca2+ fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation. PMID:24116142

  9. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    PubMed Central

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

  10. Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein

    PubMed Central

    Benito-Pescador, David; Santander, Daniela; Arranz, M.; Díaz-Mínguez, José M.; Eslava, Arturo P.; van Kan, Jan A. L.; Benito, Ernesto P.

    2016-01-01

    Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor. PMID:26952144

  11. Mitochondrial haplotype analysis for differentiation of isolates of Phytophthora cinnamomi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While Phytophthora cinnamomi is heterothallic, there are few instances of successful crossing in laboratory experiments and analysis of field populations indicates a clonally reproducing population. In the absence of sexual recombination the ability to monitor mitochondrial haplotypes may provide a...

  12. The role of myeloid differentiation factor 88 on mitochondrial dysfunction of peritoneal leukocytes during polymicrobial sepsis

    PubMed Central

    Zou, Lin; Chen, Dunjin; Chao, Wei

    2016-01-01

    Objective To investigate the role of myeloid differentiation factor 88 (MyD88) on mitochondrial dysfunction of peritoneal leukocytes during polymicrobial sepsis. Material and methods Polymicrobial peritonitis, a clinically relevant mouse model of sepsis, was generated by cecum ligation and puncture (CLP) in both male C57BL/6J wild-type (WT) and MyD88 knockout (MyD88–/–) mice. Twenty-four hours after surgeries, peritoneal leukocytes were collected and four parameters of mitochondrial function, including total intracellular and mitochondrial ROS burst, mitochondrial membrane depolarization and ATP depletion, were measured by flow cytometry or ATP assay, and then compared. Results Polymicrobial sepsis led to a marked mitochondrial dysfunction of peritoneal leukocytes with total intracellular and mitochondrial ROS overproduction, decreased mitochondrial membrane potential and reduced intracellular ATP production. In comparison, there was no significant difference in the extent of mitochondrial dysfunction of peritoneal leukocytes between WT and MyD88–/– septic mice. Conclusions MyD88 may be not sufficient to regulate mitochondrial dysfunction of peritoneal leukocytes during polymicrobial sepsis. PMID:27536200

  13. Mitochondrial and nuclear genomic responses to loss of LRPPRC expression.

    PubMed

    Gohil, Vishal M; Nilsson, Roland; Belcher-Timme, Casey A; Luo, Biao; Root, David E; Mootha, Vamsi K

    2010-04-30

    Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remain challenging. Here, we introduce a genomic strategy to characterize such genes functionally, and we apply it to LRPPRC, a poorly studied gene that is mutated in Leigh syndrome, French-Canadian type (LSFC). We utilize RNA interference to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. We then combine genome-wide expression profiling with gene set enrichment analysis to identify cellular responses that correlate with the loss of LRPPRC. Using this strategy, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA-encoded mRNAs, but not the rRNAs, providing mechanistic insights into the enzymatic defects observed in the disease. Our analysis shows that nuclear genes encoding mitochondrial proteins are not collectively affected by the loss of LRPPRC. We do observe altered expression of genes related to hexose metabolism, prostaglandin synthesis, and glycosphingolipid biology that may either play an adaptive role in cell survival or contribute to pathogenesis. The combination of genetic perturbation, genomic profiling, and pathway analysis represents a generic strategy for understanding disease pathogenesis. PMID:20220140

  14. Dihydroorotate dehydrogenase depletion hampers mitochondrial function and osteogenic differentiation in osteoblasts.

    PubMed

    Fang, JingXian; Yamaza, Haruyoshi; Uchiumi, Takeshi; Hoshino, Yoshihiro; Masuda, Keiji; Hirofuji, Yuta; Wagener, Frank A D T G; Kang, Dongchon; Nonaka, Kazuaki

    2016-06-01

    Mutation of the dihydroorotate dehydrogenase (DHODH) gene is responsible for Miller syndrome, which is characterized by craniofacial malformations with limb abnormalities. We previously demonstrated that DHODH was involved in forming a mitochondrial supercomplex and that mutated DHODH led to protein instability, loss of enzyme activity, and increased levels of reactive oxygen species in HeLa cells. To explore the etiology of Miller syndrome in more detail, we investigated the effects of DHODH inhibition in the cells involved in skeletal structure. Dihydroorotate dehydrogenase in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was knocked down by specific small interfering RNAs (siRNAs), and cell proliferation, ATP production, and expression of bone-related genes were investigated in these cells. After depletion of DHODH using specific siRNAs, inhibition of cell proliferation and cell cycle arrest occurred in MC3T3-E1 cells. In addition, ATP production was reduced in whole cells, especially in mitochondria. Furthermore, the levels of runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs were lower in DHODH siRNA-treated cells compared with controls. These data suggest that depletion of DHODH affects the differentiation and maturation of osteoblasts. This study shows that mitochondrial dysfunction by DHODH depletion in osteoblasts can be directly linked to the abnormal bone formation in Miller syndrome. PMID:27086500

  15. Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for myogenic differentiation

    PubMed Central

    De Palma, C; Falcone, S; Pisoni, S; Cipolat, S; Panzeri, C; Pambianco, S; Pisconti, A; Allevi, R; Bassi, MT; Cossu, G; Pozzan, T; Moncada, S; Scorrano, L; Brunelli, S; Clementi, E

    2011-01-01

    During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. The functional relevance and the molecular mechanisms driving the formation of this mitochondrial network are unknown. We now show that mitochondrial elongation is required for myogenesis to occur and that this event depends on the cellular generation of nitric oxide (NO). Inhibition of NO synthesis in myogenic precursor cells leads to inhibition of mitochondrial elongation and of myogenic differentiation. This is due to the enhanced activity, translocation and docking of the pro-fission GTPase dynamin-related protein-1 (Drp1) to mitochondria, leading also to a latent mitochondrial dysfunction that increased sensitivity to apoptotic stimuli. These effects of NO inhibition were not observed in myogenic precursor cells containing a dominant-negative form of Drp1. Both NO-dependent repression of Drp1 action and maintenance of mitochondrial integrity and function were mediated through the soluble guanylate cyclase. These data uncover a novel level of regulation of differentiation linking mitochondrial morphology and function to myogenic differentiation. PMID:20467441

  16. Effects of dietary fatty acids on mitochondrial phospholipid compositions, oxidative status and mitochondrial gene expression of zebrafish at different ages.

    PubMed

    Betancor, M B; Almaida-Pagán, P F; Hernández, A; Tocher, D R

    2015-10-01

    Mitochondrial decay is generally associated with impairment in the organelle bioenergetics function and increased oxidative stress, and it appears that deterioration of mitochondrial inner membrane phospholipids (PL) and accumulation of mitochondrial DNA (mtDNA) mutations are among the main mechanisms involved in this process. In the present study, mitochondrial membrane PL compositions, oxidative status (TBARS content and SOD activity) and mtDNA gene expression of muscle and liver were analyzed in zebrafish fed two diets with lipid supplied either by rapeseed oil (RO) or a blend 60:40 of RO and DHA500 TG oil (DHA). Two feeding trials were performed using zebrafish from the same population of two ages (8 and 21 months). Dietary FA composition affected fish growth in 8-month-old animals, which could be related to an increase in stress promoted by diet composition. Lipid peroxidation was considerably higher in mitochondria of 8-month-old zebrafish fed the DHA diet than in animals fed the RO diet. This could indicate higher oxidative damage to mitochondrial lipids, very likely due to increased incorporation of DHA in PL of mitochondrial membranes. Lipids would be among the first molecules affected by mitochondrial reactive oxygen species, and lipid peroxidation could propagate oxidative reactions that would damage other molecules, including mtDNA. Mitochondrial lipid peroxidation and gene expression of 21-month-old fish showed lower responsiveness to diet composition than those of younger fish. Differences found in the effect of diet composition on mitochondrial lipids between the two age groups could be indicating age-related changes in the ability to maintain structural homeostasis of mitochondrial membranes. PMID:26156499

  17. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene mcu

    PubMed Central

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E.; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E.; Hines, Kevin J.; Ragheb, Jonathan; Jog, Neelakshi R.; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett; Cheung, Joseph Y.; Kurosaki, Tomohiro; Gill, Donald L.; Madesh, Muniswamy

    2016-01-01

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function and, when cytosolic Ca2+ concentrations become too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+-permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor CREB directly bound the mcu promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulate mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+-dependent activation of CREB controls the Ca2+-uptake capability of mitochondria and hence regulates mitochondrial metabolism. PMID:25737585

  18. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU.

    PubMed

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E; Hines, Kevin J; Ragheb, Jonathan; Jog, Neelakshi R; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett A; Cheung, Joseph Y; Kurosaki, Tomohiro; Gill, Donald L; Madesh, Muniswamy

    2015-03-01

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism. PMID:25737585

  19. Mitochondrial gene expression, antioxidant responses, and histopathology after cadmium exposure.

    PubMed

    Al Kaddissi, Simone; Legeay, Alexia; Elia, Antonia Concetta; Gonzalez, Patrice; Floriani, Magali; Cavalie, Isabelle; Massabuau, Jean-Charles; Gilbin, Rodolphe; Simon, Olivier

    2014-08-01

    The present study investigates cadmium effects on the transcription of mitochondrial genes of Procambarus clarkii after acute (0.05, 0.5, and 5 mg Cd/L; 4-10 days) and chronic exposures (10 μg Cd/L; 30-60 days). Transcriptional responses of cox1, atp6, and 12S using quantitative real-time RT-PCR were assessed in gills and hepatopancreas. Additionally, the expression levels of genes involved in detoxification and/or oxidative stress responses [mt, sod(Mn)] and enzymatic activities of antioxidants (SOD, CAT, GPX, and GST) were analyzed. The histopathological effects in hepatopancreas of crayfish were evaluated by light microscopy. Relationships between endpoints at different levels of biological organization and Cd bioaccumulation were also examined. Cd induced high levels of bioaccumulation, which was followed by mitochondrial dysfunction and histological alterations in both experiments. Moreover, perturbations in the defence mechanisms against oxidative stress tended to increase with time. Results also showed that molecular responses can vary depending on the intensity and duration of the chemical stress applied to the organisms and that the study of mt gene expression levels seemed to be the best tool to assess Cd intoxication. PMID:23065898

  20. Hepatitis C Virus Attenuates Mitochondrial Lipid β-Oxidation by Downregulating Mitochondrial Trifunctional-Protein Expression

    PubMed Central

    Munakata, Tsubasa; Kohara, Michinori; Siddiqui, Aleem; Peers, Chris

    2015-01-01

    ABSTRACT The course of hepatitis C virus (HCV) infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms, including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion, and/or attenuation of lipid β-oxidation. However, little is known about the effects of HCV on lipid β-oxidation. A previous proteomics study revealed that HCV interacted with both the α- and β-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyzes the last 3 steps of mitochondrial lipid β-oxidation for cellular energy production. Here we show that in HCV-infected Huh7.5 cells, lipid β-oxidation was significantly attenuated. Consistently with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss-of-function study showed that MTP depletion rendered cells less responsive to alpha interferon (IFN-α) treatment by impairing IFN-stimulated gene expression. These aspects of host-virus interaction explain how HCV alters host energy homeostasis and how it may also contribute to the establishment of persistent infection in the liver. IMPORTANCE HCV infection triggers metabolic alterations, which lead to significant disease outcomes, such as fatty liver (steatosis). This study revealed that HCV impairs mitochondrial lipid β-oxidation, which results in low lipid combustion. On the other hand, the HCV-induced defects in metabolic status played an important role in the control of the type I interferon system. Under the conditions of impaired lipid β-oxidation, host cells were less responsive to the ability of exogenously added IFN-α to suppress HCV replication. This suggests that interference with lipid β-oxidation may assist the virus in the establishment of a long-term, persistent infection. Further understanding of this aspect of virus-host interaction may lead to improvements in the current

  1. Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype

    PubMed Central

    Branco, Ana F.; Pereira, Susana P.; Gonzalez, Susana; Gusev, Oleg; Rizvanov, Albert A.; Oliveira, Paulo J.

    2015-01-01

    H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays. PMID:26121149

  2. Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype.

    PubMed

    Branco, Ana F; Pereira, Susana P; Gonzalez, Susana; Gusev, Oleg; Rizvanov, Albert A; Oliveira, Paulo J

    2015-01-01

    H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays. PMID:26121149

  3. Inferring differentiation pathways from gene expression

    PubMed Central

    Costa, Ivan G.; Roepcke, Stefan; Hafemeister, Christoph; Schliep, Alexander

    2008-01-01

    Motivation: The regulation of proliferation and differentiation of embryonic and adult stem cells into mature cells is central to developmental biology. Gene expression measured in distinguishable developmental stages helps to elucidate underlying molecular processes. In previous work we showed that functional gene modules, which act distinctly in the course of development, can be represented by a mixture of trees. In general, the similarities in the gene expression programs of cell populations reflect the similarities in the differentiation path. Results: We propose a novel model for gene expression profiles and an unsupervised learning method to estimate developmental similarity and infer differentiation pathways. We assess the performance of our model on simulated data and compare it with favorable results to related methods. We also infer differentiation pathways and predict functional modules in gene expression data of lymphoid development. Conclusions: We demonstrate for the first time how, in principal, the incorporation of structural knowledge about the dependence structure helps to reveal differentiation pathways and potentially relevant functional gene modules from microarray datasets. Our method applies in any area of developmental biology where it is possible to obtain cells of distinguishable differentiation stages. Availability: The implementation of our method (GPL license), data and additional results are available at http://algorithmics.molgen.mpg.de/Supplements/InfDif/ Contact: filho@molgen.mpg.de, schliep@molgen.mpg.de Supplementary information: Supplementary data is available at Bioinformatics online. PMID:18586709

  4. Differential co-expression analysis of venous thromboembolism based on gene expression profile data

    PubMed Central

    MING, ZHIBING; DING, WENBIN; YUAN, RUIFAN; JIN, JIE; LI, XIAOQIANG

    2016-01-01

    The aim of the present study was to screen differentially co-expressed genes and the involved transcription factors (TFs) and microRNAs (miRNAs) in venous thromboembolism (VTE). Microarray data of GSE19151 were downloaded from Gene Expression Omnibus, including 70 patients with VTE and 63 healthy controls. Principal component analysis (PCA) was performed using R software. Differential co-expression analysis was performed using R, followed by screening of modules using Cytoscape. Functional annotation was performed using Database for Annotation, Visualization, and Integrated Discovery. Moreover, Fisher test was used to screen key TFs and miRNAs for the modules. PCA revealed the disease and healthy samples could not be distinguished at the gene expression level. A total of 4,796 upregulated differentially co-expressed genes (e.g. zinc finger protein 264, electron-transfer-flavoprotein, beta polypeptide and Janus kinase 2) and 3,629 downregulated differentially co-expressed genes (e.g. adenylate cyclase 7 and single-stranded DNA binding protein 2) were identified, which were further mined to obtain 17 and eight modules separately. Functional annotation revealed that the largest upregulated module was primarily associated with acetylation and the largest downregulated module was mainly involved in mitochondrion. Moreover, 48 TFs and 62 miRNA families were screened for the 17 upregulated modules, such as E2F transcription factor 4, miR-30 and miR-135 regulating the largest module. Conversely, 35 TFs and 18 miRNA families were identified for the 8 downregulated modules, including mitochondrial ribosomal protein S12 and miR-23 regulating the largest module. Differentially co-expressed genes regulated by TFs and miRNAs may jointly contribute to the abnormal acetylation and mitochondrion presentation in the progression of VTE. PMID:27284300

  5. Meta-analysis of differentially expressed genes in ankylosing spondylitis.

    PubMed

    Lee, Y H; Song, G G

    2015-01-01

    The purpose of this study was to identify differentially expressed (DE) genes and biological processes associated with changes in gene expression in ankylosing spondylitis (AS). We performed a meta-analysis using the integrative meta-analysis of expression data program on publicly available microarray AS Gene Expression Omnibus (GEO) datasets. We performed Gene Ontology (GO) enrichment analyses and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes. Four GEO datasets, including 31 patients with AS and 39 controls, were available for the meta-analysis. We identified 65 genes across the studies that were consistently DE in patients with AS vs controls (23 upregulated and 42 downregulated). The upregulated gene with the largest effect size (ES; -1.2628, P = 0.020951) was integral membrane protein 2A (ITM2A), which is expressed by CD4+ T cells and plays a role in activation of T cells. The downregulated gene with the largest ES (1.2299, P = 0.040075) was mitochondrial ribosomal protein S11 (MRPS11). The most significant GO enrichment was in the respiratory electron transport chain category (P = 1.67 x 10-9). Therefore, our meta-analysis identified genes that were consistently DE as well as biological pathways associated with gene expression changes in AS. PMID:26125709

  6. Low-dose ionizing radiation induces mitochondrial fusion and increases expression of mitochondrial complexes I and III in hippocampal neurons

    PubMed Central

    Chang, Chuang-Rung; Kao, Mou-Chieh; Chen, Kuan-Wei; Chiu, Shih-Che; Hsu, Ming-Ling; Hsiang, I-Chou; Chen, Yu-Jen; Chen, Linyi

    2015-01-01

    High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation. PMID:26415228

  7. Expression and Purification of Mitochondrial RNA Polymerase and Transcription Factor A from Drosophila melanogaster.

    PubMed

    Gajewski, John P; Arnold, Jamie J; Salminen, Tiina S; Kaguni, Laurie S; Cameron, Craig E

    2016-01-01

    Mitochondrial gene expression is essential in all organisms. Our understanding of mitochondrial transcription on a biochemical level has been limited by the inability to purify the individual protein components involved in mitochondrial gene expression. Recently, new systems have been identified that permit purification of these proteins from bacteria. However, the generalizability of these systems is not clear. Here, we have applied the technology from the Cameron lab to express and purify mitochondrial RNA polymerase and transcription factor A from Drosophila melanogaster. We show that the use of SUMO system to produce SUMO fusion proteins in bacteria is effective not only for the human and mouse proteins, but also for the fly proteins. The application of this system to produce the mitochondrial proteins from other organisms should permit detailed understanding of mitochondrial transcription from any organism. PMID:26530684

  8. Differential placental gene expression in severe preeclampsia.

    PubMed

    Sitras, V; Paulssen, R H; Grønaas, H; Leirvik, J; Hanssen, T A; Vårtun, A; Acharya, G

    2009-05-01

    We investigated the global placental gene expression profile in severe preeclampsia. Twenty-one women were randomly selected from 50 participants with uncomplicated pregnancies to match 21 patients with severe preeclampsia. A 30K Human Genome Survey Microarray v.2.0 (Applied Biosystems) was used to evaluate the gene expression profile. After RNA isolation, five preeclamptic placentas were excluded due to poor RNA quality. The series composed of 37 hybridizations in a one-channel detection system of chemiluminescence emitted by the microarrays. An empirical Bayes analysis was applied to find differentially expressed genes. In preeclamptic placentas 213 genes were significantly (fold-change>or=2 and pdifferentially expressed genes were associated with Alzheimer disease, angiogenesis, Notch-, TGFbeta- and VEGF-signalling pathways. Sixteen genes best discriminated preeclamptic from normal placentas. Comparison between early- (<34 weeks) and late-onset preeclampsia showed 168 differentially expressed genes with oxidative stress, inflammation, and endothelin signalling pathways mainly involved in early-onset disease. Validation of the microarray results was performed by RT-PCR, quantitative urine hCG measurement and placental histopathologic examination. In summary, placental gene expression is altered in preeclampsia and we provide a comprehensive list of the differentially expressed genes. Placental gene expression is different between early- and late-onset preeclampsia, suggesting differences in pathophysiology. PMID:19249095

  9. Introducing Knowledge into Differential Expression Analysis

    PubMed Central

    Biecek, Przemysław; Tiuryn, Jerzy; Vingron, Martin

    2010-01-01

    Abstract Gene expression measurements allow determining sets of up- or down-regulated, or unchanged genes in a particular experimental condition. Additional biological knowledge can suggest examples of genes from one of these sets. For instance, known target genes of a transcriptional activator are expected, but are not certain to go down after this activator is knocked out. Available differential expression analysis tools do not take such imprecise examples into account. Here we put forward a novel partially supervised mixture modeling methodology for differential expression analysis. Our approach, guided by imprecise examples, clusters expression data into differentially expressed and unchanged genes. The partially supervised methodology is implemented by two methods: a newly introduced belief-based mixture modeling, and soft-label mixture modeling, a method proved efficient in other applications. We investigate on synthetic data the input example settings favorable for each method. In our tests, both belief-based and soft-label methods prove their advantage over semi-supervised mixture modeling in correcting for erroneous examples. We also compare them to alternative differential expression analysis approaches, showing that incorporation of knowledge yields better performance. We present a broad range of knowledge sources and data to which our partially supervised methodology can be applied. First, we determine targets of Ste12 based on yeast knockout data, guided by a Ste12 DNA-binding experiment. Second, we distinguish miR-1 from miR-124 targets in human by clustering expression data under transfection experiments of both microRNAs, using their computationally predicted targets as examples. Finally, we utilize literature knowledge to improve clustering of time-course expression profiles. PMID:20726790

  10. Dietary fatty acids affect mitochondrial phospholipid compositions and mitochondrial gene expression of rainbow trout liver at different ages.

    PubMed

    Almaida-Pagán, P F; De Santis, C; Rubio-Mejía, O L; Tocher, D R

    2015-01-01

    Mitochondria are among the first responders to various stressors that challenge the homeostasis of cells and organisms. Mitochondrial decay is generally associated with impairment in the organelle bioenergetics function and increased oxidative stress, and it appears that deterioration of mitochondrial inner membrane phospholipids (PL), particularly cardiolipin (CL), and accumulation of mitochondrial DNA (mtDNA) mutations are among the main mechanisms involved in this process. In the present study, liver mitochondrial membrane PL compositions, lipid peroxidation, and mtDNA gene expression were analyzed in rainbow trout fed three diets with the same base formulation but with lipid supplied either by fish oil (FO), rapeseed oil (RO), or high DHA oil (DHA) during 6 weeks. Specifically, two feeding trials were performed using fish from the same population of two ages (1 and 3 years), and PL class compositions of liver mitochondria, fatty acid composition of individual PL classes, TBARS content, and mtDNA expression were determined. Dietary fatty acid composition strongly affected mitochondrial membrane composition from trout liver but observed changes did not fully reflect the diet, particularly when it contained high DHA. The changes were PL specific, CL being particularly resistant to changes in DHA. Some significant differences observed in expression of mtDNA with diet may suggest long-term dietary effects in mitochondrial gene expression which could affect electron transport chain function. All the changes were influenced by fish age, which could be related to the different growth rates observed between 1- and 3-year-old trout but that could also indicate age-related changes in the ability to maintain structural homeostasis of mitochondrial membranes. PMID:25398637

  11. Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

    PubMed Central

    Rajala, Nina; Hensen, Fenna; Wessels, Hans J. C. T.; Ives, Daniel; Gloerich, Jolein; Spelbrink, Johannes N.

    2015-01-01

    Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data. PMID:25695250

  12. Mitochondrial biogenesis during differentiation of Artemia salina cysts.

    PubMed

    Schmitt, H; Grossfeld, H; Littauer, U Z

    1973-09-01

    Mitochondria isolated from cysts of Artemia salina (brine shrimp) were found to be devoid of cristae and to possess a low respiratory capability. Hydration of the cysts induces marked biochemical and morphological changes in the mitochondria. Their biogenesis proceeds in two stages. The first stage is completed within 1 h and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c and perhaps some morphological changes. In the second stage there is an increase in the protein-synthesizing capacity of the mitochondria as well as striking changes in mitochondrial morphology leading to the formation of cristae. PMID:4355924

  13. Differential survival and reproductive performance across three mitochondrial lineages in Melita plumulosa following naphthalene exposure.

    PubMed

    Chung, Pann Pann; Ballard, J William O; Hyne, Ross V

    2013-10-01

    Populations subject to anthropogenic contaminants often display altered patterns of genetic variation, including decreased genetic variability. Selective pressures of contaminant exposure are also reflected in differential tolerance between genotypes. An industrial chemical spill in a major eastern Australian waterway in July 2006 resulted in altered patterns of genetic variability in a nearby population of the amphipod, Melita plumulosa for up to one year post-spill, despite the site being declared clean after 48 h. Here, we investigate the toxicant response of three mitochondrial lines naturally occurring at the impacted site by comparing survivorship and life-history trait variables following naphthalene exposure. Overall, M. plumulosa demonstrated differential survivorship between mitochondrial lines under exposure to high concentrations of naphthalene. In addition, we identified differential fecundity and frequencies of gravidity in female amphipods between the mitochondrial haplotypes examined. These findings suggest that the patterns of genetic variability previously identified may be linked with differential tolerance and/or reproductive performance between mitochondrial lineages. PMID:23800590

  14. Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

    PubMed

    Prowse, Andrew B J; Chong, Fenny; Elliott, David A; Elefanty, Andrew G; Stanley, Edouard G; Gray, Peter P; Munro, Trent P; Osborne, Geoffrey W

    2012-01-01

    Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag. PMID:23284940

  15. Expression of a mitochondrial progesterone receptor in human spermatozoa correlates with a progestin-dependent increase in mitochondrial membrane potential.

    PubMed

    Tantibhedhyangkul, J; Hawkins, K C; Dai, Q; Mu, K; Dunn, C N; Miller, S E; Price, T M

    2014-11-01

    The hyperactivation of human spermatozoa necessary for fertilization requires a substantial increase in cellular energy production. The factors responsible for increasing cellular energy remain poorly defined. This article proposes a role for a novel mitochondrial progesterone receptor (PR-M) in modulation of mitochondrial activity. Basic science studies demonstrate a 38 kDa protein with western blot analysis, consistent with PR-M; whereas imaging studies with confocal and immunoelectron microscopy demonstrate a PR on the mitochondria. Treatment with a PR-specific progestin shows increased mitochondrial membrane potential, not related to induction of an acrosome reaction. The increase in mitochondrial membrane potential was inhibited by a specific PR antagonist, but not affected by an inhibitor to the progesterone-dependent Catsper voltage-activated channel. In conclusion, these studies suggest expression of a novel mitochondrial PR in human spermatozoa with a progestin-dependent increase in mitochondrial activity. This mechanism may serve to enhance cellular energy production as the spermatozoa traverse the female genital tract being exposed to increasing concentrations of progesterone. PMID:25187426

  16. Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model

    PubMed Central

    Keller, Amy C.; Knaub, Leslie A.; McClatchey, P. Mason; Connon, Chelsea A.; Bouchard, Ron; Miller, Matthew W.; Geary, Kate E.; Walker, Lori A.; Klemm, Dwight J.; Reusch, Jane E. B.

    2016-01-01

    Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p < 0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p < 0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets. PMID:27034743

  17. Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model.

    PubMed

    Keller, Amy C; Knaub, Leslie A; McClatchey, P Mason; Connon, Chelsea A; Bouchard, Ron; Miller, Matthew W; Geary, Kate E; Walker, Lori A; Klemm, Dwight J; Reusch, Jane E B

    2016-01-01

    Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p < 0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p < 0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets. PMID:27034743

  18. Cigarette smoke decreases mitochondrial porin expression and steroidogenesis

    SciTech Connect

    Bose, Mahuya; Whittal, Randy M.; Gairola, C. Gary; Bose, Himangshu S.

    2008-03-01

    Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane for steroidogenesis. Here, we investigated the effect of cigarette smoke (CS) on steroidogenesis using adrenal mitochondria isolated from mice chronically exposed to CS. Steroidogenesis was decreased approximately 78% in CS-exposed mitochondria, as measured by synthesis of the steroid hormone precursor pregnenolone. This effect was accompanied by decreased mitochondrial import of {sup 35}S-StAR. Further characterization of the imported {sup 35}S-StAR by native gradient PAGE revealed the presence of a high molecular weight complex in both control and CS-exposed groups. Following density gradient fractionation of {sup 35}S-StAR that had been extracted from control mitochondria, precursor StAR could be found in fractions 2-6 and smaller-sized StAR complexes in fractions 6-13. In the CS-exposed group, the appearance of precursor shifted from fraction 1-6 and the smaller complexes in fractions 6-9 disappeared. Mass spectrometric analysis revealed that the {sup 35}S-StAR-associated protein complex was composed of several resident matrix proteins as well as the OMM resident, VDAC. VDAC expression was greatly reduced by CS, and blockage of VDAC with Koenig's polyanion decreased pregnenolone synthesis in isolated mitochondria. Taken together, these results suggest that VDAC may participate in steroidogenesis by promoting StAR interaction with the OMM and that CS may inhibit steroidogenesis by reducing VDAC-StAR interactions.

  19. Mitochondrial (Dys)function in Adipocyte (De)differentiation and Systemic Metabolic Alterations

    PubMed Central

    De Pauw, Aurélia; Tejerina, Silvia; Raes, Martine; Keijer, Jaap; Arnould, Thierry

    2009-01-01

    In mammals, adipose tissue, composed of BAT and WAT, collaborates in energy partitioning and performs metabolic regulatory functions. It is the most flexible tissue in the body, because it is remodeled in size and shape by modifications in adipocyte cell size and/or number, depending on developmental status and energy fluxes. Although numerous reviews have focused on the differentiation program of both brown and white adipocytes as well as on the pathophysiological role of white adipose tissues, the importance of mitochondrial activity in the differentiation or the dedifferentiation programs of adipose cells and in systemic metabolic alterations has not been extensively reviewed previously. Here, we address the crucial role of mitochondrial functions during adipogenesis and in mature adipocytes and discuss the cellular responses of white adipocytes to mitochondrial activity impairment. In addition, we discuss the increase in scientific knowledge regarding mitochondrial functions in the last 10 years and the recent suspicion of mitochondrial dysfunction in several 21st century epidemics (ie, obesity and diabetes), as well as in lipodystrophy found in HIV-treated patients, which can contribute to the development of new therapeutic strategies targeting adipocyte mitochondria. PMID:19700756

  20. Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

    PubMed Central

    Jokinen, Riikka; Lahtinen, Taina; Marttinen, Paula; Myöhänen, Maarit; Ruotsalainen, Pilvi; Yeung, Nicolas; Shvetsova, Antonina; Kastaniotis, Alexander J.; Hiltunen, J. Kalervo; Öhman, Tiina; Nyman, Tuula A.; Weiler, Hartmut; Battersby, Brendan J.

    2015-01-01

    Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment. PMID:25808953

  1. The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

    PubMed

    Stoebner, P E; Carayon, P; Penarier, G; Fréchin, N; Barnéon, G; Casellas, P; Cano, J P; Meynadier, J; Meunier, L

    1999-06-01

    The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure. PMID:10354064

  2. Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes

    PubMed Central

    Ziabreva, Iryna; Campbell, Graham; Rist, Julia; Zambonin, Jessica; Rorbach, Joanna; Wydro, Mateusz M; Lassmann, Hans; Franklin, Robin J M; Mahad, Don

    2010-01-01

    Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the in vitro impact of established inhibitors of mitochondrial respiratory chain complex IV or cytochrome c oxidase on oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes as well as on differentiation capacity of OPCs from P0 rat. Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders. © 2010 Wiley-Liss, Inc. PMID:20665559

  3. Gene expression profiling in mitochondrial disease: assessment of microarray accuracy by high-throughput Q-PCR.

    PubMed

    Beckman, Kenneth B; Lee, Kathleen Y; Golden, Tamara; Melov, Simon

    2004-09-01

    Mitochondrial diseases are a heterogeneous array of disorders with a complex etiology. Use of microarrays as a tool to investigate complex human disease is increasingly common, however, a principle drawback of microarrays is their limited dynamic range, due to the poor quantification of weak signals. Although it is generally understood that low-intensity microarray 'spots' may be unreliable, there exists little documentation of their accuracy. Quantitative PCR (Q-PCR) is frequently used to validate microarray data, yet few Q-PCR validation studies have focused on the accuracy of low-intensity microarray signals. Hence, we have used Q-PCR to systematically assess microarray accuracy as a function of signal strength in a mouse model of mitochondrial disease, the superoxide dismutase 2 (SOD2) nullizygous mouse. We have focused on a unique category of data--spots with only one weak signal in a two-dye comparative hybridization--and show that such 'high-low' signal intensities are common for differentially expressed genes. This category of differential expression may be more important in mitochondrial disease in which there are often mosaic expression patterns due to the idiosyncratic distribution of mutant mtDNA in heteroplasmic individuals. Using RNA from the SOD2 mouse, we found that when spotted cDNA microarray data are filtered for quality (low variance between many technical replicates) and spot intensity (above a negative control threshold in both channels), there is an excellent quantitative concordance with Q-PCR (R2 = 0.94). The accuracy of gene expression ratios from low-intensity spots (R2 = 0.27) and 'high-low' spots (R2 = 0.32) is considerably lower. Our results should serve as guidelines for microarray interpretation and the selection of genes for validation in mitochondrial disorders. PMID:16120406

  4. Impact of mitochondrial telomerase over-expression on drug resistance of hepatocellular carcinoma

    PubMed Central

    Yan, Jing; Zhou, Yuan; Chen, Daixing; Li, Lili; Yang, Xin; You, Yang; Ling, Xianlong

    2015-01-01

    Background: The efficacy of chemotherapy in patients with hepatocellular carcinomas still poor due to multidrug resistance. This study aimed to investigate the impact of the over-expressed mitochondrial human telomerase reverse transcriptase on multidrug resistance of hepatocellular carcinomas. Methods: HepG2 and SK-Hep1 cell lines were used. And sensitivity to chemotherapeutic drugs was detected. Results: Mitochondrial human telomerase reverse transcriptase over-expression in hepatocellular carcinomas cells could significantly reduce its sensitivity to multiple chemotherapeutic drugs in vitro and in vivo. Hepatocellular carcinomas cells over-expressing mitochondrial human telomerase reverse transcriptase showed a significantly higher mitochondrial membrane potential, a markedly lower activated caspase-3 after drug treatment, and an increased mtDNA copy number, which explained the drastically decreased drug-induced apoptosis of hepatocellular carcinomas cells with mitochondrial human telomerase reverse transcriptase over-expression. Conclusion: Over-expressed mitochondrial human telomerase reverse transcriptase may increase the mtDNA copy number and inhibit the activation of mitochondrial apoptotic pathway to contribute to the multidrug resistance of hepatocellular carcinomas cells. PMID:25755831

  5. Use of mitochondrial RNA genes for the differentiation of four Trichinella species by multiplex PCR amplification.

    PubMed

    Blaga, R; Fu, BaoQuan; Le Rhun, D; Le Naour, E; Heckman, A; Zocevic, A; Liu, MingYuan; Boireau, P

    2009-06-01

    Until now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species. PMID:19389269

  6. Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.

    PubMed Central

    Raza, Haider; Robin, Marie-Anne; Fang, Ji-Kang; Avadhani, Narayan G

    2002-01-01

    The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress. PMID:12020353

  7. Lipid Peroxidation-Derived Reactive Aldehydes Directly and Differentially Impair Spinal Cord and Brain Mitochondrial Function

    PubMed Central

    Vaishnav, Radhika A.; Singh, Indrapal N.; Miller, Darren M.

    2010-01-01

    Abstract Mitochondrial bioenergetic dysfunction in traumatic spinal cord and brain injury is associated with post-traumatic free radical–mediated oxidative damage to proteins and lipids. Lipid peroxidation by-products, such as 4-hydroxy-2-nonenal and acrolein, can form adducts with proteins and exacerbate the effects of direct free radical–induced protein oxidation. The aim of the present investigation was to determine and compare the direct contribution of 4-hydroxy-2-nonenal and acrolein to spinal cord and brain mitochondrial dysfunction. Ficoll gradient–isolated mitochondria from normal rat spinal cords and brains were treated with carefully selected doses of 4-hydroxy-2-nonenal or acrolein, followed by measurement of complex I– and complex II–driven respiratory rates. Both compounds were potent inhibitors of mitochondrial respiration in a dose-dependent manner. 4-Hydroxy-2-nonenal significantly compromised spinal cord mitochondrial respiration at a 0.1-μM concentration, whereas 10-fold greater concentrations produced a similar effect in brain. Acrolein was more potent than 4-hydroxy-2-nonenal, significantly decreasing spinal cord and brain mitochondrial respiration at 0.01 μM and 0.1 μM concentrations, respectively. The results of this study show that 4-hydroxy-2-nonenal and acrolein can directly and differentially impair spinal cord and brain mitochondrial function, and that the targets for the toxic effects of aldehydes appear to include pyruvate dehydrogenase and complex I–associated proteins. Furthermore, they suggest that protein modification by these lipid peroxidation products may directly contribute to post-traumatic mitochondrial damage, with spinal cord mitochondria showing a greater sensitivity than those in brain. PMID:20392143

  8. Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

    PubMed

    Consiglio, Marco; Viano, Marta; Casarin, Stefania; Castagnoli, Carlotta; Pescarmona, Gianpiero; Silvagno, Francesca

    2015-10-01

    Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin. PMID:26010336

  9. Gene Expression Profiling Specifies Chemokine, Mitochondrial and Lipid Metabolism Signatures in Leprosy

    PubMed Central

    Guerreiro, Luana Tatiana Albuquerque; Robottom-Ferreira, Anna Beatriz; Ribeiro-Alves, Marcelo; Toledo-Pinto, Thiago Gomes; Rosa Brito, Tiana; Rosa, Patrícia Sammarco; Sandoval, Felipe Galvan; Jardim, Márcia Rodrigues; Antunes, Sérgio Gomes; Shannon, Edward J.; Sarno, Euzenir Nunes; Pessolani, Maria Cristina Vidal; Williams, Diana Lynn; Moraes, Milton Ozório

    2013-01-01

    Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy. PMID:23798993

  10. A mitochondrial sirtuin, SIRT3, regulates muscle differentiation and metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    SIRT3 is a member of the sirtuin family of NAD-dependent deacetylases and is localized to the mitochondria. SIRT3 is highly expressed in brown adipose tissue, heart, muscle, and metabolically active tissue enriched with mitochondria. Recent reports found that SIRT3 is able to deacetylate and regula...

  11. Divergent branches of mitochondrial signaling regulate specific genes and the viability of specialized cell types of differentiated yeast colonies

    PubMed Central

    Rešetárová, Stanislava; Kučerová, Helena; Hlaváček, Otakar; Váchová, Libuše; Palková, Zdena

    2016-01-01

    Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation. PMID:26992228

  12. The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

    PubMed Central

    Menga, Alessio; Iacobazzi, Vito; Infantino, Vittoria; Avantaggiati, Maria Laura; Palmieri, Ferdinando

    2015-01-01

    The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. PMID:25597433

  13. Reactive Oxygen Species, Ki-Ras, and Mitochondrial Superoxide Dismutase Cooperate in Nerve Growth Factor-induced Differentiation of PC12 Cells*

    PubMed Central

    Cassano, Silvana; Agnese, Savina; D'Amato, Valentina; Papale, Massimo; Garbi, Corrado; Castagnola, Patrizio; Ruocco, Maria Rosaria; Castellano, Immacolata; De Vendittis, Emmanuele; Santillo, Mariarosaria; Amente, Stefano; Porcellini, Antonio; Avvedimento, Enrico Vittorio

    2010-01-01

    Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H2O2. Low and spatially restricted levels of H2O2 induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells. PMID:20495008

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

    PubMed

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

    2009-12-01

    Sirt1 is a NAD(+)-dependent histone deacetylase that interacts with the regulatory protein of mitochondrial biogenesis PGC-1alpha and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins, and PGC-1alpha existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days, 10 Hz; 3 h/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1alpha levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70-90% in soleus and plantaris muscles of running animals, whereas Sirt1 activity remained unchanged. In chronically stimulated muscle, cytochrome c was increased by 30% compared with nonstimulated muscle, whereas Sirt1 activity was increased modestly by 20-25%. In contrast, in denervated muscle, these markers of mitochondrial content were decreased by 30-50% compared with the control muscle, whereas Sirt1 activity was increased by 75-80%. Our data suggest that Sirt1 and PGC-1alpha expression are independently regulated and that, although Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse. PMID:19797682

  15. Yeast PPR proteins, watchdogs of mitochondrial gene expression

    PubMed Central

    Herbert, Christopher J; Golik, Pawel; Bonnefoy, Nathalie

    2013-01-01

    PPR proteins are a family of ubiquitous RNA-binding factors, found in all the Eukaryotic lineages, and are particularly numerous in higher plants. According to recent bioinformatic analyses, yeast genomes encode from 10 (in S. pombe) to 15 (in S. cerevisiae) PPR proteins. All of these proteins are mitochondrial and very often interact with the mitochondrial membrane. Apart from the general factors, RNA polymerase and RNase P, most yeast PPR proteins are involved in the stability and/or translation of mitochondrially encoded RNAs. At present, some information concerning the target RNA(s) of most of these proteins is available, the next challenge will be to refine our understanding of the function of the proteins and to resolve the yeast PPR-RNA-binding code, which might differ significantly from the plant PPR code. PMID:24184848

  16. Drosophila expressing human SOD1 successfully recapitulates mitochondrial phenotypic features of familial amyotrophic lateral sclerosis.

    PubMed

    Gallart-Palau, Xavier; Ng, Chee-Hoe; Ribera, Joan; Sze, Siu Kwan; Lim, Kah-Leong

    2016-06-15

    Mitochondrial pathology is a seminal pathogenic hallmark of familial amyotrophic lateral sclerosis (FALS) which is extensively manifested by human patients and mutant SOD1(G93A) mammalian models. Rodents expressing human FALS-associated mutations successfully mimic several human disease features; although they are not as amenable to genetic and therapeutic compound screenings as non-mammalian models. In this study, we report a newly generated and characterized Drosophila model that expresses human SOD1(G93A) in muscle fibers. Presence of SOD1(G93A) in thoracic muscles causes mitochondrial pathology and impairs normal motor behavior in these flies. Use of this new FALS-24B-SOD1(G93A) fly model holds promise for better understanding of the mitochondrial affectation process in FALS and for the discovery of novel therapeutic compounds able to reverse mitochondrial dysfunction in this fatal disease. PMID:27163198

  17. Diabetes and activation of peroxisome proliferator activated receptor alpha increases mitochondrial thioesterase I protein expression and activity in the heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mitochondrial thioesterase-I (MTE-I) catalyzes the de-esterification of fattyacyl-CoAs to fatty acid anions in the mitochondrial matrix, which are extruded to the cytosol, thus preventing the accumulation of toxic mitochondrial fattyacyl-CoAs. MTE-I mRNA expression in the heart is regulated by perox...

  18. Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

    SciTech Connect

    Kopecky, J.; Baudysova, M.; Zanotti, F.; Janikova, D.; Pavelka, S.; Houstek, J. )

    1990-12-25

    In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-(35S)methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.

  19. Abundant mitochondrial genome diversity, population differentiation and convergent evolution in pines.

    PubMed Central

    Wu, J; Krutovskii, K V; Strauss, S H

    1998-01-01

    We examined mitochondrial DNA polymorphisms via the analysis of restriction fragment length polymorphisms in three closely related species of pines from western North America: knobcone (Pinus attenuata Lemm.), Monterey (P. radiata D. Don), and bishop (P. muricata D. Don). A total of 343 trees derived from 13 populations were analyzed using 13 homologous mitochondrial gene probes amplified from three species by polymerase chain reaction. Twenty-eight distinct mitochondrial DNA haplotypes were detected and no common haplotypes were found among the species. All three species showed limited variability within populations, but strong differentiation among populations. Based on haplotype frequencies, genetic diversity within populations (HS) averaged 0.22, and population differentiation (GST and theta) exceeded 0.78. Analysis of molecular variance also revealed that >90% of the variation resided among populations. For the purposes of genetic conservation and breeding programs, species and populations could be readily distinguished by unique haplotypes, often using the combination of only a few probes. Neighbor-joining phenograms, however, strongly disagreed with those based on allozymes, chloroplast DNA, and morphological traits. Thus, despite its diagnostic haplotypes, the genome appears to evolve via the rearrangement of multiple, convergent subgenomic domains. PMID:9832536

  20. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

    NASA Astrophysics Data System (ADS)

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K.

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.

  1. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer.

    PubMed

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses. PMID:24451681

  2. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

    PubMed Central

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses. PMID:24451681

  3. Differential gene expression in ripening banana fruit.

    PubMed

    Clendennen, S K; May, G D

    1997-10-01

    During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants. PMID:9342866

  4. Global variability in gene expression and alternative splicing is modulated by mitochondrial content.

    PubMed

    Guantes, Raul; Rastrojo, Alberto; Neves, Ricardo; Lima, Ana; Aguado, Begoña; Iborra, Francisco J

    2015-05-01

    Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype. PMID:25800673

  5. Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

    SciTech Connect

    Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J.L.; Bal, Amanjit; Gill, Kiran Dip

    2013-12-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded

  6. Differentiating between monozygotic twins through next-generation mitochondrial genome sequencing.

    PubMed

    Wang, Zheng; Zhu, Ruxin; Zhang, Suhua; Bian, Yinnan; Lu, Daru; Li, Chengtao

    2015-12-01

    Monozygotic (MZ) twins, considered to be genetically identical, cannot be distinguished from one another by standard forensic DNA testing. A recent study employed whole genome sequencing to identify extremely rare mutations and reported that mutation analysis could be used to differentiate between MZ twins. Compared with nuclear DNA, mitochondrial DNA (mtDNA) has higher mutation rates; therefore, minor differences theoretically exist in MZ twins' mitochondrial genome (mtGenome). However, conventional Sanger-type sequencing (STS) is neither amenable to, nor feasible for, the detection of low-level sequence variants. The recent introduction of massively parallel sequencing (MPS) has the capability to sequence many targeted regions of multiple samples simultaneously with desirable depth of coverage. Thus, the aim of this study was to assess whether full mtGenome sequencing analysis can be used to differentiate between MZ twins. Ten sets of MZ twins provided blood samples that underwent extraction, quantification, mtDNA enrichment, library preparation, and ultra-deep sequencing. Point heteroplasmies were observed in eight sets of MZ twins, and a single nucleotide variant (nt15301) was detected in five sets of MZ twins. Thus, this study demonstrates that ultra-deep mtGenome sequencing could be used to differentiate between MZ twins. PMID:26327617

  7. Mitochondrial content is central to nuclear gene expression: Profound implications for human health.

    PubMed

    Muir, Rebecca; Diot, Alan; Poulton, Joanna

    2016-02-01

    We review a recent paper in Genome Research by Guantes et al. showing that nuclear gene expression is influenced by the bioenergetic status of the mitochondria. The amount of energy that mitochondria make available for gene expression varies considerably. It depends on: the energetic demands of the tissue; the mitochondrial DNA (mtDNA) mutant load; the number of mitochondria; stressors present in the cell. Hence, when failing mitochondria place the cell in energy crisis there are major effects on gene expression affecting the risk of degenerative diseases, cancer and ageing. In 2015 the UK parliament approved a change in the regulation of IVF techniques, allowing "Mitochondrial replacement therapy" to become a reproductive choice for women at risk of transmitting mitochondrial disease to their children. This is the first time that this technique will be available. Therefore understanding the interaction between mitochondria and the nucleus has never been more important. PMID:26725055

  8. Mitochondrial content is central to nuclear gene expression: Profound implications for human health

    PubMed Central

    Muir, Rebecca; Diot, Alan

    2016-01-01

    We review a recent paper in Genome Research by Guantes et al. showing that nuclear gene expression is influenced by the bioenergetic status of the mitochondria. The amount of energy that mitochondria make available for gene expression varies considerably. It depends on: the energetic demands of the tissue; the mitochondrial DNA (mtDNA) mutant load; the number of mitochondria; stressors present in the cell. Hence, when failing mitochondria place the cell in energy crisis there are major effects on gene expression affecting the risk of degenerative diseases, cancer and ageing. In 2015 the UK parliament approved a change in the regulation of IVF techniques, allowing “Mitochondrial replacement therapy” to become a reproductive choice for women at risk of transmitting mitochondrial disease to their children. This is the first time that this technique will be available. Therefore understanding the interaction between mitochondria and the nucleus has never been more important. PMID:26725055

  9. A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast.

    PubMed Central

    Stuart, J A; Harper, J A; Brindle, K M; Jekabsons, M B; Brand, M D

    2001-01-01

    Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the specific (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochondrial uncoupling were assessed. UCP1 provides a GDP-inhibitable model system to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1 homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1 microg/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating proton conductance that was completely GDP-sensitive. The catalytic-centre activity of UCP1 in these yeast mitochondria was similar to that in mammalian brown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11 microg/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates and an increased proton conductance that was partly GDP-insensitive. Thus, although UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is not a physiological property of the native protein and is therefore artifactual. This observation might be important in the interpretation of results from experiments in which the functions of UCP1 homologues are verified by their ability to uncouple yeast mitochondria. PMID:11389685

  10. PGC-1α/β induced expression partially compensates for respiratory chain defects in cells from patients with mitochondrial disorders

    PubMed Central

    Srivastava, Sarika; Diaz, Francisca; Iommarini, Luisa; Aure, Karine; Lombes, Anne; Moraes, Carlos T.

    2009-01-01

    Members of the peroxisome proliferator-activated receptor γ coactivator (PGC) family are potent inducers of mitochondrial biogenesis. We have tested the potential effect of increased mitochondrial biogenesis in cells derived from patients harboring oxidative phosphorylation defects due to either nuclear or mitochondrial DNA mutations. We found that the PGC-1α and/or PGC-1β expression improved mitochondrial respiration in cells harboring a complex III or IV deficiency as well as in transmitochondrial cybrids harboring mitochondrial encephalomyopathy lactic acidosis and stroke A3243G tRNA(Leu)UUR gene mutation. The respiratory function improvement was found to be associated with increased levels of mitochondrial components per cell, although this increase was not homogeneous. These results reinforce the concept that increased mitochondrial biogenesis is a promising venue for the treatment of mitochondrial diseases. PMID:19297390

  11. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

    PubMed Central

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2. PMID:26010876

  12. Differential Expression of Lipid and Carbohydrate Metabolism Genes in Upper Airway versus Diaphragm Muscle

    PubMed Central

    van Lunteren, Erik; Spiegler, Sarah; Moyer, Michelle

    2010-01-01

    Study Objectives: Contractile properties of upper airway muscles influence upper airway patency, an issue of particular importance for subjects with obstructive sleep apnea. Expression of genes related to cellular energetics is, in turn, critical for the maintenance of contractile integrity over time during repetitive activation. We tested the hypothesis that sternohyoid has lower expression of genes related to lipid and carbohydrate energetic pathways than the diaphragm. Methods: Sternohyoid and diaphragm from normal adult rats were examined with gene expression arrays. Analysis focused on genes belonging to Gene Ontology (GO) groups carbohydrate metabolism and lipid metabolism. Results: There were 433 genes with at least ± 2-fold significant differential expression between sternohyoid and diaphragm, of which 192 had sternohyoid > diaphragm and 241 had diaphragm > sternohyoid expression. Among genes with higher sternohyoid expression, there was over-representation of the GO group carbohydrate metabolism (P = 0.0053, n = 13 genes, range of differential expression 2.1- to 6.2-fold) but not lipid metabolism (P = 0.44). Conversely, among genes with higher diaphragm expression, there was over-representation of the GO group lipid metabolism (P = 0.0000065, n = 32 genes, range of differential expression 2.0- to 37.9-fold) but not carbohydrate metabolism (P = 0.23). Nineteen genes with diaphragm > sternohyoid expression were related to fatty acid metabolism (P = 0.000000058), in particular fatty acid β oxidation and biosynthesis in the mitochondria. Conclusions: Sternohyoid has much lower gene expression than diaphragm for mitochondrial enzymes that participate in fatty acid oxidation and biosynthesis. This likely contributes to the lower fatigue resistance of pharyngeal upper airway muscles compared with the diaphragm. Citation: van Lunteren E; Spiegler S; Moyer M. Differential expression of lipid and carbohydrate metabolism genes in upper airway versus diaphragm

  13. Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets.

    PubMed

    Brun, Thierry; Li, Ning; Jourdain, Alexis A; Gaudet, Pascale; Duhamel, Dominique; Meyer, Jérémy; Bosco, Domenico; Maechler, Pierre

    2015-09-15

    In pancreatic β-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mm glucose, 0.4 mm palmitate, 0.4 mm oleate and transiently to H2O2. Culture at physiological 5.6 mm glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in β-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, whereas the glutamate carrier GC1 and the aspartate/glutamate carrier AGC1 were down-regulated by palmitate and oleate, respectively. High glucose decreased mRNA levels of key transcription factors (HNF4A, IPF1, PPARA and TFAM) and energy-sensor SIRT1. High glucose also reduced expression of 11 mtDNA-encoded respiratory chain subunits. Interestingly, transcript levels of the carriers for aspartate/glutamate AGC2, malate DIC and malate/oxaloacetate/aspartate UCP2 were increased by high glucose, a profile suggesting important mitochondrial anaplerotic/cataplerotic activities and NADPH-generating shuttles. Chronic exposure to high glucose impaired glucose-stimulated insulin secretion, decreased insulin content, promoted caspase-3 cleavage and cell death, revealing glucotoxicity. Overall, expression profile of mitochondrion-associated genes was selectively modified by glucose, delineating a glucotoxic-specific signature. PMID:26123492

  14. Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number.

    PubMed Central

    Huang, J; Struck, F; Matzinger, D F; Levings, C S

    1994-01-01

    The mitochondrial Rieske iron-sulfur protein is an obligatory component of the respiratory electron transport chain that is encoded by a single-copy gene in mammals and fungi. In contrast, this protein is encoded by a small gene family in dicotyledonous tobacco and monocotyledonous maize. We cloned four cDNAs from tobacco that encode the mitochondrial Rieske iron-sulfur protein. These clones, along with a previously isolated cDNA, represent five independent members of the gene family that can be divided into three subfamilies. All of these genes were derived from the two progenitor species and were expressed in amphidiploid tobacco. The proteins encoded by these five genes are probably functional because they all contain the universally conserved hexyl peptides necessary for the 2Fe-2S cluster formation. The expression of the Rieske protein gene family is differentially regulated; a 6- to 11-fold higher level of steady state transcripts was found in flowers than in leaves, stems, and roots. Members of at least two subfamilies were preferentially expressed in flowers, indicating that they share a common cis-regulatory element(s), which can respond to a flower-specific signal(s). Although approximately 10 times more transcripts occurred in flowers than in leaves, flower and leaf mitochondria contained a similar amount of the Rieske protein. Flowers, however, contained seven times more Rieske proteins than leaves. These results indicated an increase in mitochondrion number in flowers. High-energy demands during anther development might bring about an increase in mitochondrion numbers in flowers and the flower-enhanced expression of the Rieske protein gene family. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism and/or changes in mitochondrion numbers. PMID:8180500

  15. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    SciTech Connect

    Zuo, Luning; Li, Qiang; Sun, Bei; Xu, Zhiying; Ge, Zhiming

    2013-03-29

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  16. Increased Mitochondrial Calcium Sensitivity and Abnormal Expression of Innate Immunity Genes Precede Dopaminergic Defects in Pink1-Deficient Mice

    PubMed Central

    Akundi, Ravi S.; Huang, Zhenyu; Eason, Joshua; Pandya, Jignesh D.; Zhi, Lianteng; Cass, Wayne A.; Sullivan, Patrick G.; Büeler, Hansruedi

    2011-01-01

    Background PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca2+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson's disease (PD) display altered activity in the nigrostriatal system of Pink1−/− mice. Methods and Findings Purified brain mitochondria of Pink1−/− mice showed impaired Ca2+ storage capacity, resulting in increased Ca2+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1−/− mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1−/− mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1−/− mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1−/− embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1−/− mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting. Conclusions Increased mitochondrial Ca2+ sensitivity and JNK activity are early defects in Pink1−/− mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1−/− mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant

  17. Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing

    PubMed Central

    Torres, Tatiana Teixeira; Dolezal, Marlies; Schlötterer, Christian; Ottenwälder, Birgit

    2009-01-01

    Mitochondria play an essential role in several cellular processes. Nevertheless, very little is known about patterns of gene expression of genes encoded by the mitochondrial DNA (mtDNA). In this study, we used next-generation sequencing (NGS) for transcription profiling of genes encoded in the mitochondrial genome of Drosophila melanogaster and D. pseudoobscura. The analysis of males and females in both species indicated that the expression pattern was conserved between the two species, but differed significantly between both sexes. Interestingly, mRNA levels were not only different among genes encoded by separate transcription units, but also showed significant differences among genes located in the same transcription unit. Hence, mRNA abundance of genes encoded by mtDNA seems to be heavily modulated by post-transcriptional regulation. Finally, we also identified several transcripts with a noncanonical structure, suggesting that processing of mitochondrial transcripts may be more complex than previously assumed. PMID:19843606

  18. Mitochondrial genetic differentiation and morphological difference of Miniopterus fuliginosus and Miniopterus magnater in China and Vietnam

    PubMed Central

    Li, Shi; Sun, Keping; Lu, Guanjun; Lin, Aiqing; Jiang, Tinglei; Jin, Longru; Hoyt, Joseph R; Feng, Jiang

    2015-01-01

    Because of its complicated systematics, the bent-winged bat is one of the most frequently studied bat species groups. In China, two morphologically similar bent-winged bat species, Miniopterus fuliginosus and Miniopterus magnater were identified, but their distribution range and genetic differentiation are largely unexplored. In this study, we applied DNA bar codes and two other mitochondrial DNA genes including morphological parameters to determine the phylogeny, genetic differentiation, spatial distribution, and morphological difference of the M. fuliginosus and M. magnater sampled from China and one site in Vietnam. Mitochondrial DNA gene genealogies revealed two monophyletic lineages throughout the Tropic of Cancer. According to DNA bar code divergences, one is M. fuliginosus corresponding to the Chinese mainland and the other is M. magnater corresponding to tropical regions including Hainan and Guangdong provinces of China and Vietnam. Their most recent common ancestor was dated to the early stage of the Quaternary glacial period (ca. 2.26 million years ago [Ma] on the basis of D-loop data, and ca. 1.69–2.37 Ma according to ND2). A population expansion event was inferred for populations of M. fuliginosus at 0.14 Ma. The two species probably arose in separate Pleistocene refugia under different climate zones. They significantly differed in forearm length, maxillary third molar width, and greatest length of the skull. PMID:25859327

  19. Axin is expressed in mitochondria and suppresses mitochondrial ATP synthesis in HeLa cells.

    PubMed

    Shin, Jee-Hye; Kim, Hyun-Wook; Rhyu, Im Joo; Kee, Sun-Ho

    2016-01-01

    Many recent studies have revealed that axin is involved in numerous cellular functions beyond the negative regulation of β-catenin-dependent Wnt signaling. Previously, an association of ectopic axin with mitochondria was observed. In an effort to investigate the relationship between axin and mitochondria, we found that axin expression suppressed cellular ATP production, which was more apparent as axin expression levels increased. Also, mitochondrial expression of axin was observed using two axin-expressing HeLa cell models: doxycycline-inducible ectopic axin expression (HeLa-axin) and axin expression enhanced by long-term treatment with XAV939 (HeLa-XAV). In biochemical analysis, axin is associated with oxidative phosphorylation (OXPHOS) complex IV and is involved in defects in the assembly of complex IV-containing supercomplexes. Functionally, axin expression reduced the activity of OXPHOS complex IV and the oxygen consumption rate (OCR), suggesting axin-mediated mitochondrial dysfunction. Subsequent studies using various inhibitors of Wnt signaling showed that the reduction in cellular ATP levels was weaker in cases of ICAT protein expression and treatment with iCRT3 or NSC668036 compared with XAV939 treatment, suggesting that XAV939 treatment affects ATP synthesis in addition to suppressing Wnt signaling activity. Axin-mediated regulation of mitochondrial function may be an additional mechanism to Wnt signaling for regulation of cell growth. PMID:26704260

  20. Relationship between mitochondrial DNA Copy Number and SIRT1 Expression in Porcine Oocytes

    PubMed Central

    Sato, Daichi; Itami, Nobuhiko; Tasaki, Hidetaka; Takeo, Shun; Kuwayama, Takehito; Iwata, Hisataka

    2014-01-01

    The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes. PMID:24747689

  1. Abnormal Mitochondrial Function and Impaired Granulosa Cell Differentiation in Androgen Receptor Knockout Mice

    PubMed Central

    Wang, Ruey-Sheng; Chang, Heng-Yu; Kao, Shu-Huei; Kao, Cheng-Heng; Wu, Yi-Chen; Yeh, Shuyuan; Tzeng, Chii-Reuy; Chang, Chawnshang

    2015-01-01

    In the ovary, the paracrine interactions between the oocyte and surrounded granulosa cells are critical for optimal oocyte quality and embryonic development. Mice lacking the androgen receptor (AR−/−) were noted to have reduced fertility with abnormal ovarian function that might involve the promotion of preantral follicle growth and prevention of follicular atresia. However, the detailed mechanism of how AR in granulosa cells exerts its effects on oocyte quality is poorly understood. Comparing in vitro maturation rate of oocytes, we found oocytes collected from AR−/− mice have a significantly poor maturating rate with 60% reached metaphase II and 30% remained in germinal vesicle breakdown stage, whereas 95% of wild-type AR (AR+/+) oocytes had reached metaphase II. Interestingly, we found these AR−/− female mice also had an increased frequency of morphological alterations in the mitochondria of granulosa cells with reduced ATP generation (0.18 ± 0.02 vs. 0.29 ± 0.02 µM/mg protein; p < 0.05) and aberrant mitochondrial biogenesis. Mechanism dissection found loss of AR led to a significant decrease in the expression of peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1-β (PGC1-β) and its sequential downstream genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), in controlling mitochondrial biogenesis. These results indicate that AR may contribute to maintain oocyte quality and fertility via controlling the signals of PGC1-β-mediated mitochondrial biogenesis in granulosa cells. PMID:25941928

  2. Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

    PubMed Central

    Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S. R. Murthy; Joly, Erik; Ruderman, Neil B.; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

    2016-01-01

    Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

  3. Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression.

    PubMed

    Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S R Murthy; Joly, Erik; Ruderman, Neil B; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

    2016-01-01

    Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

  4. Proinflammatory cytokines promote glial heme oxygenase-1 expression and mitochondrial iron deposition: implications for multiple sclerosis.

    PubMed

    Mehindate, K; Sahlas, D J; Frankel, D; Mawal, Y; Liberman, A; Corcos, J; Dion, S; Schipper, H M

    2001-06-01

    Proinflammatory cytokines, pathological iron deposition, and oxidative stress have been implicated in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). HO-1 mRNA levels and mitochondrial uptake of [(55)Fe]Cl(3)-derived iron were measured in rat astroglial cultures exposed to interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) alone or in combination with the heme oxygenase-1 (HO-1) inhibitors, tin mesoporphyrin (SnMP) or dexamthasone (DEX), or interferon beta1b (INF-beta). HO-1 expression in astrocytes was evaluated by immunohistochemical staining of spinal cord tissue derived from MS and control subjects. IL-1beta or TNF-alpha promoted sequestration of non-transferrin-derived (55)Fe by astroglial mitochondria. HO-1 inhibitors, mitochondrial permeability transition pore (MTP) blockers and antioxidants significantly attenuated cytokine-related mitochondrial iron sequestration in these cells. IFN-beta decreased HO-1 expression and mitochondrial iron sequestration in IL-1beta- and TNF-alpha-challenged astroglia. The percentage of astrocytes coexpressing HO-1 in affected spinal cord from MS patients (57.3% +/- 12.8%) was significantly greater (p < 0.05) than in normal spinal cord derived from controls subjects (15.4% +/- 8.4%). HO-1 is over-expressed in MS spinal cord astroglia and may promote mitochondrial iron deposition in MS plaques. In MS, IFN-beta may attenuate glial HO-1 gene induction and aberrant mitochondrial iron deposition accruing from exposure to proinflammatory cytokines. PMID:11389189

  5. Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia.

    PubMed

    Song, Wei; Su, Haixiang; Song, Sisi; Paudel, Hemant K; Schipper, Hyman M

    2006-03-01

    Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions. PMID:16222706

  6. Stress and corticosteroids regulate rat hippocampal mitochondrial DNA gene expression via the glucocorticoid receptor.

    PubMed

    Hunter, Richard G; Seligsohn, Ma'ayan; Rubin, Todd G; Griffiths, Brian B; Ozdemir, Yildirim; Pfaff, Donald W; Datson, Nicole A; McEwen, Bruce S

    2016-08-01

    Glucocorticoids (GCs) are involved in stress and circadian regulation, and produce many actions via the GC receptor (GR), which is classically understood to function as a nuclear transcription factor. However, the nuclear genome is not the only genome in eukaryotic cells. The mitochondria also contain a small circular genome, the mitochondrial DNA (mtDNA), that encodes 13 polypeptides. Recent work has established that, in the brain and other systems, the GR is translocated from the cytosol to the mitochondria and that stress and corticosteroids have a direct influence on mtDNA transcription and mitochondrial physiology. To determine if stress affects mitochondrially transcribed mRNA (mtRNA) expression, we exposed adult male rats to both acute and chronic immobilization stress and examined mtRNA expression using quantitative RT-PCR. We found that acute stress had a main effect on mtRNA expression and that expression of NADH dehydrogenase 1, 3, and 6 (ND-1, ND-3, ND-6) and ATP synthase 6 (ATP-6) genes was significantly down-regulated. Chronic stress induced a significant up-regulation of ND-6 expression. Adrenalectomy abolished acute stress-induced mtRNA regulation, demonstrating GC dependence. ChIP sequencing of GR showed that corticosterone treatment induced a dose-dependent association of the GR with the control region of the mitochondrial genome. These findings demonstrate GR and stress-dependent transcriptional regulation of the mitochondrial genome in vivo and are consistent with previous work linking stress and GCs with changes in the function of brain mitochondria. PMID:27457949

  7. TP53 status regulates ACSL5-induced expression of mitochondrial mortalin in enterocytes and colorectal adenocarcinomas.

    PubMed

    Klaus, Christina; Kaemmerer, Elke; Reinartz, Andrea; Schneider, Ursula; Plum, Patrick; Jeon, Min Kyung; Hose, Josephine; Hartmann, Franziska; Schnölzer, Martina; Wagner, Norbert; Kopitz, Jürgen; Gassler, Nikolaus

    2014-07-01

    Acyl-CoA synthetase 5 (ACSL5), a mitochondrially localized enzyme, catalyzes the synthesis of long-chain fatty acid thioesters and is physiologically involved in pro-apoptotic sensing of enterocytes. The aim of the present study is to identify an ACSL5-dependent regulation of mitochondrially expressed proteins and the characterization of related pathways in normal and diseased human intestinal mucosa. Proteomics of isolated mitochondria from ACSL5 transfectants and CaCo2 controls were performed. ACSL5-dependent protein synthesis was verified with quantitative reverse transcription plus the polymerase chain reaction, Western blotting, short-interfering-RNA-mediated gene silencing and additional cell culture experiments. Lipid changes were analyzed with tandem mass spectrometry. ACSL5-related pathways were characterized in normal mucosa and sporadic adenocarcinomas of the human intestine. In CaCo2 cells transfected with ACSL5, mortalin (HSPA9) was about two-fold increased in mitochondria, whereas cytoplasmic mortalin levels were unchanged. Disturbance of acyl-CoA/sphingolipid metabolism, induced by ACSL5 over-expression, was characterized as crucial. ACSL5-related over-expression of mitochondrial mortalin was found in HEK293 and Lovo (wild-type TP53 [tumor protein p53]) and CaCo2 (p53-negative; TP53 mutated) cells but not in Colo320DM cells (mutated TP53). In normal human intestinal mucosa, an increasing gradient of both ACSL5 and mortalin from bottom to top was observed, whereas p53 (wild-type TP53) decreased. In sporadic intestinal adenocarcinomas with strong p53 immunostaining (mutated TP53), ACSL5-related mortalin expression was heterogeneous. ACSL5-induced mitochondrial mortalin expression is assumed to be a stress response to ACSL5-related changes in lipid metabolism and is regulated by the TP53 status. Uncoupling of ACSL5 and mitochondrial mortalin by mutated TP53 could be important in colorectal carcinogenesis. PMID:24770931

  8. Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation

    PubMed Central

    Santambrogio, Paolo; Erba, Benedetta Gaia; Campanella, Alessandro; Cozzi, Anna; Causarano, Vincenza; Cremonesi, Laura; Gallì, Anna; Della Porta, Matteo Giovanni; Invernizzi, Rosangela; Levi, Sonia

    2011-01-01

    Background Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. Design and Methods We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. Results Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. Conclusions Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation. PMID:21712541

  9. Differential proteomic profiling unveils new molecular mechanisms associated with mitochondrial complex III deficiency

    PubMed Central

    Morán, María; López-Bernardo, Elia; Cadenas, Susana; Hidalgo, Beatriz; Sánchez, Ricardo; Seneca, Sara; Arenas, Joaquín; Martín, Miguel A.; Ugalde, Cristina

    2014-01-01

    We have analyzed the cellular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in BCS1L, a major genetic cause of mitochondrial complex III enzyme deficiency. Mutant fibroblasts exhibited low oxygen consumption rates and intracellular ATP levels, indicating that the main altered molecular event probably is a limited respiration-coupled ATP production through the OXPHOS system. Two-dimensional DIGE and MALDI-TOF/TOF mass spectrometry analyses unambiguously identified 39 proteins whose expression was significantly altered in complex III-deficient fibroblasts. Extensive statistical and cluster analyses revealed a protein profile characteristic for the BCS1L mutant fibroblasts that included alterations in energy metabolism, cell signaling and gene expression regulation, cytoskeleton formation and maintenance, and intracellular stress responses. The physiological validation of the predicted functional adaptations of human cultured fibroblasts to complex III deficiency confirmed the up-regulation of glycolytic enzyme activities and the accumulation of branched-chain among other amino acids, suggesting the activation of anaerobic glycolysis and cellular catabolic states, in particular protein catabolism, together with autophagy as adaptive responses to mitochondrial respiratory chain dysfunction and ATP deficiency. Our data point to an overall metabolic and genetic reprogramming that could contribute to explain the clinical manifestations of complex III deficiency in patients. PMID:25239759

  10. Genetic differentiation in pointing dog breeds inferred from microsatellites and mitochondrial DNA sequence.

    PubMed

    Parra, D; Méndez, S; Cañón, J; Dunner, S

    2008-02-01

    Recent studies presenting genetic analysis of dog breeds do not focus specifically on genetic relationships among pointing dog breeds, although hunting was among the first traits of interest when dogs were domesticated. This report compares histories with genetic relationships among five modern breeds of pointing dogs (English Setter, English Pointer, Epagneul Breton, Deutsch Drahthaar and German Shorthaired Pointer) collected in Spain using mitochondrial, autosomal and Y-chromosome information. We identified 236 alleles in autosomal microsatellites, four Y-chromosome haplotypes and 18 mitochondrial haplotypes. Average F(ST) values were 11.2, 14.4 and 13.1 for autosomal, Y-chromosome microsatellite markers and mtDNA sequence respectively, reflecting relatively high genetic differentiation among breeds. The high gene diversity observed in the pointing breeds (61.7-68.2) suggests contributions from genetically different individuals, but that these individuals originated from the same ancestors. The modern English Setter, thought to have arisen from the Old Spanish Pointer, was the first breed to cluster independently when using autosomal markers and seems to share a common maternal origin with the English Pointer and German Shorthaired Pointer, either via common domestic breed females in the British Isles or through the Old Spanish Pointer females taken to the British Isles in the 14th and 16th centuries. Analysis of mitochondrial DNA sequence indicates the isolation of the Epagneul Breton, which has been formally documented, and shows Deutsch Drahthaar as the result of crossing the German Shorthaired Pointer with other breeds. Our molecular data are consistent with historical documents. PMID:18254732

  11. Expression and properties of the mitochondrial and cytosolic forms of fumarase in germinating maize seeds.

    PubMed

    Eprintsev, Alexander T; Fedorin, Dmitry N; Starinina, Elena V; Igamberdiev, Abir U

    2014-10-01

    Fumarase (EC 4.2.1.2) catalyzes reversible interconversion of malate and fumarate. It is usually associated with the tricarboxylic acid cycle in mitochondria, although the cytosolic form has also been detected. We investigated the expression of two fumarase genes and activities of the mitochondrial and cytosolic isoforms of fumarase in maize (Zea mays) scutellum during germination. Both isoforms were purified to electrophoretic homogeneity. The cytosolic form had low optimum pH (6.5) and high affinity to malate (Km 5 μM) when compared with the mitochondrial form (optimum pH 7.0, Km 50 μM). The cytosolic form was strongly activated by Mg(2+) and even more by Mn(2+) , whereas the mitochondrial form was moderately activated by Mg(2+) and Mn(2+) was less effective. The highest fumarase activity in scutellum and a high expression of the gene encoding the cytosolic form were observed during the maximal activity of the glyoxylate cycle. In leaves, the localization of fumarase is only mitochondrial and only one fumarase gene is expressed. It is concluded that the function of cytosolic fumarase in maize scutellum can be related to metabolism of succinate formed in the glyoxylate cycle. PMID:24611547

  12. Screening of differentially expressed genes in pathological scar tissues using expression microarray.

    PubMed

    Huang, L P; Mao, Z; Zhang, L; Liu, X X; Huang, C; Jia, Z S

    2015-01-01

    Pathological scar tissues and normal skin tissues were differentiated by screening for differentially expressed genes in pathologic scar tissues via gene expression microarray. The differentially expressed gene data was analyzed by gene ontology and pathway analyses. There were 5001 up- or down-regulated genes in 2-fold differentially expressed genes, 956 up- or down-regulated genes in 5-fold differentially expressed genes, and 114 up- or down-regulated genes in 20-fold differentially expressed genes. Therefore, significant differences were observed in the gene expression in pathological scar tissues and normal foreskin tissues. The development of pathological scar tissues has been correlated to changes in multiple genes and pathways, which are believed to form a dynamic network connection. PMID:26400303

  13. Mitochondrial GPX1 silencing triggers differential photosynthesis impairment in response to salinity in rice plants.

    PubMed

    Lima-Melo, Yugo; Carvalho, Fabricio E L; Martins, Márcio O; Passaia, Gisele; Sousa, Rachel H V; Neto, Milton C Lima; Margis-Pinheiro, Márcia; Silveira, Joaquim A G

    2016-08-01

    The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2 O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2 O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency. PMID:26799169

  14. FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression.

    PubMed

    Ferber, E C; Peck, B; Delpuech, O; Bell, G P; East, P; Schulze, A

    2012-06-01

    Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase/Akt pathway, and are key regulators of the cell cycle, apoptosis and response to oxidative stress. FOXOs have been shown to have tumour suppressor function and are important for stem cell maintenance. We have performed a detailed analysis of the transcriptional programme induced in response to Forkhead-box protein O3a (FOXO3a) activation. We observed that FOXO3a activation results in the repression of a large number of nuclear-encoded genes with mitochondrial function. Repression of these genes was mediated by FOXO3a-dependent inhibition of c-Myc. FOXO3a activation also caused a reduction in mitochondrial DNA copy number, expression of mitochondrial proteins, respiratory complexes and mitochondrial respiratory activity. FOXO3a has been previously implicated in the detoxification of reactive oxygen species (ROS) through induction of manganese-containing superoxide dismutase (SOD2). We observed that reduction in ROS levels following FOXO3a activation was independent of SOD2, but required c-Myc inhibition. Hypoxia increases ROS production from the mitochondria, which is required for stabilisation of the hypoxia-inducible factor-1α (HIF-1α). FOXO3a activation blocked the hypoxia-dependent increase in ROS and prevented HIF-1α stabilisation. Our data suggest that FOXO factors regulate mitochondrial activity through inhibition of c-Myc function and alter the hypoxia response. PMID:22139133

  15. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression.

    PubMed

    Kühl, Inge; Dujeancourt, Laurent; Gaisne, Mauricette; Herbert, Christopher J; Bonnefoy, Nathalie

    2011-10-01

    Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation. PMID:21727087

  16. FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression

    PubMed Central

    Ferber, E C; Peck, B; Delpuech, O; Bell, G P; East, P; Schulze, A

    2012-01-01

    Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase/Akt pathway, and are key regulators of the cell cycle, apoptosis and response to oxidative stress. FOXOs have been shown to have tumour suppressor function and are important for stem cell maintenance. We have performed a detailed analysis of the transcriptional programme induced in response to Forkhead-box protein O3a (FOXO3a) activation. We observed that FOXO3a activation results in the repression of a large number of nuclear-encoded genes with mitochondrial function. Repression of these genes was mediated by FOXO3a-dependent inhibition of c-Myc. FOXO3a activation also caused a reduction in mitochondrial DNA copy number, expression of mitochondrial proteins, respiratory complexes and mitochondrial respiratory activity. FOXO3a has been previously implicated in the detoxification of reactive oxygen species (ROS) through induction of manganese-containing superoxide dismutase (SOD2). We observed that reduction in ROS levels following FOXO3a activation was independent of SOD2, but required c-Myc inhibition. Hypoxia increases ROS production from the mitochondria, which is required for stabilisation of the hypoxia-inducible factor-1α (HIF-1α). FOXO3a activation blocked the hypoxia-dependent increase in ROS and prevented HIF-1α stabilisation. Our data suggest that FOXO factors regulate mitochondrial activity through inhibition of c-Myc function and alter the hypoxia response. PMID:22139133

  17. Differential diagnosis of Mendelian and mitochondrial disorders in patients with suspected multiple sclerosis

    PubMed Central

    Katz Sand, Ilana B.; Honce, Justin M.; Lublin, Fred D.

    2015-01-01

    Several single gene disorders share clinical and radiologic characteristics with multiple sclerosis and have the potential to be overlooked in the differential diagnostic evaluation of both adult and paediatric patients with multiple sclerosis. This group includes lysosomal storage disorders, various mitochondrial diseases, other neurometabolic disorders, and several other miscellaneous disorders. Recognition of a single-gene disorder as causal for a patient’s ‘multiple sclerosis-like’ phenotype is critically important for accurate direction of patient management, and evokes broader genetic counselling implications for affected families. Here we review single gene disorders that have the potential to mimic multiple sclerosis, provide an overview of clinical and investigational characteristics of each disorder, and present guidelines for when clinicians should suspect an underlying heritable disorder that requires diagnostic confirmation in a patient with a definite or probable diagnosis of multiple sclerosis. PMID:25636970

  18. Ectopic expression of interferon regulatory factor-1 potentiates granulocytic differentiation.

    PubMed Central

    Coccia, E M; Stellacci, E; Valtieri, M; Masella, B; Feccia, T; Marziali, G; Hiscott, J; Testa, U; Peschle, C; Battistini, A

    2001-01-01

    Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of differentiation. PMID:11716756

  19. In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

    PubMed

    Luz, Anthony L; Lagido, Cristina; Hirschey, Matthew D; Meyer, Joel N

    2016-01-01

    Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc. PMID:27479364

  20. Over-Expressing Mitofusin-2 in Healthy Mature Mammalian Skeletal Muscle Does Not Alter Mitochondrial Bioenergetics

    PubMed Central

    Lally, James S. V.; Herbst, Eric A. F.; Matravadia, Sarthak; Maher, Amy C.; Perry, Christopher G. R.; Ventura-Clapier, Renée; Holloway, Graham P.

    2013-01-01

    The role of mitofusin-2 (MFN-2) in regulating mitochondrial dynamics has been well-characterized in lower order eukaryotic cell lines through the complete ablation of MFN-2 protein. However, to support the contractile function of mature skeletal muscle, the subcellular architecture and constituent proteins of this tissue differ substantially from simpler cellular organisms. Such differences may also impact the role of MFN-2 in mature mammalian muscle, and it is unclear if minor fluctuations in MFN-2, as observed in response to physiological perturbations, has a functional consequence. Therefore, we have transiently transfected MFN-2 cDNA into rat tibialis anterior muscle to determine the effect of physiolgically relevant increases in MFN-2 protein on mitochondrial bioenergetics. Permeabilized muscle fibres generated from muscle following MFN-2-transfection were used for functional assessments of mitochondrial bioenergetics. In addition, we have further established a novel method for selecting fibre bundles that are positively transfected, and using this approach transient transfection increased MFN-2 protein ∼2.3 fold in selected muscle fibres. However, this did not alter maximal rates of oxygen consumption or the sensitivity for ADP-stimulated respiration. In addition, MFN-2 over-expression did not alter rates of H2O2 emission. Altogether, and contrary to evidence from lower order cell lines, our results indicate that over-expressing MFN-2 in healthy muscle does not influence mitochondrial bioenergetics in mature mammalian skeletal muscle. PMID:23383258

  1. Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training

    PubMed Central

    Vincent, Grace; Lamon, Séverine; Gant, Nicholas; Vincent, Peter J.; MacDonald, Julia R.; Markworth, James F.; Edge, Johann A.; Hickey, Anthony J. R.

    2015-01-01

    Purpose: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period. Methods: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured. Results: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass. Conclusion: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression. PMID:25759671

  2. Differentially expressed genes and morphological changes during lengthened immobilization in rat soleus muscle.

    PubMed

    Kim, Ji Won; Kwon, Oh Yun; Kim, Myoung Hee

    2007-02-01

    To examine the effect of lengthened immobilization on the expression of genes and concomitant morphological changes in soleus muscle, rat hindlimbs were immobilized at the ankle in full dorsiflexion by plaster cast. After removing the muscle (after 1 hr, 1, 4, and 7 days of immobilization), morphology and differential gene expression were analyzed through electron microscopy and differential display reverse transcription-polymerase chain reaction (DDRT-PCR), respectively. At the myotendinous junction (MTJ), a large cytoplasmic space appeared after 1 hr of immobilization and became enlarged over time, together with damaged Z lines. Interfibrillar space was detected after 1 day of immobilization, but diminished after 7 days. At the muscle belly, Z-line streaming and widening were observed following 1 hr of immobilization. Disorganization of myofilaments (misalignment of adjacent sarcomeres, distortion, or absence of Z lines) was detected after 4 days. Furthermore, mitochondrial swelling and cristae disruption were observed after 1 day of stretching. A set of 15 differentially expressed candidate genes was identified through DDRT-PCR. Of 11 known genes, seven (Atp5g3, TOM22, INrf2, Slc25a4, Hdac6, Tpm1, and Sv2b) were up and three (Podxl, Myh1, and Surf1) were down-regulated following immobilization. In the case of Acyp2, 1-day stretching-specific expression was observed. Atp5g3, Slc25a4, TOM22, and Surf1 are mitochondrial proteins related to energy metabolism, except TOM22, which has a chaperone-like activity located in the mitochondrial outer membrane. Together with these, INrf2, Hdac6, Podxl, and Acyp2 are related more or less to stress-induced apoptosis, indicating the responses to apoptotic changes in mitochondria caused by stretching. The expression of both Tpm1 and Myh1, fast twitch isoforms, suggests adaption to the immobilization. These results altogether indicate that lengthened immobilization regulates the expression of several stress/apoptosis-related and

  3. Differential Effect of Endurance Training on Mitochondrial Protein Damage, Degradation, and Acetylation in the Context of Aging.

    PubMed

    Johnson, Matthew L; Irving, Brian A; Lanza, Ian R; Vendelbo, Mikkel H; Konopka, Adam R; Robinson, Matthew M; Henderson, Gregory C; Klaus, Katherine A; Morse, Dawn M; Heppelmann, Carrie; Bergen, H Robert; Dasari, Surendra; Schimke, Jill M; Jakaitis, Daniel R; Nair, K Sreekumaran

    2015-11-01

    Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise. PMID:25504576

  4. Expression of the Ciona intestinalis alternative oxidase (AOX) in Drosophila complements defects in mitochondrial oxidative phosphorylation.

    PubMed

    Fernandez-Ayala, Daniel J M; Sanz, Alberto; Vartiainen, Suvi; Kemppainen, Kia K; Babusiak, Marek; Mustalahti, Eero; Costa, Rodolfo; Tuomela, Tea; Zeviani, Massimo; Chung, Jongkyeong; O'Dell, Kevin M C; Rustin, Pierre; Jacobs, Howard T

    2009-05-01

    Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinson's disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders. PMID:19416715

  5. Tri-mean-based statistical differential gene expression detection.

    PubMed

    Ji, Zhaohua; Wu, Chunguo; Wang, Yao; Guan, Renchu; Tu, Huawei; Wu, Xiaozhou; Liang, Yanchun

    2012-01-01

    Based on the assumption that only a subset of disease group has differential gene expression, traditional detection of differentially expressed genes is under the constraint that cancer genes are up- or down-regulated in all disease samples compared with normal samples. However, in 2005, Tomlins assumed and discussed the situation that only a subset of disease samples would be activated, which are often referred to as outliers. PMID:23155761

  6. Human white adipocytes express the cold receptor TRPM8 which activation induces UCP1 expression, mitochondrial activation and heat production.

    PubMed

    Rossato, Marco; Granzotto, Marnie; Macchi, Veronica; Porzionato, Andrea; Petrelli, Lucia; Calcagno, Alessandra; Vencato, Juri; De Stefani, Diego; Silvestrin, Valentina; Rizzuto, Rosario; Bassetto, Franco; De Caro, Raffaele; Vettor, Roberto

    2014-03-01

    Mammals possess two types of adipose tissue, white (WAT) and brown (BAT). The uncoupling protein 1 (UCP1) is a hallmark of BAT, being the pivotal player for cold-induced thermogenesis. WAT can acquire BAT characteristics with up-regulation of UCP1 after cold exposure or adrenergic stimulation. In the present study we demonstrated that human white adipocytes express the cold-sensing receptor TRPM8 which activation by menthol and icilin induced a rise in [Ca²⁺](i) and UCP1 expression, increased mitochondrial membrane potential, glucose uptake and heat production. The induction of "brown-like" phenotype in human white adipocytes after TRPM8 activation was supported by ultrastructural morphological changes of mitochondrial morphology and of their intracellular localization, with no modifications of the genes regulating mitochondrial biogenesis. In conclusion human white adipocytes express the cold receptor TRPM8 which activation induces their "browning" supporting a possible role of this receptor in the control of adipose tissue metabolism and body energy balance. PMID:24342393

  7. The Expression of Ubiquitous Mitochondrial Creatine Kinase Is Downregulated as Prostate Cancer Progression

    PubMed Central

    Amamoto, Rie; Uchiumi, Takeshi; Yagi, Mikako; Monji, Keisuke; Song, YooHyun; Oda, Yoshinao; Shiota, Masaki; Yokomizo, Akira; Naito, Seiji; Kang, Dongchon

    2016-01-01

    Background: Mitochondria play crucial roles in cell signaling events, interorganellar communication, aging, cell proliferation and apoptosis, and mitochondrial impairment has been shown to accelerate or modulate cancer progression. Ubiquitous mitochondrial creatine kinase (uMtCK) is predominantly localized in the intermembrane space of mitochondria and catalyzes the reversible exchange of high-energy phosphate between adenosine tri-phosphate (ATP) and phosphocreatine. However, little is known about its expression and function in human prostate cancer progression. Method: We investigated the expression of uMtCK in 148 prostate carcinoma tissues and matched normal tissue by immunohistochemistry. The expression and localization of uMtCK and hexokinase II, a marker of glycolysis, were examined in prostate carcinoma cell lines using western blot and immunofluorescence. Results: MtCK expression was significantly lower in high Gleason grade carcinoma compared with normal prostate or low grade carcinoma. Western blot further revealed that uMtCK was highly expressed in LNCaP and 22Rv1 cell lines, as well as in the normal prostate cell line RWPE-1. However, uMtCK expression was almost absent in PC3 and DU145 cell lines, in correlation with absent or mutant p53 expression, respectively. In contrast, hexokinase II was overexpressed in PC3 cells. Moreover, in the low uMtCK expressing cell lines, glycolytic ATP production was increased, whereas mitochondrial ATP production was decreased. Conclusions: These data suggest that uMtCK is downregulated as prostate cancer progresses in correlation with a metabolic switch in ATP usage. PMID:26722360

  8. Genetic Variation and Geographic Differentiation in Mitochondrial DNA of the Horseshoe Crab, LIMULUS POLYPHEMUS

    PubMed Central

    Saunders, Nancy C.; Kessler, Louis G.; Avise, John C.

    1986-01-01

    Restriction site variation in mitochondrial DNA (mtDNA) of the horseshoe crab (Limulus polyphemus) was surveyed in populations ranging from New Hampshire to the Gulf Coast of Florida. MtDNA clonal diversity was moderately high, particularly in southern samples, and a major genetic "break" (nucleotide sequence divergence approximately 2%) distinguished all sampled individuals which were north vs. south of a region in northeastern Florida. The area of genotypic divergence in Limulus corresponds to a long-recognized zoogeographic boundary between warm-temperate and tropical marine faunas, and it suggests that selection pressures and/or gene flow barriers associated with water mass differences may also influence the evolution of species widely distributed across such transition zones. On the other hand, a comparison of the mtDNA divergence patterns in Limulus with computer models involving stochastic lineage extinction in species with limited gene flow demonstrates that deterministic explanations need not necessarily be invoked to account for the observations. Experiments to distinguish stochastic from deterministic possibilities are suggested. Overall, the pattern and magnitude of mtDNA differentiation in horseshoe crabs is very similar to that typically reported for freshwater and terrestrial species assayed over a comparable geographic range. Results demonstrate for the first time that, geographically, at least some continuously distributed marine organisms can show considerable mtDNA genetic differentiation. PMID:17246319

  9. Nuclear introgression without mitochondrial introgression in two turtle species exhibiting sex-specific trophic differentiation.

    PubMed

    Mitchell, Sarah M; Muehlbauer, Laura K; Freedberg, Steven

    2016-05-01

    Despite the presence of reproductive barriers between species, interspecific gene introgression has been documented in a range of natural systems. Comparing patterns of genetic introgression in biparental versus matrilineal markers can potentially reveal sex-specific barriers to interspecific gene flow. Hybridization has been documented in the freshwater turtles Graptemys geographica and G. pseudogeographica, whose ranges are largely sympatric. Morphological differentiation between the species is restricted to females, with female G. geographica possessing large heads and jaws compared to the narrow heads of G. pseudogeographica females. If hybrid females are morphologically intermediate, they may be less successful at exploiting parental feeding niches, thereby limiting the introgression of maternally inherited, but not biparental, molecular markers. We paired sequence data with stable isotope analysis and examined sex-specific genetic introgression and trophic differentiation in sympatric populations of G. geographica and G. pseudogeographica. We observed introgression from G. pseudogeographica into G. geographica at three nuclear loci, but not at the mitochondrial locus. Analysis of ∂(15)N and ∂(13)C was consistent with species differences in trophic positioning in females, but not males. These results suggest that ecological divergence in females may reduce the opportunity for gene flow in this system. PMID:27252833

  10. Morphological adaptation with no mitochondrial DNA differentiation in the coastal plain swamp sparrow

    USGS Publications Warehouse

    Greenberg, R.; Cordero, P.J.; Droege, S.; Fleischer, R.C.

    1998-01-01

    We estimated genetic differentiation between morphologically distinct tidal marsh populations of Swamp Sparrows (Melospiza georgiana nigrescens) and the more wide-spread inland populations (M. g. georgiana and M. g. ericrypta). The tidal marsh populations are consistently grayer with more extensive black markings (particularly in the crown), and their bills are larger. These differences are variously shared with other species of salt marsh birds and small mammals. We analyzed mitochondrial DNA sequences (5' end of control region, COII/tlys/ATPase8, and ND2) of Swamp Sparrows and found low levels of genetic variation and no evidence of geographic structure. These results suggest a rapid and recent geographic expansion of Swamp Sparrows from restricted Pleistocene populations. Morphological differentiation has occurred without long-term genetic isolation, suggesting that selection on the divergent traits is intense. The grayer and more melanistic plumage is probably cryptic coloration for foraging on tidal mud, which tends to be grayish as a result of the formation of iron sulfides, rather than iron oxides, under anaerobic conditions.

  11. Genetic diversity and differentiation of the Ryukyu endemic frog Babina holsti as revealed by mitochondrial DNA.

    PubMed

    Tominaga, Atsushi; Matsui, Masafumi; Nakata, Katsushi

    2014-02-01

    We surveyed the genetic diversity and genetic differentiation of an endangered frog, Babina holsti, endemic to Okinawajima and Tokashikijima Islands of the Ryukyus, to elucidate its divergence history and obtain basic data for its conservation. Genetic differentiation between the two island lineages is moderate (3.1% p-distance in the cyt b gene). This result suggests that the two island lineages have been isolated between the late Pliocene and the middle Pleistocene and have never migrated between the current northern part of Okinawajima and Tokashikijima Islands, which were once connected in the late Pleistocene glacial age. On Okinawajima Island, the southernmost sample was constituted by a unique haplotype, without considerable genetic distance from haplotypes detected from northern samples. This unique haplotype composition in the southernmost sample would have resulted from the restricted gene flow between the southernmost population and the other populations in Okinawajima Island. Furthermore, the absence of genetic diversity within the southernmost sample indicates that this population has recently experienced population size reduction, possibly by predation pressure from an introduced mongoose, which is more abundant in the southern part than in the northern part of the island. Lower genetic diversity in the Tokashikijima sample implies a small effective population size for mitochondrial DNA (mtDNA) in B. holsti on the island. Immediate conservation measures should be taken for the populations from the southernmost range in Okinawajima and Tokashikijima. PMID:24521314

  12. Sebocytes differentially express and secrete adipokines.

    PubMed

    Kovács, Dóra; Lovászi, Marianna; Póliska, Szilárd; Oláh, Attila; Bíró, Tamás; Veres, Imre; Zouboulis, Christos C; Ståhle, Mona; Rühl, Ralph; Remenyik, Éva; Törőcsik, Dániel

    2016-03-01

    In addition to producing sebum, sebocytes link lipid metabolism with inflammation at a cellular level and hence, greatly resemble adipocytes. However, so far no analysis was performed to identify and characterize the adipocyte-associated inflammatory proteins, the members of the adipokine family in sebocytes. Therefore, we determined the expression profile of adipokines [adiponectin, interleukin (IL) 6, resistin, leptin, serpin E1, visfatin, apelin, chemerin, retinol-binding protein 4 (RBP4) and monocyte chemoattractant protein 1 (MCP1)] in sebaceous glands of healthy and various disease-affected (acne, rosacea, melanoma and psoriasis) skin samples. Sebaceous glands in all examined samples expressed adiponectin, IL6, resistin, leptin, serpin E1 and visfatin, but not apelin, chemerin, RBP4 and MCP1. Confirming the presence of the detected adipokines in the human SZ95 sebaceous gland cell line we further characterized their expression and secretion patterns under different stimuli mimicking bacterial invasion [by using Toll-like receptor (TLR)2 and 4 activators], or by 13-cis retinoic acid (13CRA; also known as isotretinoin), a key anti-acne agent. With the exception of resistin, the expression of all of the detected adipokines (adiponectin, IL6, leptin, serpin E1 and visfatin) could be further regulated at the level of gene expression, showing a close correlation with the secreted protein levels. Besides providing further evidence on similarities between adipocytes and sebocytes, our results strongly suggest that sebocytes are not simply targets of inflammation but may exhibit initiatory and modulatory roles in the inflammatory processes of the skin through the expression and secretion of adipokines. PMID:26476096

  13. Differentially expressed genes in association with in vitro invasiveness of human epithelioid sarcoma

    PubMed Central

    Weber, A; Engers, R; Nockemann, S; Gohr, L L; Zur Hausen, A; Gabbert, H E

    2001-01-01

    Aims—Differential display reverse transcription polymerase chain reaction (RT-PCR) was performed to identify genes associated with the invasive potential of human epithelioid sarcoma. Methods—Two different clonal subpopulations, GRU-1A and GRU-1B, derived from the same human epithelioid sarcoma cell line GRU-1 and known to differ greatly in their invasive potential were compared by means of mRNA fingerprinting. Results—Using a set of 10 arbitrary upstream primers and nine anchored oligo-dT primers, 22 candidate gene fragments were identified; differential expression was confirmed in four of these fragments by northern blot analysis. At the mRNA level, apoferritin light chain was predominantly expressed by the highly invasive cell line GRU-1A. In contrast, the mitochondrial gene M1, encoding cytochrome c oxidase I, and the TI-227H gene were expressed more strongly by the low invasive cell line GRU-1B. Furthermore, a novel gene fragment was identified and cloned that was preferentially expressed in the low invasive cell line GRU-1B, and therefore might have an inhibitory role in invasion. Consequently, this gene fragment was designated as expressed in low invasive sarcoma cells (ELISC-1). Conclusions—A novel gene fragment (ELISC-1) and three known genes were identified as potential regulators of tumour invasiveness. Cloning of the entire sequence of ELISC-1 and subsequent investigations are required to establish its biological role. PMID:11577175

  14. Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.

    PubMed Central

    Voos, W; von Ahsen, O; Müller, H; Guiard, B; Rassow, J; Pfanner, N

    1996-01-01

    The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation. Images PMID:8654364

  15. Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells.

    PubMed

    Constantinescu, Andrei Alexandru; Abbas, Malak; Kassem, Mohamad; Gleizes, Céline; Kreutter, Guillaume; Schini-Kerth, Valerie; Mitrea, Ioan Liviu; Toti, Florence; Kessler, Laurence

    2016-07-01

    To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine β-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1-100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-β-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-β-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype. PMID:27344165

  16. Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure.

    PubMed

    Wu, San-Pin; Kao, Chung-Yang; Wang, Leiming; Creighton, Chad J; Yang, Jin; Donti, Taraka R; Harmancey, Romain; Vasquez, Hernan G; Graham, Brett H; Bellen, Hugo J; Taegtmeyer, Heinrich; Chang, Ching-Pin; Tsai, Ming-Jer; Tsai, Sophia Y

    2015-01-01

    Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy. PMID:26356605

  17. Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure

    PubMed Central

    Wu, San-Pin; Kao, Chung-Yang; Wang, Leiming; Creighton, Chad J.; Yang, Jin; Donti, Taraka R.; Harmancey, Romain; Vasquez, Hernan G.; Graham, Brett H.; Bellen, Hugo J.; Taegtmeyer, Heinrich; Chang, Ching-Pin; Tsai, Ming-Jer; Tsai, Sophia Y.

    2015-01-01

    Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy. PMID:26356605

  18. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle

    PubMed Central

    Winter, Lilli; Kuznetsov, Andrey V.; Grimm, Michael; Zeöld, Anikó; Fischer, Irmgard; Wiche, Gerhard

    2015-01-01

    Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion–fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways. PMID:26019234

  19. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle.

    PubMed

    Winter, Lilli; Kuznetsov, Andrey V; Grimm, Michael; Zeöld, Anikó; Fischer, Irmgard; Wiche, Gerhard

    2015-08-15

    Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion-fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways. PMID:26019234

  20. Mitochondrial p32 is upregulated in Myc expressing brain cancers and mediates glutamine addiction

    PubMed Central

    Chao, Ying; Pastorino, Sandra; Mukthavaram, Rajesh; Jiang, Pengfei; Cho, Yoon-Jae; Pingle, Sandeep C.; Crawford, John R.; Piccioni, David E.; Kesari, Santosh

    2015-01-01

    Metabolic reprogramming is a key feature of tumorigenesis that is controlled by oncogenes. Enhanced utilization of glucose and glutamine are the best-established hallmarks of tumor metabolism. The oncogene c-Myc is one of the major players responsible for this metabolic alteration. However, the molecular mechanisms involved in Myc-induced metabolic reprogramming are not well defined. Here we identify p32, a mitochondrial protein known to play a role in the expression of mitochondrial respiratory chain complexes, as a critical player in Myc-induced glutamine addiction. We show that p32 is a direct transcriptional target of Myc and that high level of Myc in malignant brain cancers correlates with high expression of p32. Attenuation of p32 expression reduced growth rate of glioma cells expressing Myc and impaired tumor formation in vivo. Loss of p32 in glutamine addicted glioma cells induced resistance to glutamine deprivation and imparted sensitivity to glucose withdrawal. Finally, we provide evidence that p32 expression contributes to Myc-induced glutamine addiction of cancer cells. Our findings suggest that Myc promotes the expression of p32, which is required to maintain sufficient respiratory capacity to sustain glutamine metabolism in Myc transformed cells. PMID:25528767

  1. Non-cytotoxic copper overload boosts mitochondrial energy metabolism to modulate cell proliferation and differentiation in the human erythroleukemic cell line K562.

    PubMed

    Ruiz, Lina M; Jensen, Erik L; Rossel, Yancing; Puas, German I; Gonzalez-Ibanez, Alvaro M; Bustos, Rodrigo I; Ferrick, David A; Elorza, Alvaro A

    2016-07-01

    Copper is integral to the mitochondrial respiratory complex IV and contributes to proliferation and differentiation, metabolic reprogramming and mitochondrial function. The K562 cell line was exposed to a non-cytotoxic copper overload to evaluate mitochondrial dynamics, function and cell fate. This induced higher rates of mitochondrial turnover given by an increase in mitochondrial fusion and fission events and in the autophagic flux. The appearance of smaller and condensed mitochondria was also observed. Bioenergetics activity included more respiratory complexes, higher oxygen consumption rate, superoxide production and ATP synthesis, with no decrease in membrane potential. Increased cell proliferation and inhibited differentiation also occurred. Non-cytotoxic copper levels can modify mitochondrial metabolism and cell fate, which could be used in cancer biology and regenerative medicine. PMID:27094959

  2. Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU).

    PubMed

    Chemello, Francesco; Mammucari, Cristina; Gherardi, Gaia; Rizzuto, Rosario; Lanfranchi, Gerolamo; Cagnin, Stefano

    2015-09-01

    The mitochondrial calcium uniporter (MCU) gene codifies for the inner mitochondrial membrane (IMM) channel responsible for mitochondrial Ca(2 +) uptake. Cytosolic Ca(2 +) transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca(2 +) regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca(2 +) transients elicit large increases in the [Ca(2 +)] of the mitochondrial matrix ([Ca(2 +)]mt). Mitochondrial Ca(2 +) uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca(2 +) uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca(2 +) uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection). Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE60931). PMID:26484227

  3. Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

    PubMed Central

    2014-01-01

    Background Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. Results We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. Conclusion Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required. PMID:24484525

  4. Differential expression of myrosinase gene families.

    PubMed Central

    Lenman, M; Falk, A; Rödin, J; Höglund, A S; Ek, B; Rask, L

    1993-01-01

    In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominatly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axiz contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of

  5. Differential Gene Expression in the Oxyntic and Pyloric Mucosa of the Young Pig

    PubMed Central

    Colombo, Michela; Priori, Davide; Trevisi, Paolo; Bosi, Paolo

    2014-01-01

    The stomach is often considered a single compartment, although morphological differences among specific areas are well known. Oxyntic mucosa (OXY) and pyloric mucosa (PYL, in other species called antral mucosa) are primarily equipped for acid secretion and gastrin production, respectively, while it is not yet clear how the remainder of genes expressed differs in these areas. Here, the differential gene expression between OXY and PYL mucosa was assessed in seven starter pigs. Total RNA expression was analyzed by whole genome Affymetrix Porcine Gene 1.1_ST array strips. Exploratory functional analysis of gene expression values was done by Gene Set Enrichment Analysis, comparing OXY and PYL. Normalized enrichment scores (NESs) were calculated for each gene (statistical significance defined when False Discovery Rate % <25 and P-values of NES<0.05). Expression values were selected for a set of 44 genes and the effect of point of gastric sample was tested by analysis of variance with the procedure for repeated measures. In OXY, HYDROGEN ION TRANSMEMBRANE TRANSPORTER ACTIVITY gene set was the most enriched set compared to PYL, including the two genes for H+/K+-ATPase. Pathways related to mitochondrial activity and feeding behavior were also enriched (primarily cholecystokinin receptors and ghrelin). Aquaporin 4 was the top-ranking gene. In PYL, two gene sets were enriched compared with OXY: LYMPHOCYTE ACTIVATION and LIPID RAFT, a gene set involved in cholesterol-rich microdomains of the plasma membrane. The single most differentially expressed genes were gastrin and secretoglobin 1A, member 1, presumably located in the epithelial line, to inactivate inflammatory mediators. Several genes related to mucosal integrity, immune response, detoxification and epithelium renewal were also enriched in PYL (P<0.05). The data indicate that there is significant differential gene expression between OXY of the young pig and PYL and further functional studies are needed to confirm their

  6. Differential global gene expression in red and white skeletal muscle

    NASA Technical Reports Server (NTRS)

    Campbell, W. G.; Gordon, S. E.; Carlson, C. J.; Pattison, J. S.; Hamilton, M. T.; Booth, F. W.

    2001-01-01

    The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

  7. MicroRNA-7 Regulates the Function of Mitochondrial Permeability Transition Pore by Targeting VDAC1 Expression.

    PubMed

    Chaudhuri, Amrita Datta; Choi, Doo Chul; Kabaria, Savan; Tran, Alan; Junn, Eunsung

    2016-03-18

    Mitochondrial dysfunction is one of the major contributors to neurodegenerative disorders including Parkinson disease. The mitochondrial permeability transition pore is a protein complex located on the mitochondrial membrane. Under cellular stress, the pore opens, increasing the release of pro-apoptotic proteins, and ultimately resulting in cell death. MicroRNA-7 (miR-7) is a small non-coding RNA that has been found to exhibit a protective role in the cellular models of Parkinson disease. In the present study, miR-7 was predicted to regulate the function of mitochondria, according to gene ontology analysis of proteins that are down-regulated by miR-7. Indeed, miR-7 overexpression inhibited mitochondrial fragmentation, mitochondrial depolarization, cytochrome c release, reactive oxygen species generation, and release of mitochondrial calcium in response to 1-methyl-4-phenylpyridinium (MPP(+)) in human neuroblastoma SH-SY5Y cells. In addition, several of these findings were confirmed in mouse primary neurons. Among the mitochondrial proteins identified by gene ontology analysis, the expression of voltage-dependent anion channel 1 (VDAC1), a constituent of the mitochondrial permeability transition pore, was down-regulated by miR-7 through targeting 3'-untranslated region of VDAC1 mRNA. Similar to miR-7 overexpression, knockdown of VDAC1 also led to a decrease in intracellular reactive oxygen species generation and subsequent cellular protection against MPP(+). Notably, overexpression of VDAC1 without the 3'-UTR significantly abolished the protective effects of miR-7 against MPP(+)-induced cytotoxicity and mitochondrial dysfunction, suggesting that the protective effect of miR-7 is partly exerted through promoting mitochondrial function by targeting VDAC1 expression. These findings point to a novel mechanism by which miR-7 accomplishes neuroprotection by improving mitochondrial health. PMID:26801612

  8. Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea

    PubMed Central

    2010-01-01

    Background The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility. Results It was shown that the ORF220 protein can be guided to the mitochondria using the mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic stem mustard plants expressed the chimeric gene containing the orf220 gene and a mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic plants were male-sterile, most being unable to produce pollen while some could only produce non-vigorous pollen. The transgenic stem mustard plants also showed aberrant floral development identical to that observed in the CMS stem mustard phenotype. Results obtained from oligooarray analysis showed that some genes related to mitochondrial energy metabolism were down-regulated, indicating a weakening of mitochondrial function in transgenic stem mustard. Some genes related to pollen development were shown to be down-regulated in transgenic stem mustard and the expression of some transcription factor genes was also altered. Conclusion The work presented furthers our understanding of how the mitochondrially-targeted expression of CMS-associated orf220 gene causes male sterility through

  9. Expression of a novel non-coding mitochondrial RNA in human proliferating cells

    PubMed Central

    Villegas, Jaime; Burzio, Veronica; Villota, Claudio; Landerer, Eduardo; Martinez, Ronny; Santander, Marcela; Martinez, Rodrigo; Pinto, Rodrigo; Vera, María I.; Boccardo, Enrique; Villa, Luisa L.; Burzio, Luis O.

    2007-01-01

    Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5′ end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5′ end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation. PMID:17962305

  10. Differentially Expressed Genes and Signature Pathways of Human Prostate Cancer

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Robbins, Charles J.; Sang, Qing-Xiang Amy

    2015-01-01

    Genomic technologies including microarrays and next-generation sequencing have enabled the generation of molecular signatures of prostate cancer. Lists of differentially expressed genes between malignant and non-malignant states are thought to be fertile sources of putative prostate cancer biomarkers. However such lists of differentially expressed genes can be highly variable for multiple reasons. As such, looking at differential expression in the context of gene sets and pathways has been more robust. Using next-generation genome sequencing data from The Cancer Genome Atlas, differential gene expression between age- and stage- matched human prostate tumors and non-malignant samples was assessed and used to craft a pathway signature of prostate cancer. Up- and down-regulated genes were assigned to pathways composed of curated groups of related genes from multiple databases. The significance of these pathways was then evaluated according to the number of differentially expressed genes found in the pathway and their position within the pathway using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. The “transforming growth factor-beta signaling” and “Ran regulation of mitotic spindle formation” pathways were strongly associated with prostate cancer. Several other significant pathways confirm reported findings from microarray data that suggest actin cytoskeleton regulation, cell cycle, mitogen-activated protein kinase signaling, and calcium signaling are also altered in prostate cancer. Thus we have demonstrated feasibility of pathway analysis and identified an underexplored area (Ran) for investigation in prostate cancer pathogenesis. PMID:26683658

  11. Differentially Expressed Genes in Hirudo medicinalis Ganglia after Acetyl-L-Carnitine Treatment

    PubMed Central

    Federighi, Giuseppe; Macchi, Monica; Bernardi, Rodolfo; Scuri, Rossana; Brunelli, Marcello; Durante, Mauro; Traina, Giovanna

    2013-01-01

    Acetyl-l-carnitine (ALC) is a naturally occurring substance that, when administered at supra-physiological concentration, is neuroprotective. It is involved in membrane stabilization and in enhancement of mitochondrial functions. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer’s disease and painful neuropathies. ALC is known to improve the cognitive capability of aged animals chronically treated with the drug and, recently, it has been reported that it impairs forms of non-associative learning in the leech. In the present study the effects of ALC on gene expression have been analyzed in the leech Hirudo medicinalis. The suppression subtractive hybridisation methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the leech nervous system after ALC treatment. The method detects differentially but also little expressed transcripts of genes whose sequence or identity is still unknown. We report that a single administration of ALC is able to modulate positively the expression of genes coding for functions that reveal a lasting effect of ALC on the invertebrate, and confirm the neuroprotective and neuromodulative role of the substance. In addition an important finding is the modulation of genes of vegetal origin. This might be considered an instance of ectosymbiotic mutualism. PMID:23308261

  12. Differential proteome and gene expression reveal response to carbon ion irradiation in pubertal mice testes.

    PubMed

    Li, Hongyan; He, Yuxuan; Zhang, Hong; Miao, Guoying

    2014-03-21

    Heavy ion radiation, a high linear energy transfer (LET) radiation, has been shown to have adverse effects on reproduction in male mice. The aim of this study was to profile and investigate the differentially expressed proteins in pubertal male mice testes following carbon ion radiation (CIR). Male mice underwent whole-body irradiation with CIR (1 and 4 Gy), and MALDI-TOF/TOF analysis was used to investigate the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 14 days. 8 differentially expressed proteins were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Our results indicated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. PMID:24440814

  13. Expression profiles for six zebrafish genes during gonadal sex differentiation

    PubMed Central

    Jørgensen, Anne; Morthorst, Jane E; Andersen, Ole; Rasmussen, Lene J; Bjerregaard, Poul

    2008-01-01

    Background The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. Results In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish. Conclusion In zebrafish, the first significant peak in gene expression during the investigated period (2–40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex

  14. Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism

    SciTech Connect

    Hals, Ingrid K.; Ogata, Hirotaka; Pettersen, Elin; Ma, Zuheng; Bjoerklund, Anneli; Skorpen, Frank; Egeberg, Kjartan Wollo; Grill, Valdemar

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The impact of UCP-2 over expression on mitochondrial function is controversial. Black-Right-Pointing-Pointer We tested mitochondrial functions at defined levels of overexpression. Black-Right-Pointing-Pointer We find minor increases of fatty acid oxidation and uncoupling. Black-Right-Pointing-Pointer Effects were seen only at high level (fourfold) of over expression. Black-Right-Pointing-Pointer Hence it is doubtful whether these effects are of importance in diabetes. -- Abstract: Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 {mu}g/ml of doxycycline (dox) induced UCP-2 fourfold (424 {+-} 113%, mean {+-} SEM) and 0.1 {mu}g/ml twofold (178 {+-} 29%, n = 3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 {+-} 11%) as well as D-[U-{sup 14}C]-glucose oxidation (+5 {+-} 9% at 11 mM glucose). Oxidation of [1-{sup 14}C]-oleate was increased from 4088 to 5797 fmol/{mu}g prot/2 h at 3.3 mM glucose, p < 0.03. Oxidation of L-[{sup 14}C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p < 0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p < 0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the

  15. Mitochondrial Reactive Oxygen Species Regulate Adipocyte Differentiation of Mesenchymal Stem Cells in Hematopoietic Stress Induced by Arabinosylcytosine

    PubMed Central

    Wang, Weimin; Zhang, Yao; Lu, Wenyi; Liu, Kaiyan

    2015-01-01

    Objective The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs) in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS) in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo. Methods The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C) and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis. Results ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ,) while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase). Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation. PMID:25768922

  16. Differential Gene Expression in Benznidazole-Resistant Trypanosoma cruzi Parasites

    PubMed Central

    Villarreal, Diana; Nirdé, Philippe; Hide, Mallorie; Barnabé, Christian; Tibayrenc, Michel

    2005-01-01

    We analyzed the differential gene expression among representative Trypanosoma cruzi stocks in relation to benznidazole exposures using a random differentially expressed sequences (RADES) technique. Studies were carried out with drug pressure both at the natural susceptibility level of the wild-type parasite (50% inhibitory concentration for the wild type) and at different resistance levels. The pattern of differential gene expression performed with resistant stocks was compared to the population structure of this parasite, established by random amplified polymorphic DNA analysis and multilocus enzyme electrophoresis. A RADES band polymorphism was observed, and over- or underexpression was linked to the resistance level of the stock. The analysis of RADES bands suggested that different products may be involved in benznidazole resistance mechanisms. No significant association was found between phylogenetic clustering and benznidazole susceptibility. Benznidazole resistance may involve several mechanisms, depending on the level of drug exposure. PMID:15980339

  17. Differential expression of pentraxin 3 in neutrophils.

    PubMed

    Razvina, Olga; Jiang, Shuying; Matsubara, Koichi; Ohashi, Riuko; Hasegawa, Go; Aoyama, Takashi; Daigo, Kenji; Kodama, Tatsuhiko; Hamakubo, Takao; Naito, Makoto

    2015-02-01

    Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense. PMID:25449330

  18. Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells

    SciTech Connect

    Franco, Maribel; Johansson, Magnus . E-mail: magnus.johansson@ki.se; Karlsson, Anna

    2007-07-15

    Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-{beta}-D-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases.

  19. Expression profile and mitochondrial colocalization of Tdp1 in peripheral human tissues.

    PubMed

    Fam, Hok Khim; Chowdhury, Miraj K; Walton, Cheryl; Choi, Kunho; Boerkoel, Cornelius F; Hendson, Glenda

    2013-08-01

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that processes blocked 3' ends of DNA breaks. Functional loss of Tdp1 causes spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1). Based on the prominent cytoplasmic expression of Tdp1 in the neurons presumably affected in SCAN1, we hypothesized that Tdp1 participates in the repair of mitochondrial DNA. As a step toward testing this hypothesis, we profiled Tdp1 expression in different human tissues by immunohistochemistry and immunofluorescence respectively and determined whether Tdp1 was expressed in the cytoplasm of tissues other than the neurons. We found that Tdp1 was ubiquitously expressed and present in the cytoplasm of many cell types. Within human skeletal muscle and multiple mouse tissues, Tdp1 partially colocalized with the mitochondria. In cultured human dermal fibroblasts, Tdp1 redistributed to the cytoplasm and partially colocalized with mitochondria following oxidative stress. These studies suggest that one role of cytoplasmic Tdp1 is the repair of mitochondrial DNA lesions arising from oxidative stress. PMID:23536040

  20. Unbiased Gene Expression Analysis Implicates the huntingtin Polyglutamine Tract in Extra-mitochondrial Energy Metabolism

    PubMed Central

    Lee, Jong-Min; Ivanova, Elena V; Seong, Ihn Sik; Cashorali, Tanya; Kohane, Isaac; Gusella, James F; MacDonald, Marcy E

    2007-01-01

    The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over ∼37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1α expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdhQ111/Q111 cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1α-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdhQ111/Q111 cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-κB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD. PMID:17708681

  1. Differential and limited expression of mutant alleles in multiple myeloma

    PubMed Central

    Rashid, Naim U.; Sperling, Adam S.; Bolli, Niccolo; Wedge, David C.; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A.; Fulciniti, Mariateresa; Samur, Mehmet K.; Richardson, Paul G.; Magrangeas, Florence; Minvielle, Stephane; Futreal, P. Andrew; Anderson, Kenneth C.; Avet-Loiseau, Herve; Parmigiani, Giovanni

    2014-01-01

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  2. Differential and limited expression of mutant alleles in multiple myeloma.

    PubMed

    Rashid, Naim U; Sperling, Adam S; Bolli, Niccolo; Wedge, David C; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A; Fulciniti, Mariateresa; Samur, Mehmet K; Richardson, Paul G; Magrangeas, Florence; Minvielle, Stephane; Futreal, P Andrew; Anderson, Kenneth C; Avet-Loiseau, Herve; Campbell, Peter J; Parmigiani, Giovanni; Munshi, Nikhil C

    2014-11-13

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

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

    SciTech Connect

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

    2007-07-06

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

  4. Modeling overdispersion heterogeneity in differential expression analysis using mixtures.

    PubMed

    Bonafede, Elisabetta; Picard, Franck; Robin, Stéphane; Viroli, Cinzia

    2016-09-01

    Next-generation sequencing technologies now constitute a method of choice to measure gene expression. Data to analyze are read counts, commonly modeled using negative binomial distributions. A relevant issue associated with this probabilistic framework is the reliable estimation of the overdispersion parameter, reinforced by the limited number of replicates generally observable for each gene. Many strategies have been proposed to estimate this parameter, but when differential analysis is the purpose, they often result in procedures based on plug-in estimates, and we show here that this discrepancy between the estimation framework and the testing framework can lead to uncontrolled type-I errors. Instead, we propose a mixture model that allows each gene to share information with other genes that exhibit similar variability. Three consistent statistical tests are developed for differential expression analysis. We show through a wide simulation study that the proposed method improves the sensitivity of detecting differentially expressed genes with respect to the common procedures, since it reaches the nominal value for the type-I error, while keeping elevate discriminative power between differentially and not differentially expressed genes. The method is finally illustrated on prostate cancer RNA-Seq data. PMID:26683201

  5. Differential Bacterial Gene Expression During Experimental Pneumococcal Endophthalmitis

    PubMed Central

    Thornton, Justin A.; Tullos, Nathan A.; Sanders, Melissa E.; Ridout, Granger; Wang, Yong-Dong; Taylor, Sidney D.; McDaniel, Larry S.; Marquart, Mary E.

    2015-01-01

    Streptococcus pneumoniae (pneumococcus) is a potential cause of bacterial endophthalmitis in humans that can result in ocular morbidity. We sought to identify pneumococcal genes that are differentially expressed during growth in the vitreous humor of the eye in an experimental endophthalmitis model. Microarray analysis was used to identify genes that were differentially expressed when pneumococci replicated in the vitreous of rabbit eyes as compared with bacteria grown in vitro in Todd Hewitt medium. Array results were verified by quantitative real-time PCR analysis of representative genes. Select genes potentially playing a role in virulence during endophthalmitis were deleted and mutants were tested for reduced eye pathogenesis and altered adhesion to host cells. Array analysis identified 134 genes that were differentially expressed during endophthalmitis. 112 genes demonstrated increased expression during growth in the eye whereas 22 were down-regulated. Real-time analysis verified increased expression of neuraminidase A (SP1693), neuraminidase B (SP1687), and serine protease (SP1954), and decreased expression of RlrA (SP0461) and choline transporter (SP1861). Mutation of neuraminidases A and B had no major effect on pathogenesis. Loss of SP1954 led to increased adherence to host cells. S. pneumoniae enhances and represses expression of a variety of genes during endophthalmitis. While some of these genes reflect changes in metabolic requirements, some appear to play a role in immune evasion and pathogenesis in the eye. PMID:25791614

  6. Differential expression of microRNAs in mouse embryonic bladder

    SciTech Connect

    Liu, Benchun; Cunha, Gerald R.; Baskin, Laurence S.

    2009-08-07

    MicroRNAs (miRNAs) are involved in several biological processes including development, differentiation and proliferation. Analysis of miRNA expression patterns in the process of embryogenesis may have substantial value in determining the mechanism of embryonic bladder development as well as for eventual therapeutic intervention. The miRNA expression profiles are distinct among the cellular types and embryonic stages as demonstrated by microarray technology and validated by quantitative real-time RT-PCR approach. Remarkably, the miRNA expression patterns suggested that unique miRNAs from epithelial and submucosal areas are responsible for mesenchymal cellular differentiation, especially regarding bladder smooth muscle cells. Our data show that miRNA expression patterns are unique in particular cell types of mouse bladder at specific developmental stages, reflecting the apparent lineage and differentiation status within the embryonic bladder. The identification of unique miRNAs expression before and after smooth muscle differentiation in site-specific area of the bladder indicates their roles in embryogenesis and may aid in future clinical intervention.

  7. What Is Mitochondrial DNA?

    MedlinePlus

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

  8. Depletion of SAM50 Specifically Targets BCR-ABL-Expressing Leukemic Stem and Progenitor Cells by Interfering with Mitochondrial Functions.

    PubMed

    Capala, Marta E; Pruis, Maurien; Vellenga, Edo; Schuringa, Jan Jacob

    2016-03-01

    A high proliferation rate of malignant cells requires an increased energy production, both by anaerobic glucose metabolism and mitochondrial respiration. Moreover, increased levels of mitochondria-produced reactive oxygen species (ROS) promote survival of transformed cells and contribute to the disease progression both in solid tumors and leukemia. Consequently, interfering with mitochondrial metabolism has been used as a strategy to specifically target leukemic cells. SAM50 is a mitochondrial outer membrane protein involved in the formation of mitochondrial intermembrane space bridging (MIB) complex. Although the importance of SAM50 in maintaining MIB integrity and in the assembly of mitochondrial respiratory chain complexes has been described, its specific role in the normal and leukemic hematopoietic cells remains unknown. We observed that human leukemic cells display a specific dependency on SAM50 expression, as downregulation of SAM50 in BCR-ABL-expressing, but not normal CD34(+) human hematopoietic stem and progenitor cells (HSPCs) caused a significant decrease in growth, colony formation, and replating capacity. Mitochondrial functions of BCR-ABL-expressing HSPCs were compromised, as seen by a decreased mitochondrial membrane potential and respiration. This effect of SAM50 downregulation was recapitulated in normal HSPCs exposed to cytokine-rich culture conditions that stimulate proliferation. Both oncogene-transduced and cytokine-stimulated HSPCs showed increased mitochondrial membrane potential and increased ROS levels compared to their normal counterparts. Therefore, we postulate that human leukemic HSPCs are highly dependent on the proper functioning of mitochondria and that disruption of mitochondrial integrity may aid in targeting leukemic cells. PMID:26855047

  9. Expression of mitochondrial transcription factor A in endometrial carcinomas: clinicopathologic correlations and prognostic significance.

    PubMed

    Toki, Naoyuki; Kagami, Seiji; Kurita, Tomoko; Kawagoe, Toshinori; Matsuura, Yusuke; Hachisuga, Toru; Matsuyama, Atsuji; Hashimoto, Hiroshi; Izumi, Hiroto; Kohno, Kimitoshi

    2010-04-01

    Mitochondrial transcription factor A (mtTFA) is necessary for both transcription and maintenance of mitochondrial DNA. This study was conducted to elucidate the clinicopathologic and prognostic significance of mtTFA in patients with endometrial carcinoma. This study investigated the relationship between the immunohistochemical expression of mtTFA and various clinicopathological variables in 276 endometrial carcinomas, including 245 endometrioid adenocarcinomas and 31 nonendometrioid carcinomas (21 serous carcinomas and 10 clear cell adenocarcinomas). Both uni- and multivariate regression analyses were performed. The mtTFA labeling index of endometrioid adenocarcinomas ranged from 0% to 98%, with a median value of 32%, which was selected as the cut-off point for mtTFA expression. The mtTFA expression in endometrioid adenocarcinomas was significantly associated with the surgical stage, myometrial invasion, lymphovascular space invasion, cervical invasion, and lymph node metastasis. In contrast, no correlation between clinicopathologic variables and mtTFA expression was found in nonendometrioid carcinomas. Correlation analysis between mtTFA and p53 expression by using the Pearson test showed significant correlation in endometrioid adenocarcinomas (P = 0.007), but no significant correlation in nonendometrioid carcinomas (P = 0.947). A univariate survival analysis showed that the 10-year overall survival rate of the patients with mtTFA-positive endometrioid adenocarcinoma was significantly worse than that of patients with mtTFA-negative endometrioid adenocarcinoma (80.8% vs. 93.8%, P = 0.012). However, the multivariate analysis revealed that mtTFA expression in endometrioid adenocarcinomas was no independent prognostic factor. The positive mtTFA expression is a useful maker for progression of the tumors and the poor prognosis of the patients in endometrioid adenocarcinomas. PMID:20232213

  10. Population-level expression variability of mitochondrial DNA-encoded genes in humans

    PubMed Central

    Wang, Gang; Yang, Ence; Mandhan, Ishita; Brinkmeyer-Langford, Candice L; Cai, James J

    2014-01-01

    Human mitochondria contain multiple copies of a circular genome made up of double-stranded DNA (mtDNA) that encodes proteins involved in cellular respiration. Transcript abundance of mtDNA-encoded genes varies between human individuals, yet the level of variation in the general population has not been systematically assessed. In the present study, we revisited large-scale RNA sequencing data generated from lymphoblastoid cell lines of HapMap samples of European and African ancestry to estimate transcript abundance and quantify expression variation for mtDNA-encoded genes. In both populations, we detected up to over 100-fold difference in mtDNA gene expression between individuals. The marked variation was not due to differences in mtDNA copy number between individuals, but was shaped by the transcription of hundreds of nuclear genes. Many of these nuclear genes were co-expressed with one another, resulting in a module-enriched co-expression network. Significant correlations in expression between genes of the mtDNA and nuclear genomes were used to identify factors involved with the regulation of mitochondrial functions. In conclusion, we determined the baseline amount of variability in mtDNA gene expression in general human populations and cataloged a complete set of nuclear genes whose expression levels are correlated with those of mtDNA-encoded genes. Our findings will enable the integration of information from both mtDNA and nuclear genetic systems, and facilitate the discovery of novel regulatory pathways involving mitochondrial functions. PMID:24398800

  11. Expression of Molecular Differentiation Markers Does Not Correlate with Histological Differentiation Grade in Intrahepatic Cholangiocarcinoma

    PubMed Central

    Demarez, Céline; Hubert, Catherine; Sempoux, Christine; Lemaigre, Frédéric P.

    2016-01-01

    The differentiation status of tumor cells, defined by histomorphological criteria, is a prognostic factor for survival of patients affected with intrahepatic cholangiocarcinoma (ICC). To strengthen the value of morphological differentiation criteria, we wished to correlate histopathological differentiation grade with expression of molecular biliary differentiation markers and of microRNAs previously shown to be dysregulated in ICC. We analysed a series of tumors that were histologically classified as well, moderately or poorly differentiated, and investigated the expression of cytokeratin 7, 19 and 903 (CK7, CK19, CK903), SRY-related HMG box transcription factors 4 and 9 (SOX4, SOX9), osteopontin (OPN), Hepatocyte Nuclear Factor-1 beta (HNF1β), Yes-associated protein (YAP), Epithelial cell adhesion molecule (EPCAM), Mucin 1 (MUC1) and N-cadherin (NCAD) by qRT-PCR and immunostaining, and of miR-31, miR-135b, miR-132, miR-200c, miR-221 and miR-222. Unexpectedly, except for subcellular location of SOX9 and OPN, no correlation was found between the expression levels of these molecular markers and histopathological differentiation grade. Therefore, our data point toward necessary caution when investigating the evolution and prognosis of ICC on the basis of cell differentiation criteria. PMID:27280413

  12. The effects of aging, physical training, and a single bout of exercise on mitochondrial protein expression in human skeletal muscle

    PubMed Central

    Bori, Zoltan; Zhao, Zhongfu; Koltai, Erika; Fatouros, Ioannis G.; Jamurtas, Athanasios Z.; Douroudos, Ioannis I.; Terzis, Gerasimos; Chatzinikolaou, Athanasios; Sovatzidis, Apostolos; Draganidis, Dimitrios; Boldogh, Istvan; Radak, Zsolt

    2016-01-01

    Aging results in a significant decline in aerobic capacity and impaired mitochondrial function. We have tested the effects of moderate physical activity on aerobic capacity and a single bout of exercise on the expression profile of mitochondrial biogenesis, and fusion and fission related genes in skeletal muscle of human subjects. Physical activity attenuated the aging-associated decline in VO2 max (p<0.05). Aging increased and a single exercise bout decreased the expression of nuclear respiratory factor-1 (NRF1), while the transcription factor A (TFAM) expression showed a strong relationship with VO2max and increased significantly in the young physically active group. Mitochondrial fission representing FIS1 was induced by regular physical activity, while a bout of exercise decreased fusion-associated gene expression. The expression of polynucleotide phosphorylase (PNPase) changed inversely in young and old groups and decreased with aging. The A2 subunit of cyclic AMP-activated protein kinase (AMPK) was induced by a single bout of exercise in skeletal muscle samples of both young and old subjects (p<0.05). Our data suggest that moderate levels of regular physical activity increases a larger number of mitochondrial biogenesis-related gene expressions in young individuals than in aged subjects. Mitochondrial fission is impaired by aging and could be one of the most sensitive markers of the age-associated decline in the adaptive response to physical activity. PMID:22449457

  13. PPR (pentatricopeptide repeat) proteins in mammals: important aids to mitochondrial gene expression.

    PubMed

    Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M A

    2008-11-15

    Genes encoding PPR (pentatricopeptide repeat)-containing proteins constitute one of the largest gene families in plants. The majority of these proteins are predicted to target organelles and to bind to RNA. Strikingly, there is a dearth of these proteins in mammals, although genomic searches reveal six candidates, all of which are also predicted to target the mitochondrion. Two of these proteins, POLRMT (the mitochondrial RNA polymerase) and MRPS27, a mitoribosomal protein, are involved in transcription and translation respectively. PTCD1 (pentatricopeptide repeat domain protein 1) and PTCD3 are predicted to be involved in the assembly of respiratory chain complexes, whereas mutations in one other protein, LRPPRC (leucine-rich pentatricopeptide repeat cassette), have been shown to cause defects in the levels of cytochrome c oxidase, the terminal member of the respiratory chain. In this issue of the Biochemical Journal, Xu et al. turn their attention to the remaining candidate, PTCD2. Depletion in a mouse model led to deficiencies of the third complex of the respiratory chain that caused profound ultrastructural changes in the heart. The exact molecular function of PTCD2 remains unclear, but depletion leads to an apparent lack of processing of the mitochondrial transcript encoding apocytochrome b, a critical member of complex III. These data are consistent with PTCD2 playing an important role in the post-transcriptional expression of the mitochondrial genome. PMID:18939947

  14. Partial kinetoplast-mitochondrial gene organization and expression in the respiratory deficient plant trypanosomatid Phytomonas serpens.

    PubMed

    Maslov, D A; Nawathean, P; Scheel, J

    1999-04-30

    In plant-dwelling trypanosomatids from the genus Phytomonas, mitochondrial functions, such as cytochrome mediated respiration, ATP production and Krebs cycle, are missing, and cell energetics is based on the glycolysis. Using Blue Native/Tricine-SDS two-dimensional gel electrophoretic analysis, we observed that mitochondrial respiratory Complexes III (cytochrome bc1) and IV (cytochrome c oxidase) were absent in Phytomonas serpens; however, Complex V (ATPase) was present. A deletion of the genes for cytochrome c oxidase subunit III (COIII) and apocytochrome b (Cyb) was identified within the 6234 bp sequenced region of the 31 kb maxicircle kinetoplast DNA. Genes, found in this region, include 12S and 9S ribosomal RNAs, subunits 7, 8 and 9 of NADH dehydrogenase (ND7, ND8 and ND9) and subunit 6 of ATPase (A6 or MURF4), as well as the genes (MURF1, MURF5 and G3) with unknown function. Most genes are actively transcribed and some mRNAs are edited. Fully edited mRNAs for A6 and G3 were abundant, while edited ND7 transcripts were rare, and only partially edited and pre-edited transcripts for ND8 were detected. The data show that the mitochondrial genome of P. serpens is functional, although its functions may be limited to expressing the ATPase and, possibly, NADH dehydrogenase complexes. PMID:10340485

  15. Modulation of adipogenesis-related gene expression by estrogen-related receptor gamma during adipocytic differentiation.

    PubMed

    Kubo, Mayumi; Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Takeda, Satoru; Inoue, Satoshi

    2009-02-01

    Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRgamma in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRgamma was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRgamma mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRgamma-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARgamma, and PGC-1beta in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRgamma in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRgamma positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes. PMID:18809516

  16. Differentially-Expressed Pseudogenes in HIV-1 Infection

    PubMed Central

    Gupta, Aditi; Brown, C. Titus; Zheng, Yong-Hui; Adami, Christoph

    2015-01-01

    Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit. PMID:26426037

  17. Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

    PubMed

    Li, Chun Ge; Wang, Hui; Chen, Hong Ju; Zhao, Yan; Fu, Pei Sheng; Ji, Xiang Shan

    2014-01-01

    Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHβ and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species. PMID:25199961

  18. Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

    PubMed

    Zhao, Yanhong; Liao, Xiaofang; Huang, Zhipeng; Chen, Peng; Zhou, Bujin; Liu, Dongmei; Kong, Xiangjun; Zhou, Ruiyang

    2015-08-01

    Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In

  19. A high throughput screening for rarely transcribed differentially expressed genes.

    PubMed Central

    von Stein, O D; Thies, W G; Hofmann, M

    1997-01-01

    A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined. PMID:9185570

  20. The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

    PubMed Central

    Grönke, Sebastian; Stewart, James B.; Mourier, Arnaud; Ruzzenente, Benedetta; Kukat, Christian; Wibom, Rolf; Habermann, Bianca; Partridge, Linda; Larsson, Nils-Göran

    2011-01-01

    The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation. PMID:22022283

  1. Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

    PubMed Central

    Eya, Jonathan C.; Ukwuaba, Vitalis O.; Yossa, Rodrigue; Gannam, Ann L.

    2015-01-01

    A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish. PMID:25853266

  2. Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation

    PubMed Central

    Panek, Cecilia Analia; Ramos, Maria Victoria; Mejias, Maria Pilar; Abrey-Recalde, Maria Jimena; Fernandez-Brando, Romina Jimena; Gori, Maria Soledad; Salamone, Gabriela Verónica; Palermo, Marina Sandra

    2015-01-01

    Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1. PMID:25502213

  3. Age-related changes of mitochondrial transcription factor a expression in rotator cuff degeneration

    PubMed Central

    Ichiseki, Toru; Ueda, Shusuke; Ueda, Yoshimichi; Kaneuji, Ayumi; Kawahara, Norio; Matsumoto, Tadami

    2015-01-01

    One cause of rotator cuff tears is thought to be age-related degenerative changes occurring in the rotator cuff. Using Rat rotator cuff we determined age-related changes in mitochondrial transcription factor A (TFAM) expression in rotator cuff degeneration to clarify the presence/absence of mitochondrial stress. The materials used were rotator cuffs (supraspinatus) of 5-, 24-, 48-, and 100-week-old Wistar Rats (five animals each). Histopathological study revealed a 4-layer structure consisting of a bone layer, calcified cartilage layer, non-calcified cartilage layer, and tendinous component), with age-related narrowing of the non-calcified cartilage layer confirmed to be present. In an immunohistochemical TFAM study positive findings of the non-calcified cartilage layer were less prominent in the 100-week-old group. In an Enzyme-Linked Immunosorbent Assay (ELISA) study, these were more prominent in the 5-week-old to 24-week-old groups, and slightly less so in the 48-week-old group as compared to the 24-week-old one. In the 100-week-old group as compared to the 24-week-old one they were significantly less prominent (p<0.05). The non-calcified cartilage layer is a major site for the dispersion of mechanical energy, and the change in TFAM expression noted at the same site in the present study and the results of ELISA suggest that age-related changes in mitochondrial stress may be one cause of rotator cuff degeneration. PMID:26692954

  4. Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

    SciTech Connect

    Villarroya, Joan; Lara, Mari-Carmen; Dorado, Beatriz; Garrido, Marta; Garcia-Arumi, Elena; Meseguer, Anna; Hirano, Michio; Vila, Maya R.

    2011-04-08

    Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity

  5. PHGDH Expression Is Required for Mitochondrial Redox Homeostasis, Breast Cancer Stem Cell Maintenance, and Lung Metastasis.

    PubMed

    Samanta, Debangshu; Park, Youngrok; Andrabi, Shaida A; Shelton, Laura M; Gilkes, Daniele M; Semenza, Gregg L

    2016-08-01

    Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor-positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers. Cancer Res; 76(15); 4430-42. ©2016 AACR. PMID:27280394

  6. Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2.

    PubMed

    Avogadri, Francesca; Gnjatic, Sacha; Tassello, Jodie; Frosina, Denise; Hanson, Nicole; Laudenbach, Megan; Ritter, Erika; Merghoub, Taha; Busam, Klaus J; Jungbluth, Achim A

    2016-03-01

    Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100. PMID:26894771

  7. Orostachys japonicus Suppresses Osteoclast Differentiation by Inhibiting NFATc1 Expression.

    PubMed

    Shim, Ki-Shuk; Ha, Hyunil; Kim, Taesoo; Lee, Chung-Jo; Ma, Jin Yeul

    2015-01-01

    The herb Orostachys japonicus has been traditionally used to treat chronic diseases, such as hepatitis, hemorrhoids, and cancer, in Asia. In this study, we investigated the effect of Orostachys japonicus water extract (OJWE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone loss. We found that OJWE inhibited RANKL-induced osteoclast differentiation in a dose-dependent manner without affecting bone resorption in bone marrow-derived macrophage cells. Interestingly, OJWE significantly reduced serum levels of C-terminal telopeptide of type 1 collagen and tartrate-resistant acid phosphatase (TRAP) 5b, markers of bone resorption and osteoclast number, respectively, in an animal model of bone loss. Furthermore, OJWE suppressed the RANKL-induced up-regulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression, and activation of the p38 signaling pathway, but prevented the RANKL-mediated down-regulation of interferon regulatory factor-8 (IRF-8), which is known to be an anti-osteoclastogenic factor that represses NFATc1 expression. We also identified gallic acid and quercetin-3-O-β-D-glucoside as the OJWE components that inhibit RANKL-induced osteoclast differentiation. These results suggest that OJWE inhibits osteoclast differentiation by inhibiting RANKL-induced NFATc1 expression, which prevents osteoclast differentiation and bone loss. The present study elucidated a mechanism of action underlying the inhibitory effect of OJWE on osteoclast differentiation. Our findings suggest that O. japonicus has therapeutic potential for use in the treatment of bone diseases. PMID:26205967

  8. Dynamin-related Protein 1 Inhibition Mitigates Bisphenol A-mediated Alterations in Mitochondrial Dynamics and Neural Stem Cell Proliferation and Differentiation.

    PubMed

    Agarwal, Swati; Yadav, Anuradha; Tiwari, Shashi Kant; Seth, Brashket; Chauhan, Lalit Kumar Singh; Khare, Puneet; Ray, Ratan Singh; Chaturvedi, Rajnish Kumar

    2016-07-29

    The regulatory dynamics of mitochondria comprises well orchestrated distribution and mitochondrial turnover to maintain the mitochondrial circuitry and homeostasis inside the cells. Several pieces of evidence suggested impaired mitochondrial dynamics and its association with the pathogenesis of neurodegenerative disorders. We found that chronic exposure of synthetic xenoestrogen bisphenol A (BPA), a component of consumer plastic products, impaired autophagy-mediated mitochondrial turnover, leading to increased oxidative stress, mitochondrial fragmentation, and apoptosis in hippocampal neural stem cells (NSCs). It also inhibited hippocampal derived NSC proliferation and differentiation, as evident by the decreased number of BrdU- and β-III tubulin-positive cells. All these effects were reversed by the inhibition of oxidative stress using N-acetyl cysteine. BPA up-regulated the levels of Drp-1 (dynamin-related protein 1) and enhanced its mitochondrial translocation, with no effect on Fis-1, Mfn-1, Mfn-2, and Opa-1 in vitro and in the hippocampus. Moreover, transmission electron microscopy studies suggested increased mitochondrial fission and accumulation of fragmented mitochondria and decreased elongated mitochondria in the hippocampus of the rat brain. Impaired mitochondrial dynamics by BPA resulted in increased reactive oxygen species and malondialdehyde levels, disruption of mitochondrial membrane potential, and ATP decline. Pharmacological (Mdivi-1) and genetic (Drp-1siRNA) inhibition of Drp-1 reversed BPA-induced mitochondrial dysfunctions, fragmentation, and apoptosis. Interestingly, BPA-mediated inhibitory effects on NSC proliferation and neuronal differentiations were also mitigated by Drp-1 inhibition. On the other hand, Drp-1 inhibition blocked BPA-mediated Drp-1 translocation, leading to decreased apoptosis of NSC. Overall, our studies implicate Drp-1 as a potential therapeutic target against BPA-mediated impaired mitochondrial dynamics and

  9. The defective expression of gtpbp3 related to tRNA modification alters the mitochondrial function and development of zebrafish.

    PubMed

    Chen, Danni; Li, Feng; Yang, Qingxian; Tian, Miao; Zhang, Zengming; Zhang, Qinghai; Chen, Ye; Guan, Min-Xin

    2016-08-01

    Human mitochondrial DNA (mtDNA) mutations have been associated with a wide spectrum of clinical abnormalities. However, nuclear modifier gene(s) modulate the phenotypic expression of pathogenic mtDNA mutations. In our previous investigation, we identified the human GTPBP3 related to mitochondrial tRNA modification, acting as a modifier to influence of deafness-associated mtDNA mutation. Mutations in GTPBP3 have been found to be associated with other human diseases. However, the pathophysiology of GTPBP3-associated disorders is still not fully understood. Here, we reported the generation and characterization of Gtpbp3 depletion zebrafish model using antisense morpholinos. Zebrafish gtpbp3 has three isoforms localized at mitochondria. Zebrafish gtpbp3 is expressed at various embryonic stages and in multiple tissues. In particular, the gtpbp3 was expressed more abundantly in adult zebrafish ovary and testis. The expression of zebrafish gtpbp3 can functionally restore the growth defects caused by the mss1/gtpbp3 mutation in yeast. A marked decrease of mitochondrial ATP generation accompanied by increased levels of apoptosis and reactive oxygen species were observed in gtpbp3 knockdown zebrafish embryos. The Gtpbp3 morphants exhibited defective in embryonic development including bleeding, melenin, oedema and curved tails within 5days post fertilization, as compared with uninjected controls. The co-injection of wild type gtpbp3 mRNA partially rescued these defects in Gtpbp3 morphants. These data suggest that zebrafish Gtpbp3 is a structural and functional homolog of human and yeast GTPBP3. The mitochondrial dysfunction caused by defective Gtpbp3 may alter the embryonic development in the zebrafish. In addition, this zebrafish model of mitochondrial disease may provide unique opportunities for studying defective tRNA modification, mitochondrial biogenesis, and pathophysiology of mitochondrial disorders. PMID:27184967

  10. [Differential gene expression in the jellyfish Aurelia aurita].

    PubMed

    Matveev, I V

    2005-01-01

    The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium. PMID:16706147

  11. Stable over-expression of the 2-oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl-2-dependent mitochondrial GSH transport.

    PubMed

    Wilkins, Heather M; Brock, Samantha; Gray, Josie J; Linseman, Daniel A

    2014-07-01

    Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron-like cell lines over-expressing the mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up-regulation of the anti-apoptotic protein, B cell lymphoma 2 (Bcl-2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl-2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co-immunoprecipitated with Bcl-2 from rat brain lysates in a GSH-dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl-2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels. Stable over-expression of the 2-oxoglutarate carrier (OGC) in a motor neuronal cell line induced a specific increase in mitochondrial GSH and markedly enhanced resistance to oxidative stress. Over-expression of OGC also induced Bcl-2 expression which was owing to the specific increase in mitochondrial GSH. Intriguingly, enhanced expression of Bcl-2 was required to sustain OGC-dependent GSH transport into the mitochondria. Thus, OGC and Bcl-2 work in a concerted manner to maintain the

  12. Differential proteome and gene expression for testis of mice exposed to carbon ion radiation

    NASA Astrophysics Data System (ADS)

    Zhang, Hong; Li, Hongyan

    Objective To investigate the effect and mechanism of high linear energy transfer (LET) carbon ion irradiation (CIR) on reproduction in the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Male mice underwent whole-body irradiation with CIR (0.5, 1 and 4Gy), and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis was used to determine the alteration in protein expression in 2-DE (two-dimensional gel electrophoresis) gels of testes caused by irradiation after 7, 14 days. Results 15 differentially expressed proteins, such as glucose-regulated protein(GRP78), aconitate hydratase-mitochondrial precursor (ACO), pyruvate kinase isozymes M1/M2 (PKM1/M2), glutathione-S-transferaseA3 (GSTA3), glutathione S-transferase Pi 1 (GSTP1), Cu/Zn super-oxide dismutase (SOD1), Peptidyl-prolyl cis-trans isomerase (Pin1) and Heat shock 70 kDa protein 4L (HSPa4L), were identified and these proteins were mainly involved in energy supply, the endoplasmic reticulum, cell proliferation, cell cycle, antioxidant capacity and mitochondrial respiration, which play important roles in the inhibition of testicular function in response to CIR. Furthermore, we confirmed the relationship between transcription of mRNA and the abundance of proteins. Conclusion The findings of the present study demonstrated that these proteins may lead to new insights into the molecular mechanism of CIR toxicity, and suggested that the gene expression response to CIR involves diverse regulatory mechanisms from transcription of mRNA to the formation of functional proteins. These data also may provide a scientific basis for protecting astronauts and space traveler’s health and safety.

  13. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    PubMed

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis. PMID:26410814

  14. Analysis of differentially expressed proteins in colorectal cancer using hydroxyapatite column and SDS-PAGE.

    PubMed

    Lim, Shi-Rou; Gooi, Boon-Hui; Singh, Manjit; Gam, Lay-Harn

    2011-11-01

    Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method. PMID:21863284

  15. Identification of differentially expressed genes in rat aortic allograft vasculopathy.

    PubMed Central

    Chen, J.; Myllärniemi, M.; Akyürek, L. M.; Häyry, P.; Marsden, P. A.; Paul, L. C.

    1996-01-01

    Graft vasculopathy is an important complication of long-surviving organ transplants, but its pathogenesis has remained elusive. We investigated rat aortic transplants with vasculopathy, aortic transplants without vasculopathy, and normal aortas for differentially expressed mRNA transcripts to gain further insight into the molecular mechanisms involved. Aortic transplants were performed in allogeneic or syngeneic recipients followed by removal after 1 or 5 months, RNA isolation, and differential display to identify mRNA transcripts the expression of which was modulated in conjunction with the transplant procedure and the development of vasculopathy. Using 80 random primers, 57 differentially displayed polymerase chain reaction products were identified, 18 of which were found in allografts but not in syngeneic grafts or normal vessels, whereas 15 were expressed in normal vessels and syngeneic grafts but not in allografts. Of the differentially displayed amplicons, 13 were successfully reamplified and used as probes for Northern analysis; differential expression was confirmed in 6 instances. DNA sequence analysis of these PCR products revealed identity with the immunoglobulin J chain in 2 instances, the ferritin heavy chain, a sequence related but not identical with Ras, and an established sequence tag recently isolated from a human fetal heart library; 1 sequence was not related to any known gene. To assess whether differential mRNA expression of the J-chain gene, a gene expressed in cells of B lymphocyte lineage, was associated with infiltration of the graft by B lymphocytes, tissue sections were stained with an antibody against the B cell marker CD45RA. Although the number of CD45RA-positive cells was low, there was a significant increase in the number of CD45RA-positive cells in the adventitia and intima of grafts with vasculopathy. Furthermore, immunostaining with anti-ferritin antiserum confirmed the presence of ferritin-positive cells within the inner layer of

  16. DEEP--a tool for differential expression effector prediction.

    PubMed

    Degenhardt, Jost; Haubrock, Martin; Dönitz, Jürgen; Wingender, Edgar; Crass, Torsten

    2007-07-01

    High-throughput methods for measuring transcript abundance, like SAGE or microarrays, are widely used for determining differences in gene expression between different tissue types, dignities (normal/malignant) or time points. Further analysis of such data frequently aims at the identification of gene interaction networks that form the causal basis for the observed properties of the systems under examination. To this end, it is usually not sufficient to rely on the measured gene expression levels alone; rather, additional biological knowledge has to be taken into account in order to generate useful hypotheses about the molecular mechanism leading to the realization of a certain phenotype. We present a method that combines gene expression data with biological expert knowledge on molecular interaction networks, as described by the TRANSPATH database on signal transduction, to predict additional--and not necessarily differentially expressed--genes or gene products which might participate in processes specific for either of the examined tissues or conditions. In a first step, significance values for over-expression in tissue/condition A or B are assigned to all genes in the expression data set. Genes with a significance value exceeding a certain threshold are used as starting points for the reconstruction of a graph with signaling components as nodes and signaling events as edges. In a subsequent graph traversal process, again starting from the previously identified differentially expressed genes, all encountered nodes 'inherit' all their starting nodes' significance values. In a final step, the graph is visualized, the nodes being colored according to a weighted average of their inherited significance values. Each node's, or sub-network's, predominant color, ranging from green (significant for tissue/condition A) over yellow (not significant for either tissue/condition) to red (significant for tissue/condition B), thus gives an immediate visual clue on which molecules--differentially

  17. ATF3 represses PPARγ expression and inhibits adipocyte differentiation

    SciTech Connect

    Jang, Min-Kyung; Jung, Myeong Ho

    2014-11-07

    Highlights: • ATF3 decrease the expression of PPARγ and its target gene in 3T3-L1 adipocytes. • ATF3 represses the promoter activity of PPARγ2 gene. • ATF/CRE (−1537/−1530) is critical for ATF3-mediated downregulation of PPARγ. • ATF3 binds to the promoter region containing the ATF/CRE. • ER stress inhibits adipocyte differentiation through downregulation of PPARγ by ATF3. - Abstract: Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3 in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of −2.6 Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of −2037 bp promoter, whereas it did not affect the activity of −1458 bp promoter, suggesting that ATF3 responsive element is located between the −2037 and −1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5′-TGACGTTT-3′) between −1537 and −1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated

  18. Effect of ambient PM(2.5) on lung mitochondrial damage and fusion/fission gene expression in rats.

    PubMed

    Li, Ruijin; Kou, Xiaojing; Geng, Hong; Xie, Jingfang; Yang, Zhenhua; Zhang, Yuexia; Cai, Zongwei; Dong, Chuan

    2015-03-16

    Exposure to ambient fine particulate matter (PM2.5) increases the risk of respiratory disease. Although previous mitochondrial research has provided new information about PM toxicity in the lung, the exact mechanism of PM2.5-mediated structural and functional damage of lung mitochondria remains unclear. In this study, changes in lung mitochondrial morphology, expression of mitochondrial fission/fusion markers, lipid peroxidation, and transport ATPase activity in SD rats exposed to ambient PM2.5 at different dosages were investigated. Also, the release of reactive oxygen species (ROS) via the respiratory burst in rat alveolar macrophages (AMs) exposed to PM2.5 was examined by luminol-dependent chemiluminescence (CL). The results showed that (1) PM2.5 deposited in the lung and induced pathological damage, particularly causing abnormal alterations of mitochondrial structure, including mitochondrial swelling and cristae disorder or even fragmentation in the presence of higher doses of PM2.5; (2) PM2.5 significantly affected the expression of specific mitochondrial fission/fusion markers (OPA1, Mfn1, Mfn2, Fis1, and Drp1) in rat lung; (3) PM2.5 inhibited Mn superoxide dismutase (MnSOD), Na(+)K(+)-ATPase, and Ca(2+)-ATPase activities and elevated malondialdehyde (MDA) content in rat lung mitochondria; and (4) PM2.5 induced rat AMs to produce ROS, which was inhibited by about 84.1% by diphenyleneiodonium chloride (DPI), an important ROS generation inhibitor. It is suggested that the pathological injury observed in rat lung exposed to PM2.5 is associated with mitochondrial fusion-fission dysfunction, ROS generation, mitochondrial lipid peroxidation, and cellular homeostasis imbalance. Damage to lung mitochondria may be one of the important mechanisms by which PM2.5 induces lung injury, contributing to respiratory diseases. PMID:25560372

  19. Complete mitochondrial genome of Helicoverpa zea (Boddie) and expression profiles of mitochondrial-encoded genes in early and late embryos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mitochondrial genome of the bollworm, Helicoverpa zea, was assembled using paired-end nucleotide sequence reads generated with a next-generation sequencing platform. Assembly resulted in a mitogenome of 15,348 bp with greater than 17,000-fold average coverage. Organization of the H. zea mitogen...

  20. Screening for Differentially Expressed Proteins Relevant to the Differential Diagnosis of Sarcoidosis and Tuberculosis

    PubMed Central

    Hu, Yang; Wang, Liu-Sheng; Zhou, Ying; Li, Qiu-Hong; Li, Yan; Du, Yu-Kui; He, Xian; Li, Nan; Yin, Zhao-Fang; Wei, Ya-Ru; Weng, Dong; Li, Hui-Ping

    2015-01-01

    Background In this study, we sought to identify differentially expressed proteins in the serum of patients with sarcoidosis or tuberculosis and to evaluate these proteins as markers for the differential diagnosis of sarcoidosis and sputum-negative tuberculosis. Methods Using protein microarrays, we identified 3 proteins exhibiting differential expression between patients with sarcoidosis and tuberculosis. Elevated expression of these proteins was verified using the enzyme-linked immunosorbent assay (ELISA) and was further confirmed by immunohistochemistry. Receiver operating characteristic (ROC) curve, logistic regression analysis, parallel, and serial tests were used to evaluate the diagnostic efficacy of the proteins. Results Intercellular Adhesion Molecule 1(ICAM-1) and leptin were screened for differentially expressed proteins relevant to sarcoidosis and tuberculosis. Using ROC curves, we found that ICAM-1 (cutoff value: 57740 pg/mL) had an area under the curve (AUC), sensitivity, and specificity of 0.718, 62.3%, and 79.5% respectively, while leptin (cutoff value: 1193.186 pg/mL) had an AUC, sensitivity, and specificity of 0.763, 88.3%, and 65.8%, respectively. Logistic regression analysis revealed that the AUC, sensitivity, and specificity of combined leptin and ICAM-1 were 0.787, 89.6%, and 65.8%, respectively, while those of combined leptin, ICAM-1, and body mass index (BMI) were 0.837, 90.9%, and 64.4%, respectively, which had the greatest diagnostic value. Parallel and serial tests indicated that the BMI-leptin parallel with the ICAM-1 serial was the best diagnostic method, achieving a sensitivity and specificity of 86.5% and 73.1%, respectively. Thus, our results identified elevated expression of ICAM-1 and leptin in serum and granulomas of sarcoidosis patients. Conclusions ICAM-1 and leptin were found to be potential markers for the diagnosis of sarcoidosis and differential diagnosis of sarcoidosis and sputum-negative tuberculosis. PMID:26368286

  1. Differentially Expressed Genes Associated with Low-Dose Gamma Radiation

    NASA Astrophysics Data System (ADS)

    Hegyesi, Hargita; Sándor, Nikolett; Schilling, Boglárka; Kis, Enikő; Lumniczky, Katalin; Sáfrány, Géza

    We have studied low dose radiation induced gene expression alterations in a primary human fibroblast cell line using Agilent's whole human genome microarray. Cells were irradiated with 60Co γ-rays (0; 0.1; 0.5 Gy) and 2 hours later total cellular RNA was isolated. We observed differential regulation of approximately 300-500 genes represented on the microarray. Of these, 126 were differentially expressed at both doses, among them significant elevation of GDF-15 and KITLG was confirmed by qRT-PCR. Based on the transcriptional studies we selected GDF-15 to assess its role in radiation response, since GDF-15 is one of the p53 gene targets and is believed to participate in mediating p53 activities. First we confirmed gamma-radiation induced dose-dependent changes in GDF-15 expression by qRT-PCR. Next we determined the effect of GDF-15 silencing on radiosensitivity. Four GDF-15 targeting shRNA expressing lentiviral vectors were transfected into immortalized human fibroblast cells. We obtained efficient GDF-15 silencing in one of the four constructs. RNA interference inhibited GDF-15 gene expression and enhanced the radiosensitivity of the cells. Our studies proved that GDF-15 plays an essential role in radiation response and may serve as a promising target in radiation therapy.

  2. Differentiation of embryonic stem cells conditionally expressing neurogenin 3.

    PubMed

    Treff, Nathan R; Vincent, Robert K; Budde, Melisa L; Browning, Victoria L; Magliocca, Joseph F; Kapur, Vivek; Odorico, Jon S

    2006-11-01

    Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression. PMID:16809427

  3. Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP).

    PubMed

    Ali, Shahnaz; Ghosh, Kanjaksha; Shetty, Shrimati

    2015-12-01

    Bengal macrothrombocytopenia (BMTCP) is a giant platelet disorder with mild to moderate thrombocytopenia, clinically characterized by mild bleeding symptoms to totally asymptomatic condition. The pathophysiological mechanism of this condition is not fully understood yet. In the present study, 5 subjects (P1-P5) with BMTCP whose platelet counts ranged between 36140X10(9)/l and mean platelet volume (MPV)13.5-16.1fl were analyzed for differential gene expression of platelets by suppressive subtractive hybridization (SSH) technique. Four genes i.e. myotubularin related protein 9 (MTMR9), iron responsive element binding protein 2 (IREB2), alpha tubulin(TUBA) and tyrosine kinase ligand (TKL) were found to be differentially expressed in patient platelets as compared to that of normal healthy controls which was further confirmed by quantitative RT PCR analysis. The study highlights a multi-factorial etiology for BMTCP which is widely prevalent in the northeastern region of the Indian subcontinent. PMID:26460267

  4. Strontium promotes cementoblasts differentiation through inhibiting sclerostin expression in vitro.

    PubMed

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan; Hu, Min

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. PMID:25003114

  5. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro

    PubMed Central

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. PMID:25003114

  6. Mitochondrial dysfunction induces SESN2 gene expression through Activating Transcription Factor 4.

    PubMed

    Garaeva, Alisa A; Kovaleva, Irina E; Chumakov, Peter M; Evstafieva, Alexandra G

    2016-01-01

    We found that inhibitors of mitochondrial respiratory chain complexes III (myxothiazol) and I (piericidin A) in some epithelial carcinoma cell lines induce transcription of the p53-responsive SESN2 gene that plays an important role in stress response and homeostatic regulation. However, the effect did not depend on p53 because i) there was no induction of p53 after the treatment with piericidin A; ii) after the treatment with myxothiazol the peak of SESN2 gene upregulation occurred as early as 5h, before the onset of p53 activation (13h); iii) a supplementation with uridine that abolishes the p53 activation in response to myxothiazol did not abrogate the induction of SESN2 transcripts; iv) in the p53 negative HCT116 p53 -/- cells SESN2 transcription could be also induced by myxothiazol. In response to the respiratory chain inhibitors we observed an induction of ATF4, the key transcription factor of the integrated stress response (ISR). We found that the induction of SESN2 transcripts could be prevented by the ISR inhibitory small molecule ISRIB. Also, by inhibiting or overexpressing ATF4 with specific shRNA or ATF4-expressing constructs, respectively, we have confirmed the role of ATF4 in the SESN2 gene upregulation induced by mitochondrial dysfunction. At a distance of 228 bp upstream from the SESN2 transcription start site we found a candidate sequence for the ATF4 binding site and confirmed its requirement for the induction of SESN2 in luciferase reporter experiments. We suggest that the upregulation of SESN2 by mitochondrial dysfunction provides a homeostatic feedback that attenuates biosynthetic processes during temporal losses of energy supply from mitochondria thereby assisting better adaptation and viability of cells in hostile environments. PMID:26771712

  7. Reference genes for accessing differential expression among developmental stages and analysis of differential expression of OBP genes in Anastrepha obliqua

    PubMed Central

    Nakamura, Aline Minali; Chahad-Ehlers, Samira; Lima, André Luís A.; Taniguti, Cristiane Hayumi; Sobrinho Jr., Iderval; Torres, Felipe Rafael; de Brito, Reinaldo Alves

    2016-01-01

    The West Indian fruit fly, Anastrepha obliqua, is an important agricultural pest in the New World. The use of pesticide-free methods to control invasive species such as this reinforces the search for genes potentially useful in their genetic control. Therefore, the study of chemosensory proteins involved with a range of responses to the chemical environment will help not only on the understanding of the species biology but may also help the development of environmentally friendly pest control strategies. Here we analyzed the expression patterns of three OBP genes, Obp19d_2, Obp56a and Obp99c, across different phases of A. obliqua development by qPCR. In order to do so, we tested eight and identified three reference genes for data normalization, rpl17, rpl18 and ef1a, which displayed stability for the conditions here tested. All OBPs showed differential expression on adults and some differential expression among adult stages. Obp99c had an almost exclusive expression in males and Obp56a showed high expression in virgin females. Thereby, our results provide relevant data not only for other gene expression studies in this species, as well as for the search of candidate genes that may help in the development of new pest control strategies. PMID:26818909

  8. PPARδ Agonism Activates Fatty Acid Oxidation via PGC-1α but Does Not Increase Mitochondrial Gene Expression and Function

    PubMed Central

    Kleiner, Sandra; Nguyen-Tran, Van; Baré, Olivia; Huang, Xueming; Spiegelman, Bruce; Wu, Zhidan

    2009-01-01

    PPARδ (peroxisome proliferator-activated receptor δ) is a regulator of lipid metabolism and has been shown to induce fatty acid oxidation (FAO). PPARδ transgenic and knock-out mice indicate an involvement of PPARδ in regulating mitochondrial biogenesis and oxidative capacity; however, the precise mechanisms by which PPARδ regulates these pathways in skeletal muscle remain unclear. In this study, we determined the effect of selective PPARδ agonism with the synthetic ligand, GW501516, on FAO and mitochondrial gene expression in vitro and in vivo. Our results show that activation of PPARδ by GW501516 led to a robust increase in mRNA levels of key lipid metabolism genes. Mitochondrial gene expression and function were not induced under the same conditions. Additionally, the activation of Pdk4 transcription by PPARδ was coactivated by PGC-1α. PGC-1α, but not PGC-1β, was essential for full activation of Cpt-1b and Pdk4 gene expression via PPARδ agonism. Furthermore, the induction of FAO by PPARδ agonism was completely abolished in the absence of both PGC-1α and PGC-1β. Conversely, PGC-1α-driven FAO was independent of PPARδ. Neither GW501516 treatment nor knockdown of PPARδ affects PGC-1α-induced mitochondrial gene expression in primary myotubes. These results demonstrate that pharmacological activation of PPARδ induces FAO via PGC-1α. However, PPARδ agonism does not induce mitochondrial gene expression and function. PGC-1α-induced FAO and mitochondrial biogenesis appear to be independent of PPARδ. PMID:19435887

  9. Thyroid hormone-regulated brain mitochondrial genes revealed by differential cDNA cloning.

    PubMed Central

    Vega-Núñez, E; Menéndez-Hurtado, A; Garesse, R; Santos, A; Perez-Castillo, A

    1995-01-01

    Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit III). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain. Images PMID:7635984

  10. Unraveling the Limits of Mitochondrial Control Region to Estimate the Fine Scale Population Genetic Differentiation in Anadromous Fish Tenualosa ilisha

    PubMed Central

    Verma, Rashmi; Singh, Mahender; Kumar, Sudhir

    2016-01-01

    The mitochondrial control region has been the first choice for examining the population structure but hypervariability and homoplasy have reduced its suitability. We analysed eight populations using control region for examining the population structure of Hilsa. Although the control region analysis revealed broad structuring between the Arabian Sea and Bay of Bengal (FST  0.0441, p < 0.001) it was unable to detect structure among riverine populations. These results suggest that the markers used must be able to distinguish populations and control region has led to an underestimation of genetic differentiation among populations of Hilsa. PMID:27313951