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Sample records for human mitochondrial transcription

  1. Mitochondrial transcription factor A protects human retinal ...

    African Journals Online (AJOL)

    Purpose: To investigate the impact of mitochondrial transcription factor A (TFAM), as a modulator of NF-κB, on proliferation of hypoxia-induced human retinal endothelial cell (HREC), and the probable mechanism. Methods: After exposure to hypoxia (1 % O2) for 5 days, cell proliferation and cell cycle of HREC were ...

  2. Cloning and functional analysis of human mTERFL encoding a novel mitochondrial transcription termination factor-like protein

    International Nuclear Information System (INIS)

    Chen Yao; Zhou Guangjin; Yu Min; He Yungang; Tang Wei; Lai Jianhua; He Jie; Liu Wanguo; Tan Deyong

    2005-01-01

    Serum plays an important role in the regulation of cell cycle and cell growth. To identify novel serum-inhibitory factors and study their roles in cell cycle regulation, we performed mRNA differential display analysis of U251 cells in the presence or absence of serum and cloned a novel gene encoding the human mitochondrial transcription termination factor-like protein (mTERFL). The full-length mTERFL cDNA has been isolated and the genomic structure determined. The mTERFL gene consists of three exons and encodes 385 amino acids with 52% sequence similarity to the human mitochondrial transcription termination factor (mTERF). However, mTERFL and mTERF have an opposite expression pattern in response to serum. The expression of mTERFL is dramatically inhibited by the addition of serum in serum-starved cells while the mTERF is rather induced. Northern blot analysis detected three mTERFL transcripts of 1.7, 3.2, and 3.5 kb. Besides the 3.2 kb transcript that is unique to skeletal muscle, other two transcripts express predominant in heart, liver, pancreas, and skeletal muscle. Expression of the GFP-mTERFL fusion protein in HeLa cells localized it to the mitochondria. Furthermore, ectopic expression of mTERFL suppresses cell growth and arrests cells in the G1 stage demonstrated by MTT and flow cytometry analysis. Collectively, our data suggest that mTERFL is a novel mTERF family member and a serum-inhibitory factor probably participating in the regulation of cell growth through the modulation of mitochondrial transcription

  3. Structural Basis of Mitochondrial Transcription Initiation.

    Science.gov (United States)

    Hillen, Hauke S; Morozov, Yaroslav I; Sarfallah, Azadeh; Temiakov, Dmitry; Cramer, Patrick

    2017-11-16

    Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. The Relationship Between Transcript Expression Levels of Nuclear Encoded (TFAM, NRF1 and Mitochondrial Encoded (MT-CO1 Genes in Single Human Oocytes During Oocyte Maturation

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    Ghaffari Novin M.

    2015-06-01

    Full Text Available In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA, copied in oocytes, is essential for providing adenosine triphosphate (ATP during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1 and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1 and mitochondrial transcription factor A (TFAM in various stages of human oocyte maturation. Nine consenting patients, age 21-35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI procedures. mRNA levels of mitochondrial- related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR. There was no significant relationship between the relative expression levels in germinal vesicle (GV stage oocytes (p = 0.62. On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI and MII (p = 0.03 and p = 0.002. A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation.

  5. Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

    Science.gov (United States)

    Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

    2017-12-08

    Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

  6. Perfluorooctanoic acid stimulated mitochondrial biogenesis and gene transcription in rats

    International Nuclear Information System (INIS)

    Walters, M.W.; Bjork, J.A.; Wallace, K.B.

    2009-01-01

    Perfluorooctanoic acid (PFOA), used in the production of non-stick surface compounds, exhibits a worldwide distribution in the serum of humans and wildlife. In rodents PFOA transactivates PPARα and PPARγ nuclear receptors and increases mitochondrial DNA (mtDNA) copy number, which may be critical to the altered metabolic state of affected animals. A key regulator of mitochondrial biogenesis and transcription of mitochondrial genes is the PPARγ coactivator-1α (Pgc-1α) protein. The purpose of this study was to determine if Pgc-1α is implicated in the stimulation of mitochondrial biogenesis that occurs following the treatment of rats with PFOA. Livers from adult male Sprague-Dawley rats that received a 30 mg/kg daily oral dose of PFOA for 28 days were used for all experiments. Analysis of mitochondrial replication and transcription was performed by real time PCR, and proteins were detected using western blotting. PFOA treatment caused a transcriptional activation of the mitochondrial biogenesis pathway leading to a doubling of mtDNA copy number. Further, transcription of OXPHOS genes encoded by mtDNA was 3-4 times greater than that of nuclear encoded genes, suggestive of a preferential induction of mtDNA transcription. Western blot analysis revealed an increase in Pgc-1α, unchanged Tfam and decreased Cox II and Cox IV subunit protein expression. We conclude that PFOA treatment in rats induces mitochondrial biogenesis at the transcriptional level with a preferential stimulation of mtDNA transcription and that this occurs by way of activation of the Pgc-1α pathway. Implication of the Pgc-1α pathway is consistent with PPARγ transactivation by PFOA and reveals new understanding and possibly new critical targets for assessing or averting the associated metabolic disease.

  7. The mitochondrial transcription factor A functions in mitochondrial base excision repair

    DEFF Research Database (Denmark)

    Canugovi, Chandrika; Maynard, Scott; Bayne, Anne-Cécile V

    2010-01-01

    Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids. TFAM plays an important role in mitochondrial transcription and replication. TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected i...

  8. Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

    International Nuclear Information System (INIS)

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

    2014-01-01

    Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations

  9. Thyrotropin-releasing hormone controls mitochondrial biology in human epidermis.

    Science.gov (United States)

    Knuever, Jana; Poeggeler, Burkhard; Gáspár, Erzsébet; Klinger, Matthias; Hellwig-Burgel, Thomas; Hardenbicker, Celine; Tóth, Balázs I; Bíró, Tamás; Paus, Ralf

    2012-03-01

    Mitochondrial capacity and metabolic potential are under the control of hormones, such as thyroid hormones. The most proximal regulator of the hypothalamic-pituitary-thyroid (HPT) axis, TRH, is the key hypothalamic integrator of energy metabolism via its impact on thyroid hormone secretion. Here, we asked whether TRH directly modulates mitochondrial functions in normal, TRH-receptor-positive human epidermis. Organ-cultured human skin was treated with TRH (5-100 ng/ml) for 12-48 h. TRH significantly increased epidermal immunoreactivity for the mitochondria-selective subunit I of respiratory chain complex IV (MTCO1). This resulted from an increased MTCO1 transcription and protein synthesis and a stimulation of mitochondrial biogenesis as demonstrated by transmission electron microscopy and TRH-enhanced mitochondrial DNA synthesis. TRH also significantly stimulated the transcription of several other mitochondrial key genes (TFAM, HSP60, and BMAL1), including the master regulator of mitochondrial biogenesis (PGC-1α). TRH significantly enhanced mitochondrial complex I and IV enzyme activity and enhanced the oxygen consumption of human skin samples, which shows that the stimulated mitochondria are fully vital because the main source for cellular oxygen consumption is mitochondrial endoxidation. These findings identify TRH as a potent, novel neuroendocrine stimulator of mitochondrial activity and biogenesis in human epidermal keratinocytes in situ. Thus, human epidermis offers an excellent model for dissecting neuroendocrine controls of human mitochondrial biology under physiologically relevant conditions and for exploring corresponding clinical applications.

  10. Characterization of Bombyx mori mitochondrial transcription factor A, a conserved regulator of mitochondrial DNA.

    Science.gov (United States)

    Sumitani, Megumi; Kondo, Mari; Kasashima, Katsumi; Endo, Hitoshi; Nakamura, Kaoru; Misawa, Toshihiko; Tanaka, Hiromitsu; Sezutsu, Hideki

    2017-04-15

    In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Evidence for site-specific occupancy of the mitochondrial genome by nuclear transcription factors.

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    Georgi K Marinov

    Full Text Available Mitochondria contain their own circular genome, with mitochondria-specific transcription and replication systems and corresponding regulatory proteins. All of these proteins are encoded in the nuclear genome and are post-translationally imported into mitochondria. In addition, several nuclear transcription factors have been reported to act in mitochondria, but there has been no comprehensive mapping of their occupancy patterns and it is not clear how many other factors may also be found in mitochondria. Here we address these questions by using ChIP-seq data from the ENCODE, mouseENCODE and modENCODE consortia for 151 human, 31 mouse and 35 C. elegans factors. We identified 8 human and 3 mouse transcription factors with strong localized enrichment over the mitochondrial genome that was usually associated with the corresponding recognition sequence motif. Notably, these sites of occupancy are often the sites with highest ChIP-seq signal intensity within both the nuclear and mitochondrial genomes and are thus best explained as true binding events to mitochondrial DNA, which exist in high copy number in each cell. We corroborated these findings by immunocytochemical staining evidence for mitochondrial localization. However, we were unable to find clear evidence for mitochondrial binding in ENCODE and other publicly available ChIP-seq data for most factors previously reported to localize there. As the first global analysis of nuclear transcription factors binding in mitochondria, this work opens the door to future studies that probe the functional significance of the phenomenon.

  12. Transcription profiles of mitochondrial genes correlate with mitochondrial DNA haplotypes in a natural population of Silene vulgaris

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    Olson Matthew S

    2010-01-01

    Full Text Available Abstract Background Although rapid changes in copy number and gene order are common within plant mitochondrial genomes, associated patterns of gene transcription are underinvestigated. Previous studies have shown that the gynodioecious plant species Silene vulgaris exhibits high mitochondrial diversity and occasional paternal inheritance of mitochondrial markers. Here we address whether variation in DNA molecular markers is correlated with variation in transcription of mitochondrial genes in S. vulgaris collected from natural populations. Results We analyzed RFLP variation in two mitochondrial genes, cox1 and atp1, in offspring of ten plants from a natural population of S. vulgaris in Central Europe. We also investigated transcription profiles of the atp1 and cox1 genes. Most DNA haplotypes and transcription profiles were maternally inherited; for these, transcription profiles were associated with specific mitochondrial DNA haplotypes. One individual exhibited a pattern consistent with paternal inheritance of mitochondrial DNA; this individual exhibited a transcription profile suggestive of paternal but inconsistent with maternal inheritance. We found no associations between gender and transcript profiles. Conclusions Specific transcription profiles of mitochondrial genes were associated with specific mitochondrial DNA haplotypes in a natural population of a gynodioecious species S. vulgaris. Our findings suggest the potential for a causal association between rearrangements in the plant mt genome and transcription product variation.

  13. Mitochondrial transcripts and associated heteroplasmies of Ancistrus spp. (Siluriformes: Loricariidae

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    Daniel A. Moreira

    2015-12-01

    Full Text Available This data-set complements our paper entitled “The use of transcriptomic next-generation sequencing data to assembly mitochondrial genomes of Ancistrus spp. (Loricariidae” [6]. Here, we present the nucleotide sequences of each transcript used for mitogenomes assembly, as well as tables presenting the location of each transcript in the mitogenomes; the frequency, location and codon position of the detected heteroplasmic sites; and the start/stop codons usage, UTR, CDS and poliA-tail length for each protein coding gene. Readers are referred to the paper cited above for data interpretation and discussion.

  14. Coordinated Evolution of Transcriptional and Post-Transcriptional Regulation for Mitochondrial Functions in Yeast Strains.

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    Xuepeng Sun

    Full Text Available Evolution of gene regulation has been proposed to play an important role in environmental adaptation. Exploring mechanisms underlying coordinated evolutionary changes at various levels of gene regulation could shed new light on how organism adapt in nature. In this study, we focused on regulatory differences between a laboratory Saccharomyces cerevisiae strain BY4742 and a pathogenic S. cerevisiae strain, YJM789. The two strains diverge in many features, including growth rate, morphology, high temperature tolerance, and pathogenicity. Our RNA-Seq and ribosomal footprint profiling data showed that gene expression differences are pervasive, and genes functioning in mitochondria are mostly divergent between the two strains at both transcriptional and translational levels. Combining functional genomics data from other yeast strains, we further demonstrated that significant divergence of expression for genes functioning in the electron transport chain (ETC was likely caused by differential expression of a transcriptional factor, HAP4, and that post-transcriptional regulation mediated by an RNA-binding protein, PUF3, likely led to expression divergence for genes involved in mitochondrial translation. We also explored mito-nuclear interactions via mitochondrial DNA replacement between strains. Although the two mitochondrial genomes harbor substantial sequence divergence, neither growth nor gene expression were affected by mitochondrial DNA replacement in both fermentative and respiratory growth media, indicating compatible mitochondrial and nuclear genomes between these two strains in the tested conditions. Collectively, we used mitochondrial functions as an example to demonstrate for the first time that evolution at both transcriptional and post-transcriptional levels could lead to coordinated regulatory changes underlying strain specific functional variations.

  15. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

    International Nuclear Information System (INIS)

    Dement, Gregory A.; Maloney, Scott C.; Reeves, Raymond

    2007-01-01

    We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential (ΔΨ m ). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression

  16. Human skeletal muscle mitochondrial capacity.

    Science.gov (United States)

    Rasmussen, U F; Rasmussen, H N

    2000-04-01

    Under aerobic work, the oxygen consumption and major ATP production occur in the mitochondria and it is therefore a relevant question whether the in vivo rates can be accounted for by mitochondrial capacities measured in vitro. Mitochondria were isolated from human quadriceps muscle biopsies in yields of approximately 45%. The tissue content of total creatine, mitochondrial protein and different cytochromes was estimated. A number of activities were measured in functional assays of the mitochondria: pyruvate, ketoglutarate, glutamate and succinate dehydrogenases, palmitoyl-carnitine respiration, cytochrome oxidase, the respiratory chain and the ATP synthesis. The activities involved in carbohydrate oxidation could account for in vivo oxygen uptakes of 15-16 mmol O2 min-1 kg-1 or slightly above the value measured at maximal work rates in the knee-extensor model of Saltin and co-workers, i.e. without limitation from the cardiac output. This probably indicates that the maximal oxygen consumption of the muscle is limited by the mitochondrial capacities. The in vitro activities of fatty acid oxidation corresponded to only 39% of those of carbohydrate oxidation. The maximal rate of free energy production from aerobic metabolism of glycogen was calculated from the mitochondrial activities and estimates of the DeltaG or ATP hydrolysis and the efficiency of the actin-myosin reaction. The resultant value was 20 W kg-1 or approximately 70% of the maximal in vivo work rates of which 10-20% probably are sustained by the anaerobic ATP production. The lack of aerobic in vitro ATP synthesis might reflect termination of some critical interplay between cytoplasm and mitochondria.

  17. Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Transcription

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    Chandel, N. S.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T.

    1998-09-01

    Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (ρ 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) ρ 0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in ρ 0 cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

  18. Nucleotide sequence preservation of human mitochondrial DNA

    International Nuclear Information System (INIS)

    Monnat, R.J. Jr.; Loeb, L.A.

    1985-01-01

    Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately = to 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms

  19. Increased intrinsic mitochondrial function in humans with mitochondrial haplogroup H

    DEFF Research Database (Denmark)

    Larsen, Steen; Díez-Sánchez, Carmen; Rabøl, Rasmus

    2014-01-01

    and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30......% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present....

  20. Mitochondrial dysfunction and human immunodeficiency virus ...

    African Journals Online (AJOL)

    Human immunodeficiency virus (HIV) infection and the pharmacological treatment thereof have both been shown to affect mitochondrial function in a number of tissues, and each may cause specific organ pathology through specific mitochondrial pathways. HIV has been shown to kill various tissue cells by activation of ...

  1. Melatonin and human mitochondrial diseases

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    Reza Sharafati-Chaleshtori

    2017-01-01

    Full Text Available Mitochondrial dysfunction is one of the main causative factors in a wide variety of complications such as neurodegenerative disorders, ischemia/reperfusion, aging process, and septic shock. Decrease in respiratory complex activity, increase in free radical production, increase in mitochondrial synthase activity, increase in nitric oxide production, and impair in electron transport system and/or mitochondrial permeability are considered as the main factors responsible for mitochondrial dysfunction. Melatonin, the pineal gland hormone, is selectively taken up by mitochondria and acts as a powerful antioxidant, regulating the mitochondrial bioenergetic function. Melatonin increases the permeability of membranes and is the stimulator of antioxidant enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. It also acts as an inhibitor of lipoxygenase. Melatonin can cause resistance to oxidation damage by fixing the microsomal membranes. Melatonin has been shown to retard aging and inhibit neurodegenerative disorders, ischemia/reperfusion, septic shock, diabetes, cancer, and other complications related to oxidative stress. The purpose of the current study, other than introducing melatonin, was to present the recent findings on clinical effects in diseases related to mitochondrial dysfunction including diabetes, cancer, gastrointestinal diseases, and diseases related to brain function.

  2. Mitochondrial Fusion Proteins and Human Diseases

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    Michela Ranieri

    2013-01-01

    Full Text Available Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1 and 2 (MFN2, located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1, in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer’s and Parkinson’s diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.

  3. Loss of the SIN3 transcriptional corepressor results in aberrant mitochondrial function

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    Hüttemann Maik

    2010-07-01

    Full Text Available Abstract Background SIN3 is a transcriptional repressor protein known to regulate many genes, including a number of those that encode mitochondrial components. Results By monitoring RNA levels, we find that loss of SIN3 in Drosophila cultured cells results in up-regulation of not only nuclear encoded mitochondrial genes, but also those encoded by the mitochondrial genome. The up-regulation of gene expression is accompanied by a perturbation in ATP levels in SIN3-deficient cells, suggesting that the changes in mitochondrial gene expression result in altered mitochondrial activity. In support of the hypothesis that SIN3 is necessary for normal mitochondrial function, yeast sin3 null mutants exhibit very poor growth on non-fermentable carbon sources and show lower levels of ATP and reduced respiration rates. Conclusions The findings that both yeast and Drosophila SIN3 affect mitochondrial activity suggest an evolutionarily conserved role for SIN3 in the control of cellular energy production.

  4. A human transcription factor in search mode.

    Science.gov (United States)

    Hauser, Kevin; Essuman, Bernard; He, Yiqing; Coutsias, Evangelos; Garcia-Diaz, Miguel; Simmerling, Carlos

    2016-01-08

    Transcription factors (TF) can change shape to bind and recognize DNA, shifting the energy landscape from a weak binding, rapid search mode to a higher affinity recognition mode. However, the mechanism(s) driving this conformational change remains unresolved and in most cases high-resolution structures of the non-specific complexes are unavailable. Here, we investigate the conformational switch of the human mitochondrial transcription termination factor MTERF1, which has a modular, superhelical topology complementary to DNA. Our goal was to characterize the details of the non-specific search mode to complement the crystal structure of the specific binding complex, providing a basis for understanding the recognition mechanism. In the specific complex, MTERF1 binds a significantly distorted and unwound DNA structure, exhibiting a protein conformation incompatible with binding to B-form DNA. In contrast, our simulations of apo MTERF1 revealed significant flexibility, sampling structures with superhelical pitch and radius complementary to the major groove of B-DNA. Docking these structures to B-DNA followed by unrestrained MD simulations led to a stable complex in which MTERF1 was observed to undergo spontaneous diffusion on the DNA. Overall, the data support an MTERF1-DNA binding and recognition mechanism driven by intrinsic dynamics of the MTERF1 superhelical topology. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Mitochondrial transcription factor A protects human retinal ...

    African Journals Online (AJOL)

    Finally, real-time PCR results showed that mtDNA and targeted genes of NF-κB were upregulated 3-fold in HREC after TFAM .... expression, real-time PCR mixture system containing primers ... operating as a previously reported method with.

  6. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    International Nuclear Information System (INIS)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.

    2013-01-01

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease

  7. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G., E-mail: deborah.murdock@vanderbilt.edu

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  8. Transcriptional and phylogenetic analysis of five complete ambystomatid salamander mitochondrial genomes.

    Science.gov (United States)

    Samuels, Amy K; Weisrock, David W; Smith, Jeramiah J; France, Katherine J; Walker, John A; Putta, Srikrishna; Voss, S Randal

    2005-04-11

    We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger

  9. Transcriptional changes of mitochondrial genes in irradiated cells ...

    Indian Academy of Sciences (India)

    to the increased oxidative stress in the mitochondria, DNA ... Keywords. mitochondrial gene expression; TK6 cells; radiation-induced effects. ..... This work was supported by an endowment fund, College of Nursing and Health Sciences,.

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

    Science.gov (United States)

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

    2015-05-19

    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. © 2015 Authors.

  11. Mitochondrial transcription factor A (Tfam) gene sequencing and mitochondrial evaluation in inherited retinal dysplasia in miniature schnauzer dogs.

    Science.gov (United States)

    Bauer, Bianca S; Forsyth, George W; Sandmeyer, Lynne S; Grahn, Bruce H

    2011-04-01

    Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm²) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

  12. Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation.

    Science.gov (United States)

    Ngo, Huu B; Lovely, Geoffrey A; Phillips, Rob; Chan, David C

    2014-01-01

    TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

  13. Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

    Science.gov (United States)

    Sirey, Tamara M; Ponting, Chris P

    2016-10-15

    The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

  14. Mapping 3 ' transcript ends in the bank vole (Clethrionomys glareolus) mitochondrial genome with RNA-Seq

    Czech Academy of Sciences Publication Activity Database

    Marková, Silvia; Filipi, Karolína; Searle, J. B.; Kotlík, Petr

    2015-01-01

    Roč. 16, č. 870 (2015) ISSN 1471-2164 R&D Projects: GA ČR GAP506/11/1872 Institutional support: RVO:67985904 Keywords : bicistronic transcript * mitochondrial genome * Myodes glareolus * transcriptome * polyadenylation * stop codon Subject RIV: EG - Zoology Impact factor: 3.867, year: 2015

  15. In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription

    NARCIS (Netherlands)

    Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

    2014-01-01

    The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A

  16. Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Chunhua [Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226019 Jiangsu (China); Ma, Xa; Shi, Shangshi [Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Zhao, Jianya; Nie, Xiaoke [Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Han, Jingling; Xiao, Jing; Wang, Xiaoke [Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiang, Shengyang [Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226019 Jiangsu (China); Jiang, Junkang, E-mail: Jiang_junkang@163.com [Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226019 Jiangsu (China)

    2014-12-15

    Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H{sub 2}O{sub 2} production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death. - Highlights: • p53 is

  17. Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis

    International Nuclear Information System (INIS)

    Wan, Chunhua; Ma, Xa; Shi, Shangshi; Zhao, Jianya; Nie, Xiaoke; Han, Jingling; Xiao, Jing; Wang, Xiaoke; Jiang, Shengyang; Jiang, Junkang

    2014-01-01

    Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H 2 O 2 production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death. - Highlights: • p53 is robustly

  18. Overlapping transcription structure of human cytomegalovirus

    Indian Academy of Sciences (India)

    Transcription of human cytomegalovirus UL/b′ region has been studied extensively for some genes. In this study, transcripts of the UL140 and UL141, two of the UL/b′ genes, were identified in late RNAs of three HCMV isolates using Northern blot hybridization, cDNA library screening and RACE-PCR. At least three ...

  19. Overlapping transcription structure of human cytomegalovirus ...

    Indian Academy of Sciences (India)

    2013-01-21

    Jan 21, 2013 ... Transcription of human cytomegalovirus UL/b′ region has been studied extensively for some genes. In this study, transcripts of the UL140 and UL141, two of the UL/b′ genes, were identified in late RNAs of three HCMV isolates using Northern blot hybridization, cDNA library screening and RACE-PCR.

  20. Abnormal mitochondrial respiration in failed human myocardium.

    Science.gov (United States)

    Sharov, V G; Todor, A V; Silverman, N; Goldstein, S; Sabbah, H N

    2000-12-01

    Chronic heart failure (HF) is associated with morphologic abnormalities of cardiac mitochondria including hyperplasia, reduced organelle size and compromised structural integrity. In this study, we examined whether functional abnormalities of mitochondrial respiration are also present in myocardium of patients with advanced HF. Mitochondrial respiration was examined using a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles obtained from myocardium of failed explanted human hearts due to ischemic (ICM, n=9) or idiopathic dilated (IDC, n=9) cardiomyopathy. Myocardial specimens from five normal donor hearts served as controls (CON). Basal respiratory rate, respiratory rate after addition of the substrates glutamate and malate (V(SUB)), state 3 respiration (after addition of ADP, V(ADP)) and respiration after the addition of atractyloside (V(AT)) were measured in scar-free muscle bundles obtained from the subendocardial (ENDO) and subepicardial (EPI) thirds of the left ventricular (LV) free wall, interventricular septum and right ventricular (RV) free wall. There were no differences in basal and substrate-supported respiration between CON and HF regardless of etiology. V(ADP)was significantly depressed both in ICM and IDC compared to CON in all the regions studied. The respiratory control ratio, V(ADP)/V(AT), was also significantly decreased in HF compared to CON. In both ICM and IDC, V(ADP)was significantly lower in ENDO compared to EPI. The results indicate that mitochondrial respiration is abnormal in the failing human heart. The findings support the concept of low myocardial energy production in HF via oxidative phosphorylation, an abnormality with a potentially impact on global cardiac performance. Copyright 2000 Academic Press.

  1. Impaired mitochondrial function in chronically ischemic human heart

    DEFF Research Database (Denmark)

    Stride, Nis Ottesen; Larsen, Steen; Hey-Mogensen, Martin

    2013-01-01

    , and finally to assess myocardial antioxidant levels. Mitochondrial respiration in biopsies from ischemic and nonischemic regions from the left ventricle of the same heart was compared in nine human subjects. Maximal oxidative phosphorylation capacity in fresh muscle fibers was lower in ischemic compared.......05), and the levels of antioxidant protein expression was lower. Diminished mitochondrial respiration capacity and excessive ROS production demonstrate an impaired mitochondrial function in ischemic human heart muscle. No chronic ischemic preconditioning effect was found....

  2. StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

    Science.gov (United States)

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

    2014-01-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

  3. Towards a functional definition of the mitochondrial human proteome

    Directory of Open Access Journals (Sweden)

    Mauro Fasano

    2016-03-01

    Full Text Available The mitochondrial human proteome project (mt-HPP was initiated by the Italian HPP group as a part of both the chromosome-centric initiative (C-HPP and the “biology and disease driven” initiative (B/D-HPP. In recent years several reports highlighted how mitochondrial biology and disease are regulated by specific interactions with non-mitochondrial proteins. Thus, it is of great relevance to extend our present view of the mitochondrial proteome not only to those proteins that are encoded by or transported to mitochondria, but also to their interactors that take part in mitochondria functionality. Here, we propose a graphical representation of the functional mitochondrial proteome by retrieving mitochondrial proteins from the NeXtProt database and adding to the network their interactors as annotated in the IntAct database. Notably, the network may represent a reference to map all the proteins that are currently being identified in mitochondrial proteomics studies.

  4. Mitochondrial DNA mutations in human tumor cells

    OpenAIRE

    LI, HUI; HONG, ZE-HUI

    2012-01-01

    Mitochondria play significant roles in cellular energy metabolism, free radical generation and apoptosis. The dysfunction of mitochondria is correlated with the origin and progression of tumors; thus, mutations in the mitochondrial genome that affect mitochondrial function may be one of the causal factors of tumorigenesis. Although the role of mitochondrial DNA (mtDNA) mutations in carcinogenesis has been investigated extensively by various approaches, the conclusions remain controversial to ...

  5. Non-electron transfer chain mitochondrial defects differently regulate HIF-1α degradation and transcription

    Directory of Open Access Journals (Sweden)

    Antonina N. Shvetsova

    2017-08-01

    Full Text Available Mitochondria are the main consumers of molecular O2 in a cell as well as an abundant source of reactive oxygen species (ROS. Both, molecular oxygen and ROS are powerful regulators of the hypoxia-inducible factor-1α-subunit (HIF-α. While a number of mechanisms in the oxygen-dependent HIF-α regulation are quite well known, the view with respect to mitochondria is less clear. Several approaches using pharmacological or genetic tools targeting the mitochondrial electron transport chain (ETC indicated that ROS, mainly formed at the Rieske cluster of complex III of the ETC, are drivers of HIF-1α activation. However, studies investigating non-ETC located mitochondrial defects and their effects on HIF-1α regulation are scarce, if at all existing. Thus, in the present study we examined three cell lines with non-ETC mitochondrial defects and focused on HIF-1α degradation and transcription, target gene expression, as well as ROS levels. We found that cells lacking the key enzyme 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (MECR, and cells lacking manganese superoxide dismutase (MnSOD showed a reduced induction of HIF-1α under long-term (20 h hypoxia. By contrast, cells lacking the mitochondrial DNA depletion syndrome channel protein Mpv17 displayed enhanced levels of HIF-1α already under normoxic conditions. Further, we show that ROS do not exert a uniform pattern when mediating their effects on HIF-1α, although all mitochondrial defects in the used cell types increased ROS formation. Moreover, all defects caused a different HIF-1α regulation via promoting HIF-1α degradation as well as via changes in HIF-1α transcription. Thereby, MECR- and MnSOD-deficient cells showed a reduction in HIF-1α mRNA levels whereas the Mpv17 lacking cells displayed enhanced HIF-1α mRNA levels under normoxia and hypoxia. Altogether, our study shows for the first time that mitochondrial defects which are not related to the ETC and Krebs cycle

  6. Colon cancer associated transcripts in human cancers.

    Science.gov (United States)

    Chen, Yincong; Xie, Haibiao; Gao, Qunjun; Zhan, Hengji; Xiao, Huizhong; Zou, Yifan; Zhang, Fuyou; Liu, Yuchen; Li, Jianfa

    2017-10-01

    Long non-coding RNAs serve as important regulators in complicated cellular activities, including cell differentiation, proliferation and death. Dysregulation of long non-coding RNAs occurs in the formation and progression of cancers. The family of colon cancer associated transcripts, long non-coding RNAs colon cancer associated transcript-1 and colon cancer associated transcript-2 are known as oncogenes involved in various cancers. Colon cancer associated transcript-1 is a novel lncRNA located in 8q24.2, and colon cancer associated transcript-2 maps to the 8q24.21 region encompassing rs6983267. Colon cancer associated transcripts have close associations with clinical characteristics, such as lymph node metastasis, high TNM stage and short overall survival. Knockdown of them can reverse the malignant phenotypes of cancer cells, including proliferation, migration, invasion and apoptosis. Moreover, they can increase the expression level of c-MYC and oncogenic microRNAs via activating a series of complex mechanisms. In brief, the family of colon cancer associated transcripts may serve as potential biomarkers or therapeutic targets for human cancers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Muscle mitochondrial capacity exceeds maximal oxygen delivery in humans

    DEFF Research Database (Denmark)

    Boushel, Robert Christopher; Gnaiger, Erich; Calbet, Jose A L

    2011-01-01

    Across a wide range of species and body mass a close matching exists between maximal conductive oxygen delivery and mitochondrial respiratory rate. In this study we investigated in humans how closely in-vivo maximal oxygen consumption (VO(2) max) is matched to state 3 muscle mitochondrial respira...

  8. In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription.

    Science.gov (United States)

    Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

    2014-07-10

    The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Transcutaneous application of carbon dioxide (CO2 induces mitochondrial apoptosis in human malignant fibrous histiocytoma in vivo.

    Directory of Open Access Journals (Sweden)

    Yasuo Onishi

    Full Text Available Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO(2 to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO(2 exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO(2 induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO(2 treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO(2 treated tumors, highlighting the involvement of mitochondria in apoptosis

  10. An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

    Science.gov (United States)

    Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

    2009-01-01

    Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

  11. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    Science.gov (United States)

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Involvement of the mitochondrial compartment in human NCL fibroblasts

    International Nuclear Information System (INIS)

    Pezzini, Francesco; Gismondi, Floriana; Tessa, Alessandra; Tonin, Paola; Carrozzo, Rosalba; Mole, Sara E.; Santorelli, Filippo M.; Simonati, Alessandro

    2011-01-01

    Highlights: ► Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. ► Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. ► Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  13. A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

    Science.gov (United States)

    Lorimer, H E; Brewer, B J; Fangman, W L

    1995-09-01

    Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

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

    Science.gov (United States)

    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.

  15. Three dimensional reconstruction of the human skeletal muscle mitochondrial network as a tool to assess mitochondrial content and structural organization

    DEFF Research Database (Denmark)

    Dahl, Rannvá; Larsen, Steen; Dohlmann, Tine L

    2015-01-01

    a method to visualize mitochondrial networks in high resolution and assess mitochondrial volume. Methods: Confocal fluorescence microscopy imaging of mitochondrial network stains in human vastus lateralis single muscle fibers and, focused ion beam scanning electron microscopy (FIB/SEM) imaging, combined...... mitochondrial dynamics in response to life-style interventions and/or in certain pathologies. Our results question the classification of mitochondria into subsarcolemmal and intermyofibrillar pools, since they are physically interconnected. This article is protected by copyright. All rights reserved....

  16. Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine

    International Nuclear Information System (INIS)

    Velsor, Leonard W.; Kovacevic, Miro; Goldstein, Mark; Leitner, Heather M.; Lewis, William; Day, Brian J.

    2004-01-01

    The toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is linked to altered mitochondrial DNA (mtDNA) replication and subsequent disruption of cellular energetics. This manifests clinically as elevated concentrations of lactate in plasma. The mechanism(s) underlying how the changes in mtDNA replication lead to lactic acidosis remains unclear. It is hypothesized that mitochondrial oxidative stress links the changes in mtDNA replication to mitochondrial dysfunction and ensuing NRTIs toxicity. To test this hypothesis, changes in mitochondrial function, mtDNA amplification efficiency, and oxidative stress were assessed in HepG2-cultured human hepatoblasts treated with the NRTI stavudine (2',3'-didehydro-2',3'-deoxythymidine or d4T) for 48 h. d4T produced significant mitochondrial dysfunction with a 1.5-fold increase in cellular lactate to pyruvate ratios. In addition, d4T caused a dose-dependent decrease in mtDNA amplification and a correlative increase in abundance of markers of mitochondrial oxidative stress. Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, MnTBAP, a catalytic antioxidant, ameliorated or reversed d4T-induced changes in cell injury, energetics, mtDNA amplification, and mitochondrial oxidative stress. In conclusion, d4T treatment elevates mitochondrial reactive oxygen species (ROS), enhances mitochondrial oxidative stress, and contributes mechanistically to NRTI-induced toxicity. These deleterious events may be potentiated in acquired immunodeficiency syndrome (AIDS) by human immunodeficiency virus (HIV) infection itself, coinfection (e.g., viral hepatitis), aging, substance, and alcohol use

  17. The HO-1/CO system regulates mitochondrial-capillary density relationships in human skeletal muscle.

    Science.gov (United States)

    Pecorella, Shelly R H; Potter, Jennifer V F; Cherry, Anne D; Peacher, Dionne F; Welty-Wolf, Karen E; Moon, Richard E; Piantadosi, Claude A; Suliman, Hagir B

    2015-10-15

    The heme oxygenase-1 (HO-1)/carbon monoxide (CO) system induces mitochondrial biogenesis, but its biological impact in human skeletal muscle is uncertain. The enzyme system generates CO, which stimulates mitochondrial proliferation in normal muscle. Here we examined whether CO breathing can be used to produce a coordinated metabolic and vascular response in human skeletal muscle. In 19 healthy subjects, we performed vastus lateralis muscle biopsies and tested one-legged maximal O2 uptake (V̇o2max) before and after breathing air or CO (200 ppm) for 1 h daily for 5 days. In response to CO, there was robust HO-1 induction along with increased mRNA levels for nuclear-encoded mitochondrial transcription factor A (Tfam), cytochrome c, cytochrome oxidase subunit IV (COX IV), and mitochondrial-encoded COX I and NADH dehydrogenase subunit 1 (NDI). CO breathing did not increase V̇o2max (1.96 ± 0.51 pre-CO, 1.87 ± 0.50 post-CO l/min; P = not significant) but did increase muscle citrate synthase, mitochondrial density (139.0 ± 34.9 pre-CO, 219.0 ± 36.2 post-CO; no. of mitochondrial profiles/field), myoglobin content and glucose transporter (GLUT4) protein level and led to GLUT4 localization to the myocyte membrane, all consistent with expansion of the tissue O2 transport system. These responses were attended by increased cluster of differentiation 31 (CD31)-positive muscle capillaries (1.78 ± 0.16 pre-CO, 2.37 ± 0.59 post-CO; capillaries/muscle fiber), implying the enrichment of microvascular O2 reserve. The findings support that induction of the HO-1/CO system by CO not only improves muscle mitochondrial density, but regulates myoglobin content, GLUT4 localization, and capillarity in accordance with current concepts of skeletal muscle plasticity. Copyright © 2015 the American Physiological Society.

  18. A Saccharomyces cerevisiae mitochondrial DNA fragment activates Reg1p-dependent glucose-repressible transcription in the nucleus.

    Science.gov (United States)

    Santangelo, G M; Tornow, J

    1997-12-01

    As part of an effort to identify random carbon-source-regulated promoters in the Saccharomyces cerevisiae genome, we discovered that a mitochondrial DNA fragment is capable of directing glucose-repressible expression of a reporter gene. This fragment (CR24) originated from the mitochondrial genome adjacent to a transcription initiation site. Mutational analyses identified a GC cluster within the fragment that is required for transcriptional induction. Repression of nuclear CR24-driven transcription required Reg1p, indicating that this mitochondrially derived promoter is a member of a large group of glucose-repressible nuclear promoters that are similarly regulated by Reg1p. In vivo and in vitro binding assays indicated the presence of factors, located within the nucleus and the mitochondria, that bind to the GC cluster. One or more of these factors may provide a regulatory link between the nucleus and mitochondria.

  19. Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Andersen, Kasper Røjkjær; Kjær, Karina Hansen

    2011-01-01

    . Interestingly, 2′-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease–endonuclease–phosphatase family of deadenylases. Here we show that 2′-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3′–5′ exoribonuclease exhibiting a preference...

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  1. Curated compendium of human transcriptional biomarker data.

    Science.gov (United States)

    Golightly, Nathan P; Bell, Avery; Bischoff, Anna I; Hollingsworth, Parker D; Piccolo, Stephen R

    2018-04-17

    One important use of genome-wide transcriptional profiles is to identify relationships between transcription levels and patient outcomes. These translational insights can guide the development of biomarkers for clinical application. Data from thousands of translational-biomarker studies have been deposited in public repositories, enabling reuse. However, data-reuse efforts require considerable time and expertise because transcriptional data are generated using heterogeneous profiling technologies, preprocessed using diverse normalization procedures, and annotated in non-standard ways. To address this problem, we curated 45 publicly available, translational-biomarker datasets from a variety of human diseases. To increase the data's utility, we reprocessed the raw expression data using a uniform computational pipeline, addressed quality-control problems, mapped the clinical annotations to a controlled vocabulary, and prepared consistently structured, analysis-ready data files. These data, along with scripts we used to prepare the data, are available in a public repository. We believe these data will be particularly useful to researchers seeking to perform benchmarking studies-for example, to compare and optimize machine-learning algorithms' ability to predict biomedical outcomes.

  2. The transcriptional co-repressor myeloid translocation gene 16 inhibits glycolysis and stimulates mitochondrial respiration.

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    Full Text Available The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4, and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1 was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2 oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect.

  3. Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

    Directory of Open Access Journals (Sweden)

    Heyman Yvan

    2005-11-01

    Full Text Available Abstract Background Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. Methods We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1, and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1, and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA. Results Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. Conclusion Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

  4. Mitochondrial genome inheritance and replacement in the human germline.

    Science.gov (United States)

    Wolf, Don P; Hayama, Tomonari; Mitalipov, Shoukhrat

    2017-08-01

    Mitochondria, the ubiquitous power packs in nearly every eukaryotic cell, contain their own DNA, known as mtDNA, which is inherited exclusively from the mother. The number of mitochondrial genomes varies depending on the cell's energy needs. The mature oocyte contains the highest number of mitochondria of any cell type, although there is little if any mtDNA replication after fertilization until the embryo implants. This has potential repercussions for mitochondrial replacement therapy (MRT; see description of currently employed methods below) used to prevent the transmission of mtDNA-based disorders. If only a few mitochondria with defective mtDNA are left in the embryo and undergo extensive replication, it might therefore thwart the purpose of MRT In order to improve the safety and efficacy of this experimental therapy, we need a better understanding of how and which mtDNA is tagged for replication versus transcription after fertilization of the oocyte. © 2017 The Authors.

  5. Cryopreservation of human skeletal muscle impairs mitochondrial function

    DEFF Research Database (Denmark)

    Larsen, Steen; Wright-Paradis, C; Gnaiger, E

    2012-01-01

    functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity...

  6. Erythropoietin treatment enhances muscle mitochondrial capacity in humans

    DEFF Research Database (Denmark)

    Plenge, Ulla; Belhage, Bo; Guadalupe-Grau, Amelia

    2012-01-01

    in humans. In six healthy volunteers rhEpo was administered by sub-cutaneous injection over 8 weeks with oral iron (100 mg) supplementation taken daily. Mitochondrial OXPHOS was quantified by high-resolution respirometry in saponin-permeabilized muscle fibers obtained from biopsies of the vastus lateralis...

  7. The Mitochondrial Protein Atlas: A Database of Experimentally Verified Information on the Human Mitochondrial Proteome.

    Science.gov (United States)

    Godin, Noa; Eichler, Jerry

    2017-09-01

    Given its central role in various biological systems, as well as its involvement in numerous pathologies, the mitochondrion is one of the best-studied organelles. However, although the mitochondrial genome has been extensively investigated, protein-level information remains partial, and in many cases, hypothetical. The Mitochondrial Protein Atlas (MPA; URL: lifeserv.bgu.ac.il/wb/jeichler/MPA ) is a database that provides a complete, manually curated inventory of only experimentally validated human mitochondrial proteins. The MPA presently contains 911 unique protein entries, each of which is associated with at least one experimentally validated and referenced mitochondrial localization. The MPA also contains experimentally validated and referenced information defining function, structure, involvement in pathologies, interactions with other MPA proteins, as well as the method(s) of analysis used in each instance. Connections to relevant external data sources are offered for each entry, including links to NCBI Gene, PubMed, and Protein Data Bank. The MPA offers a prototype for other information sources that allow for a distinction between what has been confirmed and what remains to be verified experimentally.

  8. An anatomic transcriptional atlas of human glioblastoma.

    Science.gov (United States)

    Puchalski, Ralph B; Shah, Nameeta; Miller, Jeremy; Dalley, Rachel; Nomura, Steve R; Yoon, Jae-Guen; Smith, Kimberly A; Lankerovich, Michael; Bertagnolli, Darren; Bickley, Kris; Boe, Andrew F; Brouner, Krissy; Butler, Stephanie; Caldejon, Shiella; Chapin, Mike; Datta, Suvro; Dee, Nick; Desta, Tsega; Dolbeare, Tim; Dotson, Nadezhda; Ebbert, Amanda; Feng, David; Feng, Xu; Fisher, Michael; Gee, Garrett; Goldy, Jeff; Gourley, Lindsey; Gregor, Benjamin W; Gu, Guangyu; Hejazinia, Nika; Hohmann, John; Hothi, Parvinder; Howard, Robert; Joines, Kevin; Kriedberg, Ali; Kuan, Leonard; Lau, Chris; Lee, Felix; Lee, Hwahyung; Lemon, Tracy; Long, Fuhui; Mastan, Naveed; Mott, Erika; Murthy, Chantal; Ngo, Kiet; Olson, Eric; Reding, Melissa; Riley, Zack; Rosen, David; Sandman, David; Shapovalova, Nadiya; Slaughterbeck, Clifford R; Sodt, Andrew; Stockdale, Graham; Szafer, Aaron; Wakeman, Wayne; Wohnoutka, Paul E; White, Steven J; Marsh, Don; Rostomily, Robert C; Ng, Lydia; Dang, Chinh; Jones, Allan; Keogh, Bart; Gittleman, Haley R; Barnholtz-Sloan, Jill S; Cimino, Patrick J; Uppin, Megha S; Keene, C Dirk; Farrokhi, Farrokh R; Lathia, Justin D; Berens, Michael E; Iavarone, Antonio; Bernard, Amy; Lein, Ed; Phillips, John W; Rostad, Steven W; Cobbs, Charles; Hawrylycz, Michael J; Foltz, Greg D

    2018-05-11

    Glioblastoma is an aggressive brain tumor that carries a poor prognosis. The tumor's molecular and cellular landscapes are complex, and their relationships to histologic features routinely used for diagnosis are unclear. We present the Ivy Glioblastoma Atlas, an anatomically based transcriptional atlas of human glioblastoma that aligns individual histologic features with genomic alterations and gene expression patterns, thus assigning molecular information to the most important morphologic hallmarks of the tumor. The atlas and its clinical and genomic database are freely accessible online data resources that will serve as a valuable platform for future investigations of glioblastoma pathogenesis, diagnosis, and treatment. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. Mitochondrial regulation of epigenetics and its role in human diseases

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Tollefsbol, Trygve O; Singh, Keshav K

    2012-01-01

    as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction....... In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead...... to epigenetic changes causing genomic instability in the nuclear genome. We propose that "mitocheckpoint" mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role....

  10. Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes

    International Nuclear Information System (INIS)

    Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L.; Berrueta, Lisbeth; Salmen, Siham

    2016-01-01

    Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. - Highlights: • The expression and subcellular distribution of Foxp3, is modulated by PMA and preS1/2. • PMA and preS1/2 increase Foxp3 expression on HepG2. • PMA and preS1/2 induce foxp3 enrichment at mitochondrial, microsomal and nuclear compartments. • Results suggest a non-canonical function of Foxp3 or a mitochondrial transcriptional activity.

  11. Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa [Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of); Peterson, Darrell L. [Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA (United States); Berrueta, Lisbeth, E-mail: lberruet@ula.ve [Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of); Division of Preventive Medicine, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Salmen, Siham, E-mail: sihamsa@ula.ve [Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of)

    2016-05-01

    Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. - Highlights: • The expression and subcellular distribution of Foxp3, is modulated by PMA and preS1/2. • PMA and preS1/2 increase Foxp3 expression on HepG2. • PMA and preS1/2 induce foxp3 enrichment at mitochondrial, microsomal and nuclear compartments. • Results suggest a non-canonical function of Foxp3 or a mitochondrial transcriptional activity.

  12. Continued colonization of the human genome by mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Miria Ricchetti

    2004-09-01

    Full Text Available Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4-6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

  13. A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast.

    NARCIS (Netherlands)

    Blakely, E.L.; Mitchell, A.L.; Fisher, N.; Meunier, B.; Nijtmans, L.G.J.; Schaefer, A.M.; Jackson, M.J.; Turnbull, D.M.; Taylor, R.W.

    2005-01-01

    Whereas the majority of disease-related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical

  14. From reverse transcription to human brain tumors

    Directory of Open Access Journals (Sweden)

    Dmitrenko V. V.

    2013-05-01

    Full Text Available Reverse transcriptase from avian myeloblastosis virus (AMV was the subject of the study, from which the investi- gations of the Department of biosynthesis of nucleic acids were started. Production of AMV in grams quantities and isolation of AMV reverse transcriptase were established in the laboratory during the seventies of the past cen- tury and this initiated research on the cDNA synthesis, cloning and investigation of the structure and functions of the eukaryotic genes. Structures of salmon insulin and insulin-like growth factor (IGF family genes and their transcripts were determined during long-term investigations. Results of two modern techniques, microarray-ba- sed hybridization and SAGE, were used for the identification of the genes differentially expressed in astrocytic gliomas and human normal brain. Comparison of SAGE results on the genes overexpressed in glioblastoma with the results of microarray analysis revealed a limited number of common genes. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of glioblastoma. The first experiments on the classification of glioblastomas based on the data of the 20 genes expression were conducted by using of artificial neural network analysis. The results of these experiments showed that the expression profiles of these genes in 224 glioblastoma samples and 74 normal brain samples could be according to the Koho- nen’s maps. The CHI3L1 and CHI3L2 genes of chitinase-like cartilage protein were revealed among the most overexpressed genes in glioblastoma, which could have prognostic and diagnostic potential. Results of in vitro experiments demonstrated that both proteins, CHI3L1 and CHI3L2, may initiate the phosphorylation of ERK1/ ERK2 and AKT kinases leading to the activation of MAPK/ERK1/2 and PI3K/AKT signaling cascades in human embryonic kidney 293 cells, human glioblastoma U87MG, and U373 cells. The new human cell line

  15. Cholesterol can modulate mitochondrial aquaporin-8 expression in human hepatic cells.

    Science.gov (United States)

    Danielli, Mauro; Capiglioni, Alejo M; Marrone, Julieta; Calamita, Giuseppe; Marinelli, Raúl A

    2017-05-01

    Hepatocyte mitochondrial aquaporin-8 (mtAQP8) works as a multifunctional membrane channel protein that facilitates the uptake of ammonia for its detoxification to urea as well as the mitochondrial release of hydrogen peroxide. Since early oligonucleotide microarray studies in liver of cholesterol-fed mice showed an AQP8 downregulation, we tested whether alterations of cholesterol content per se modulate mtAQP8 expression in human hepatocyte-derived Huh-7 cells. Cholesterol loading with methyl-β-cyclodextrin (mβCD):cholesterol complexes downregulated the proteolytic activation of cholesterol-responsive sterol regulatory element-binding protein (SREBP) transcriptions factors 1 and 2, and the expression of the target gene 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Under such conditions, mtAQP8 mRNA and protein expressions were significantly reduced. In contrast, cholesterol depletion using mβCD alone increased SREBP-1 and 2 activation and upregulated HMGCR and mtAQP8 mRNA and protein expressions. The results suggest that cholesterol can regulate transcriptionally human hepatocyte mtAQP8 expression likely via SREBPs. The functional implications of our findings are discussed. © 2017 IUBMB Life, 69(5):341-346, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  16. Calorie restriction increases muscle mitochondrial biogenesis in healthy humans.

    Directory of Open Access Journals (Sweden)

    Anthony E Civitarese

    2007-03-01

    Full Text Available Caloric restriction without malnutrition extends life span in a range of organisms including insects and mammals and lowers free radical production by the mitochondria. However, the mechanism responsible for this adaptation are poorly understood.The current study was undertaken to examine muscle mitochondrial bioenergetics in response to caloric restriction alone or in combination with exercise in 36 young (36.8 +/- 1.0 y, overweight (body mass index, 27.8 +/- 0.7 kg/m(2 individuals randomized into one of three groups for a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric restriction; and CREX, caloric restriction with exercise (CREX, 12.5% CR + 12.5% increased energy expenditure (EE. In the controls, 24-h EE was unchanged, but in CR and CREX it was significantly reduced from baseline even after adjustment for the loss of metabolic mass (CR, -135 +/- 42 kcal/d, p = 0.002 and CREX, -117 +/- 52 kcal/d, p = 0.008. Participants in the CR and CREX groups had increased expression of genes encoding proteins involved in mitochondrial function such as PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05. In parallel, mitochondrial DNA content increased by 35% +/- 5% in the CR group (p = 0.005 and 21% +/- 4% in the CREX group (p < 0.004, with no change in the control group (2% +/- 2%. However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic acid cycle (citrate synthase, beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase, and electron transport chain (cytochrome C oxidase II was unchanged. DNA damage was reduced from baseline in the CR (-0.56 +/- 0.11 arbitrary units, p = 0.003 and CREX (-0.45 +/- 0.12 arbitrary units, p = 0.011, but not in the controls. In primary cultures of human myotubes, a nitric oxide donor (mimicking eNOS signaling induced mitochondrial biogenesis but failed to induce SIRT1 protein expression, suggesting that additional factors may regulate SIRT1 content during CR.The observed increase in

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...... culture and rodent skeletal muscle. To determine whether PGC-1a transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two......-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl...

  18. Major genomic mitochondrial lineages delineate early human expansions

    Directory of Open Access Journals (Sweden)

    Flores Carlos

    2001-08-01

    Full Text Available Abstract Background The phylogeographic distribution of human mitochondrial DNA variations allows a genetic approach to the study of modern Homo sapiens dispersals throughout the world from a female perspective. As a new contribution to this study we have phylogenetically analysed complete mitochondrial DNA(mtDNA sequences from 42 human lineages, representing major clades with known geographic assignation. Results We show the relative relationships among the 42 lineages and present more accurate temporal calibrations than have been previously possible to give new perspectives as how modern humans spread in the Old World. Conclusions The first detectable expansion occurred around 59,000–69,000 years ago from Africa, independently colonizing western Asia and India and, following this southern route, swiftly reaching east Asia. Within Africa, this expansion did not replace but mixed with older lineages detectable today only in Africa. Around 39,000–52,000 years ago, the western Asian branch spread radially, bringing Caucasians to North Africa and Europe, also reaching India, and expanding to north and east Asia. More recent migrations have entangled but not completely erased these primitive footprints of modern human expansions.

  19. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  20. Novel transcriptional networks regulated by CLOCK in human neurons.

    Science.gov (United States)

    Fontenot, Miles R; Berto, Stefano; Liu, Yuxiang; Werthmann, Gordon; Douglas, Connor; Usui, Noriyoshi; Gleason, Kelly; Tamminga, Carol A; Takahashi, Joseph S; Konopka, Genevieve

    2017-11-01

    The molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to human cognition and disease. In this study, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin immunoprecipitation sequencing for endogenous CLOCK in adult neocortices and RNA sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates the expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts coexpressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks is driven by hub genes with human-specific patterns of expression. These data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function. © 2017 Fontenot et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Human cytomegalovirus (HCMV) induces human endogenous retrovirus (HERV) transcription.

    Science.gov (United States)

    Assinger, Alice; Yaiw, Koon-Chu; Göttesdorfer, Ingmar; Leib-Mösch, Christine; Söderberg-Nauclér, Cecilia

    2013-11-12

    Emerging evidence suggests that human cytomegalovirus (HCMV) is highly prevalent in tumours of different origin. This virus is implied to have oncogenic and oncomodulatory functions, through its ability to control host gene expression. Human endogenous retroviruses (HERV) are also frequently active in tumours of different origin, and are supposed to contribute as cofactors to cancer development. Due to the high prevalence of HCMV in several different tumours, and its ability to control host cell gene expression, we sought to define whether HCMV may affect HERV transcription. Infection of 3 established cancer cell lines, 2 primary glioblastoma cells, endothelial cells from 3 donors and monocytes from 4 donors with HCMV (strains VR 1814 or TB40/F) induced reverse transcriptase (RT) activity in all cells tested, but the response varied between donors. Both, gammaretrovirus-related class I elements HERV-T, HERV-W, HERV-F and ERV-9, and betaretrovirus-related class II elements HML-2 - 4 and HML-7 - 8, as well as spuma-virus related class III elements of the HERV-L group were up-regulated in response to HCMV infection in GliNS1 cells. Up-regulation of HERV activity was more pronounced in cells harbouring active HCMV infection, but was also induced by UV-inactivated virus. The effect was only slightly affected by ganciclovir treatment and was not controlled by the IE72 or IE86 HCMV genes. Within this brief report we show that HCMV infection induces HERV transcriptional activity in different cell types.

  2. Characterization of human mesothelin transcripts in ovarian and pancreatic cancer

    International Nuclear Information System (INIS)

    Muminova, Zhanat E; Strong, Theresa V; Shaw, Denise R

    2004-01-01

    Mesothelin is an attractive target for cancer immunotherapy due to its restricted expression in normal tissues and high level expression in several tumor types including ovarian and pancreatic adenocarcinomas. Three mesothelin transcript variants have been reported, but their relative expression in normal tissues and tumors has been poorly characterized. The goal of the present study was to clarify which mesothelin transcript variants are commonly expressed in human tumors. Human genomic and EST nucleotide sequences in the public databases were used to evaluate sequences reported for the three mesothelin transcript variants in silico. Subsequently, RNA samples from normal ovary, ovarian and pancreatic carcinoma cell lines, and primary ovarian tumors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing to directly identify expressed transcripts. In silico comparisons of genomic DNA sequences with available EST sequences supported expression of mesothelin transcript variants 1 and 3, but there were no sequence matches for transcript variant 2. Newly-derived nucleotide sequences of RT-PCR products from tissues and cell lines corresponded to mesothelin transcript variant 1. Mesothelin transcript variant 2 was not detected. Transcript variant 3 was observed as a small percentage of total mesothelin amplification products from all studied cell lines and tissues. Fractionation of nuclear and cytoplasmic RNA indicated that variant 3 was present primarily in the nuclear fraction. Thus, mesothelin transcript variant 3 may represent incompletely processed hnRNA. Mesothelin transcript variant 1 represents the predominant mature mRNA species expressed by both normal and tumor cells. This conclusion should be important for future development of cancer immunotherapies, diagnostic tests, and gene microarray studies targeting mesothelin

  3. Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

    DEFF Research Database (Denmark)

    Larsen, Steen; Nielsen, Joachim; Hansen, Christina Neigaard

    2012-01-01

    Key points  Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects.  The most commonly used...... markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein, and complex I-IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres......).  Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content.  mtDNA was found to be a poor biomarker of mitochondrial content.  Complex IV activity was closely associated with mitochondrial oxidative...

  4. Mitochondrial Damage-Associated Molecular Patterns: From Inflammatory Signaling to Human Diseases

    Directory of Open Access Journals (Sweden)

    Serge Grazioli

    2018-05-01

    Full Text Available Over the recent years, much has been unraveled about the pro-inflammatory properties of various mitochondrial molecules once they are leaving the mitochondrial compartment. On entering the cytoplasm or the extracellular space, mitochondrial DAMPs (also known as mitochondrial alarmins can become pro-inflammatory and initiate innate and adaptive immune responses by activating cell surface and intracellular receptors. Current evidence indicates that uncontrolled and excessive release of mitochondrial DAMPs is associated with severity, has prognosis value in human diseases, and contributes to the dysregulated process observed in numerous inflammatory and autoimmune conditions, as well as in ischemic heart disease and cancer. Herein, we review that the expanding research field of mitochondrial DAMPs in innate immune responses and the current knowledge on the association between mitochondrial DAMPs and human diseases.

  5. Dynamic regulation of genes involved in mitochondrial DNA replication and transcription during mouse brown fat cell differentiation and recruitment

    DEFF Research Database (Denmark)

    Murholm, Maria; Dixen, Karen; Qvortrup, Klaus

    2009-01-01

    BACKGROUND: Brown adipocytes are specialised in dissipating energy through adaptive thermogenesis, whereas white adipocytes are specialised in energy storage. These essentially opposite functions are possible for two reasons relating to mitochondria, namely expression of uncoupling protein 1 (UCP1...... and brown fat, brown adipose tissue fractions and in selected adipose tissues during cold exposure. We find a massive induction of the majority of such genes during brown adipocyte differentiation and recruitment, e.g. of the mitochondrial transcription factors A (Tfam) and B2 (Tfb2m), whereas only a subset...

  6. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

    2015-06-15

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

  7. Study on differential transcriptional profile in human hepatocyte exposed to different doses γ ray

    International Nuclear Information System (INIS)

    Li Jianguo; Wen Jianhua; Duan Zhikai; Tian Yu; Wang Fang; Zuo Yahui

    2009-01-01

    The study analyzed the differential transcriptional profile of normal human hepatic cell and human hepatic cell radiated with three different doses (0.5 Gy, 2 Gy, 4 Gy γ ray) by gene chip technique. The results showed that the whole differentially expressed genes of three different doses have 284 in 14112 human genes analyzed, in which 261 genes were up-regulated and 23 genes were down-regulated. These genes are mainly associated with interferon receptor, mitochondrial regulation, homo sapiens hepatitis A virus cellular receptor, cell cycle regulation, kinase and zinc finger protein etc. RT-PCR results indicated that up-regulated expression of gene HAVcr-1, HAVcr-2, MFTC, MOAP1 and down-regulated expression of gene TRIP12, DCN are consistent with gene chip data. (authors)

  8. Mitochondrial respiratory efficiency is positively correlated with human sperm motility.

    Science.gov (United States)

    Ferramosca, Alessandra; Provenzano, Sara Pinto; Coppola, Lamberto; Zara, Vincenzo

    2012-04-01

    To correlate sperm mitochondrial respiratory efficiency with variations in sperm motility and with sperm morphologic anomalies. Sperm mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically-treated sperm cells. A possible relationship among sperm mitochondrial respiratory efficiency, sperm motility, and morphologic anomalies was investigated. Mitochondrial respiratory efficiency was positively correlated with sperm motility and negatively correlated with the percentage of immotile spermatozoa. Moreover, midpiece defects impaired mitochondrial functionality. Our data indicate that an increase in sperm motility requires a parallel increase in mitochondrial respiratory capacity, thereby supporting the fundamental role played by mitochondrial oxidative phosphorylation in sperm motility of normozoospermic subjects. These results are of physiopathological relevance because they suggest that disturbances of sperm mitochondrial function and of energy production could be responsible for asthenozoospermia. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

    Science.gov (United States)

    Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

    2014-01-01

    Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

  10. Cyclin D3 interacts with human activating transcription factor 5 and potentiates its transcription activity

    International Nuclear Information System (INIS)

    Liu Wenjin; Sun Maoyun; Jiang Jianhai; Shen Xiaoyun; Sun Qing; Liu Weicheng; Shen Hailian; Gu Jianxin

    2004-01-01

    The Cyclin D3 protein is a member of the D-type cyclins. Besides serving as cell cycle regulators, D-type cyclins have been reported to be able to interact with several transcription factors and modulate their transcriptional activations. Here we report that human activating transcription factor 5 (hATF5) is a new interacting partner of Cyclin D3. The interaction was confirmed by in vivo coimmunoprecipitation and in vitro binding analysis. Neither interaction between Cyclin D1 and hATF5 nor interaction between Cyclin D2 and hATF5 was observed. Confocal microscopy analysis showed that Cyclin D3 could colocalize with hATF5 in the nuclear region. Cyclin D3 could potentiate hATF5 transcriptional activity independently of its Cdk4 partner. But Cyclin D1 and Cyclin D2 had no effect on hATF5 transcriptional activity. These data provide a new clue to understand the new role of Cyclin D3 as a transcriptional regulator

  11. Oxidative stress negatively affects human sperm mitochondrial respiration.

    Science.gov (United States)

    Ferramosca, Alessandra; Pinto Provenzano, Sara; Montagna, Daniela Domenica; Coppola, Lamberto; Zara, Vincenzo

    2013-07-01

    To correlate the level of oxidative stress in serum and seminal fluid and the level of sperm deoxyribonucleic acid (DNA) fragmentation with sperm mitochondrial respiratory efficiency. Sperm mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. A possible relationship between sperm mitochondrial respiratory efficiency, the level of oxidative stress, and the level of sperm DNA fragmentation was investigated. Sperm motility was positively correlated with mitochondrial respiration but negatively correlated with oxidative stress and DNA fragmentation. Interestingly, sperm mitochondrial respiratory activity was negatively affected by oxidative stress and DNA fragmentation. Our data indicate that sperm mitochondrial respiration is decreased in patients with high levels of reactive oxygen species by an uncoupling between electron transport and adenosine triphosphate synthesis. This reduction in mitochondrial functionality might be 1 of the reasons responsible for the decrease in spermatozoa motility. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

    Science.gov (United States)

    Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

    2010-10-15

    Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

  13. Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage

    DEFF Research Database (Denmark)

    Sjövall, Fredrik; Ehinger, Johannes K H; Marelsson, Sigurður E

    2013-01-01

    Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source...... of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further...

  14. Phylogenetic Reconstruction Shows Independent Evolutionary Origins of Mitochondrial Transcription Factors from an Ancient Family of RNA Methyltransferase Proteins.

    Science.gov (United States)

    Aj Harris; Goldman, Aaron David

    2018-04-25

    Here, we generate a robust phylogenetic framework for the rRNA adenine N(6)-methyltransferase (RAMTase) protein family that shows a more ancient and complex evolutionary history within the family than previously reported. RAMTases occur universally by descent across the three domains of life, and typical orthologs within the family perform methylation of the small subunits of ribosomal RNA (rRNA). However, within the RAMTase family, two different groups of mitochondrial transcription factors, mtTFB1 and mtTFB2, have evolved in eukaryotes through neofunctionalization. Previous phylogenetic analyses have suggested that mtTFB1 and mtTFB2 comprise sister clades that arose via gene duplication, which occurred sometime following the endosymbiosis event that produced the mitochondrion. Through dense and taxonomically broad sampling of RAMTase family members especially within bacteria, we found that these eukaryotic mitochondrial transcription factors, mtTFB1 and mtTFB2, have independent origins in phylogenetically distant clades such that their divergence most likely predates the last universal common ancestor of life. The clade of mtTFB2s comprises orthologs in Opisthokonts and the clade of mtTFB1s includes orthologs in Amoebozoa and Metazoa. Thus, we clearly demonstrate that the neofunctionalization producing the transcription factor function evolved twice independently within the RAMTase family. These results are consistent with and help to elucidate outcomes from prior experimental studies, which found that some members of mtTFB1 still perform the ancestral rRNA methylation function, and the results have broader implications for understanding the evolution of new protein functions. Our phylogenetic reconstruction is also in agreement with prior studies showing two independent origins of plastid RAMTases in Viridiplantae and other photosynthetic autotrophs. We believe that this updated phylogeny of RAMTases should provide a robust evolutionary framework for ongoing

  15. Sensitive detection of viral transcripts in human tumor transcriptomes.

    Directory of Open Access Journals (Sweden)

    Sven-Eric Schelhorn

    Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

  16. Mitochondrial Effects of PGC-1alpha Silencing in MPP+ Treated Human SH-SY5Y Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Qinyong Ye

    2017-05-01

    Full Text Available The dopaminergic neuron degeneration and loss that occurs in Parkinson’s disease (PD has been tightly linked to mitochondrial dysfunction. Although the aged-related cause of the mitochondrial defect observed in PD patients remains unclear, nuclear genes are of potential importance to mitochondrial function. Human peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α is a multi-functional transcription factor that tightly regulates mitochondrial biogenesis and oxidative capacity. The goal of the present study was to explore the potential pathogenic effects of interference by the PGC-1α gene on N-methyl-4-phenylpyridinium ion (MPP+-induced SH-SY5Y cells. We utilized RNA interference (RNAi technology to probe the pathogenic consequences of inhibiting PGC-1α in the SH-SY5Y cell line. Remarkably, a reduction in PGC-1α resulted in the reduction of mitochondrial membrane potential, intracellular ATP content and intracellular H2O2 generation, leading to the translocation of cytochrome c (cyt c to the cytoplasm in the MPP+-induced PD cell model. The expression of related proteins in the signaling pathway (e.g., estrogen-related receptor α (ERRα, nuclear respiratory factor 1 (NRF-1, NRF-2 and Peroxisome proliferator-activated receptor γ (PPARγ also decreased. Our finding indicates that small interfering RNA (siRNA interference targeting the PGC-1α gene could inhibit the function of mitochondria in several capacities and that the PGC-1α gene may modulate mitochondrial function by regulating the expression of ERRα, NRF-1, NRF-2 and PPARγ. Thus, PGC-1α can be considered a potential therapeutic target for PD.

  17. [Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

    Science.gov (United States)

    Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

    2016-01-01

    For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

  18. Bacteria, Yeast, Worms, and Flies: Exploiting Simple Model Organisms to Investigate Human Mitochondrial Diseases

    Science.gov (United States)

    Rea, Shane L.; Graham, Brett H.; Nakamaru-Ogiso, Eiko; Kar, Adwitiya; Falk, Marni J.

    2010-01-01

    The extensive conservation of mitochondrial structure, composition, and function across evolution offers a unique opportunity to expand our understanding of human mitochondrial biology and disease. By investigating the biology of much simpler model organisms, it is often possible to answer questions that are unreachable at the clinical level.…

  19. Signatures of Climatic Change In Human Mitochondrial Dna From Europe

    Science.gov (United States)

    Richards, M. B.; Macaulay, V. A.; Torroni, A.; Bandelt, H.-J.

    Founder analysis is an approach to analysing non-recombining DNA sequence data, such as variation in the mitochondrial DNA (mtDNA), which aims at identifying and dating migrations into new territory. We applied the approach to about 4,000 human mtDNA sequences from Europe and the Near East, in order to estimate the proportion of modern lineages whose ancestors arrived at various times during the continent's past. We found that the major signal dates to about 15,000 years ago, at the time of rewarming following the Last Glacial Maximum (LGM). There is little or no archaeological evidence for immigration into Europe at this time, and the record indicates that at least parts of southern Europe remained populated during the LGM. Therefore, we interpret this signal as the trace of a bottleneck at the time of the LGM, as a result of the retreat from northern Europe during the peak of the glaciation, followed by a re-expansion from one or more refugial zones. Immigration episodes then figure at the beginning of the Early Upper Palaeolithic, during the Middle Upper Palaeolithic, and with the Neolithic. The impact of the latter on the composition of the European mtDNA pool was evidently rather minor. This result implies that climate is likely to have been a major force shaping human demographic history in Europe.

  20. Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes*

    Science.gov (United States)

    Villota, Claudio; Campos, América; Vidaurre, Soledad; Oliveira-Cruz, Luciana; Boccardo, Enrique; Burzio, Verónica A.; Varas, Manuel; Villegas, Jaime; Villa, Luisa L.; Valenzuela, Pablo D. T.; Socías, Miguel; Roberts, Sally; Burzio, Luis O.

    2012-01-01

    The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis. PMID:22539350

  1. Expression of mitochondrial non-coding RNAs (ncRNAs) is modulated by high risk human papillomavirus (HPV) oncogenes.

    Science.gov (United States)

    Villota, Claudio; Campos, América; Vidaurre, Soledad; Oliveira-Cruz, Luciana; Boccardo, Enrique; Burzio, Verónica A; Varas, Manuel; Villegas, Jaime; Villa, Luisa L; Valenzuela, Pablo D T; Socías, Miguel; Roberts, Sally; Burzio, Luis O

    2012-06-15

    The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

  2. Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Michał Rurek

    2018-04-01

    Full Text Available Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS. Respiratory (e.g., complex II, IV (CII, CIV and ATP synthase subunits, transporter (including diverse porin isoforms and matrix multifunctional proteins (e.g., components of RNA editing machinery were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.

  3. Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins

    Science.gov (United States)

    Rurek, Michał; Czołpińska, Magdalena; Staszak, Aleksandra Maria; Nowak, Witold; Krzesiński, Włodzimierz; Spiżewski, Tomasz

    2018-01-01

    Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation. PMID:29642585

  4. V-MitoSNP: visualization of human mitochondrial SNPs

    Directory of Open Access Journals (Sweden)

    Tsui Ke-Hung

    2006-08-01

    Full Text Available Abstract Background Mitochondrial single nucleotide polymorphisms (mtSNPs constitute important data when trying to shed some light on human diseases and cancers. Unfortunately, providing relevant mtSNP genotyping information in mtDNA databases in a neatly organized and transparent visual manner still remains a challenge. Amongst the many methods reported for SNP genotyping, determining the restriction fragment length polymorphisms (RFLPs is still one of the most convenient and cost-saving methods. In this study, we prepared the visualization of the mtDNA genome in a way, which integrates the RFLP genotyping information with mitochondria related cancers and diseases in a user-friendly, intuitive and interactive manner. The inherent problem associated with mtDNA sequences in BLAST of the NCBI database was also solved. Description V-MitoSNP provides complete mtSNP information for four different kinds of inputs: (1 color-coded visual input by selecting genes of interest on the genome graph, (2 keyword search by locus, disease and mtSNP rs# ID, (3 visualized input of nucleotide range by clicking the selected region of the mtDNA sequence, and (4 sequences mtBLAST. The V-MitoSNP output provides 500 bp (base pairs flanking sequences for each SNP coupled with the RFLP enzyme and the corresponding natural or mismatched primer sets. The output format enables users to see the SNP genotype pattern of the RFLP by virtual electrophoresis of each mtSNP. The rate of successful design of enzymes and primers for RFLPs in all mtSNPs was 99.1%. The RFLP information was validated by actual agarose electrophoresis and showed successful results for all mtSNPs tested. The mtBLAST function in V-MitoSNP provides the gene information within the input sequence rather than providing the complete mitochondrial chromosome as in the NCBI BLAST database. All mtSNPs with rs number entries in NCBI are integrated in the corresponding SNP in V-MitoSNP. Conclusion V-MitoSNP is a web

  5. Mitochondrial dysfunction in human skeletal muscle biopsies of lipid storage disorder.

    Science.gov (United States)

    Debashree, Bandopadhyay; Kumar, Manish; Keshava Prasad, Thottethodi Subrahmanya; Natarajan, Archana; Christopher, Rita; Nalini, Atchayaram; Bindu, Parayil Sankaran; Gayathri, Narayanappa; Srinivas Bharath, Muchukunte Mukunda

    2018-02-09

    Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSDs) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSDs (n = 7), compared to controls (n = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n = 5) (vs. control, n = 5) by high-throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSDs. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSDs. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases. © 2018 International Society for Neurochemistry.

  6. Mitochondrial mass is inversely correlated to complete lipid oxidation in human myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2011-01-01

    Exercise increases while physical inactivity decrease mitochondrial content and oxidative capacity of skeletal muscles in vivo. It is unknown whether mitochondrial mass and substrate oxidation are related in non-contracting skeletal muscle. Mitochondrial mass, ATP, ADP, AMP, glucose and lipid......, basal glucose oxidation and incomplete lipid oxidation were significantly increased while complete lipid oxidation was lower. Mitochondrial mass was not correlated to glucose oxidation or incomplete lipid oxidation in human myotubes but inversely correlated to complete lipid oxidation. Thus within...... a stable energetic background, an increased mitochondrial mass in human myotubes was not positive correlated to an increased substrate oxidation as expected from skeletal muscles in vivo but surprisingly with a reduced complete lipid oxidation....

  7. Oxidative stress and mitochondrial impairment can be separated from lipofuscin accumulation in aged human skeletal muscle

    DEFF Research Database (Denmark)

    Hütter, Eveline; Skovbro, Mette; Lener, Barbara

    2007-01-01

    According to the free radical theory of aging, reactive oxygen species (ROS) act as a driving force of the aging process, and it is generally believed that mitochondrial dysfunction is a major source of increased oxidative stress in tissues with high content of mitochondria, such as muscle or brain....... However, recent experiments in mouse models of premature aging have questioned the role of mitochondrial ROS production in premature aging. To address the role of mitochondrial impairment and ROS production for aging in human muscles, we have analyzed mitochondrial properties in muscle fibres isolated...... from the vastus lateralis of young and elderly donors. Mitochondrial respiratory functions were addressed by high-resolution respirometry, and ROS production was analyzed by in situ staining with the redox-sensitive dye dihydroethidium. We found that aged human skeletal muscles contain fully functional...

  8. Glutaredoxin-2 controls cardiac mitochondrial dynamics and energetics in mice, and protects against human cardiac pathologies

    Directory of Open Access Journals (Sweden)

    Georges N. Kanaan

    2018-04-01

    Full Text Available Glutaredoxin 2 (GRX2, a mitochondrial glutathione-dependent oxidoreductase, is central to glutathione homeostasis and mitochondrial redox, which is crucial in highly metabolic tissues like the heart. Previous research showed that absence of Grx2, leads to impaired mitochondrial complex I function, hypertension and cardiac hypertrophy in mice but the impact on mitochondrial structure and function in intact cardiomyocytes and in humans has not been explored. We hypothesized that Grx2 controls cardiac mitochondrial dynamics and function in cellular and mouse models, and that low expression is associated with human cardiac dysfunction. Here we show that Grx2 absence impairs mitochondrial fusion, ultrastructure and energetics in primary cardiomyocytes and cardiac tissue. Moreover, provision of the glutathione precursor, N-acetylcysteine (NAC to Grx2-/- mice did not restore glutathione redox or prevent impairments. Using genetic and histopathological data from the human Genotype-Tissue Expression consortium we demonstrate that low GRX2 is associated with fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, GRX2 is important in the control of cardiac mitochondrial structure and function, and protects against human cardiac pathologies. Keywords: Human heart, Mitochondria, Oxidative stress, Redox, Cardiac metabolism, Cardiac hypertrophy

  9. Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: Modulation by antioxidants

    International Nuclear Information System (INIS)

    Perez-de-Arce, Karen; Foncea, Rocio; Leighton, Federico

    2005-01-01

    It has been proposed that homocysteine (Hcy)-induces endothelial dysfunction and atherosclerosis by generation of reactive oxygen species (ROS). A previous report has shown that Hcy promotes mitochondrial damage. Considering that oxidative stress can affect mitochondrial biogenesis, we hypothesized that Hcy-induced ROS in endothelial cells may lead to increased mitochondrial biogenesis. We found that Hcy-induced ROS (1.85-fold), leading to a NF-κB activation and increase the formation of 3-nitrotyrosine. Furthermore, expression of the mitochondrial biogenesis factors, nuclear respiratory factor-1 and mitochondrial transcription factor A, was significantly elevated in Hcy-treated cells. These changes were accompanied by increase in mitochondrial mass and higher mRNA and protein expression of the subunit III of cytochrome c oxidase. These effects were significantly prevented by pretreatment with the antioxidants, catechin and trolox. Taken together, our results suggest that ROS is an important mediator of mitochondrial biogenesis induced by Hcy, and that modulation of oxidative stress by antioxidants may protect against the adverse vascular effects of Hcy

  10. Ionizing radiation induces PI3K-dependent JNK activation for amplifying mitochondrial dysfunction in human cervical cancer cells

    International Nuclear Information System (INIS)

    Kim, Min Jung; Choi, Soon Young; Bae, Sang Woo; Kang, Chang Mo; Lee, Yun Sil; Lee, Su Jae

    2005-01-01

    Ionizing radiation is one of the most commonly used treatments for a wide variety of tumors. Exposure of cells to ionizing radiation results in the simultaneous activation or down regulation of multiple signaling pathways, which play critical role in controlling cell death and cell survival after irradiation in a cell type specific manner. The molecular mechanism by which apoptotic cell death occurs in response to ionizing radiation has been widely explored but not precisely deciphered. Therefore an improved understanding of the mechanisms involved in radiation-induced apoptosis may ultimately provide novel strategies of intervention in specific signal transduction pathways to favorably alter the therapeutic ratio in the treatment of human malignancies. The aim of our investigation was to elucidate molecular mechanisms of the mitochondrial dysfunction mediated apoptotic cell death triggered by ionizing radiation in human cervical cancer cells. We demonstrated that ionizing radiation utilizes PI3K-JNK signaling pathway for amplifying mitochondrial dysfunction and susequent apoptotic cell death: We showed that PI3K-dependent JNK activation leads to transcriptional upregulation of Fas and the phosphorylation/inactivation of Bcl-2, resulting in mitochondrial dysfunction-mediated apoptotic cell death in response to ionizing radiation

  11. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  12. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  13. Mitochondrial biogenesis in brown adipose tissue is associated with differential expression of transcription regulatory factors

    Czech Academy of Sciences Publication Activity Database

    Villena, J. A.; Carmona, M. C.; Rodriguez de la Concepción, M.; Rossmeisl, Martin; Vinas, O.; Mampel, T.; Iglesias, R.; Giralt, M.; Villarroya, F.

    2002-01-01

    Roč. 59, č. 11 (2002), s. 1934-1944 ISSN 1420-682X Grant - others:Ministerio de Ciencia y Tecnología (ES) PM98.0188 Institutional research plan: CEZ:AV0Z5011922 Keywords : brown adipose tissue * mitochondria * transcription factors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.259, year: 2002

  14. Optimizing the measurement of mitochondrial protein synthesis in human skeletal muscle.

    Science.gov (United States)

    Burd, Nicholas A; Tardif, Nicolas; Rooyackers, Olav; van Loon, Luc J C

    2015-01-01

    The measurement of mitochondrial protein synthesis after food ingestion, contractile activity, and/or disease is often used to provide insight into skeletal muscle adaptations that occur in the longer term. Studies have shown that protein ingestion stimulates mitochondrial protein synthesis in human skeletal muscle. Minor differences in the stimulation of mitochondrial protein synthesis occur after a single bout of resistance or endurance exercise. There appear to be no measurable differences in mitochondrial protein synthesis between critically ill patients and aged-matched controls. However, the mitochondrial protein synthetic response is reduced at a more advanced age. In this paper, we discuss the challenges involved in the measurement of human skeletal muscle mitochondrial protein synthesis rates based on stable isotope amino acid tracer methods. Practical guidelines are discussed to improve the reliability of the measurement of mitochondrial protein synthesis rates. The value of the measurement of mitochondrial protein synthesis after a single meal or exercise bout on the prediction of the longer term skeletal muscle mass and performance outcomes in both the healthy and disease populations requires more work, but we emphasize that the measurements need to be reliable to be of any value to the field.

  15. Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression

    DEFF Research Database (Denmark)

    Rocha, Nuno M; Bulger, David A; Frontini, Andrea

    2017-01-01

    body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial...... network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were...

  16. 5' diversity of human hepatic PXR (NR1I2) transcripts and identification of the major transcription initiation site.

    Science.gov (United States)

    Kurose, Kouichi; Koyano, Satoru; Ikeda, Shinobu; Tohkin, Masahiro; Hasegawa, Ryuichi; Sawada, Jun-Ichi

    2005-05-01

    The human pregnane X receptor (PXR) is a crucial regulator of the genes encoding several major cytochrome P450 enzymes and transporters, such as CYP3A4 and MDR1, but its own transcriptional regulation remains unclear. To elucidate the transcriptional mechanisms of human PXR gene, we first endeavored to identify the transcription initiation site of human PXR using 5'-RACE. Five types of 5'-variable transcripts (a, b, c, d, and e) with common exon 2 sequence were found, and comparison of these sequences with the genomic sequence suggested that their 5' diversity is derived from initiation by alternative promoters and alternative splicing. None of the exons found in our study contain any new in-frame coding regions. Newly identified introns IVS-a and IVS-b were found to have CT-AC splice sites that do not follow the GT-AG rule of conventional donor and acceptor splice sites. Of the five types of 5' variable transcripts identified, RT-PCR showed that type-a was the major transcript type. Four transcription initiation sites (A-D) for type-a transcript were identified by 5'-RACE using GeneRacer RACE Ready cDNA (human liver) constructed by the oligo-capping method. Putative TATA boxes were located approximately 30 bp upstream from the transcriptional start sites of the major transcript (C) and the longest minor transcript (A) expressed in the human liver. These results indicate that the initiation of transcription of human PXR is more complex than previously reported.

  17. Changes in mitochondrial respiration in the human placenta over gestation.

    Science.gov (United States)

    Holland, Olivia J; Hickey, Anthony J R; Alvsaker, Anna; Moran, Stephanie; Hedges, Christopher; Chamley, Lawrence W; Perkins, Anthony V

    2017-09-01

    Placental mitochondria are subjected to micro-environmental changes throughout gestation, in particular large variations in oxygen. How placental mitochondrial respiration adapts to changing oxygen concentrations remains unexplored. Additionally, placental tissue is often studied in culture; however, the effect of culture on placental mitochondria is unclear. Placental tissue was obtained from first trimester and term (laboured and non-laboured) pregnancies, and selectively permeabilized to access mitochondria. Respirometry was used to compare respiration states and substrate use in mitochondria. Additionally, explants of placental tissue were cultured for four, 12, 24, 48, or 96 h and respiration measured. Mitochondrial respiration decreased at 11 weeks compared to earlier gestations (p = 0.05-0.001), and mitochondrial content increased at 12-13 weeks compared to 7-10 weeks (p = 0.042). In term placentae, oxidative phosphorylation (OXPHOS) through mitochondrial complex IV (p Respiration was increased (p ≤ 0.006-0.001) in laboured compared to non-laboured placenta. After four hours of culture, respiration was depressed compared to fresh tissue from the same placenta and continued to decline with time in culture. Markers of apoptosis were increased, while markers of autophagy, mitochondrial biogenesis, and mitochondrial membrane potential were decreased after four hours of culture. Respiration and mitochondrial content alter over gestation/with labour. Decreased respiration at 11 weeks and increased mitochondrial content at 12-13 weeks may relate to onset of maternal blood flow, and increased respiration as a result of labour may be an adaptation to ischaemia-reperfusion. At term, mitochondria were more susceptible to changes in respiratory function relative to first trimester when cultured in vitro, perhaps reflecting changes in metabolic demands as gestation progresses. Metabolic plasticity of placental mitochondria has relevance to placenta

  18. Defect in mitochondrial functions in damaged human mitral valve

    OpenAIRE

    Shinde, Santosh; Kumar, Pawan; Mishra, Kaushala; Patil, Neela

    2006-01-01

    Mitochondrial diseases are a heterogeneous group of disorders in which a primary mitochondrial dysfunction is proven by morphological, biochemical, and genetic examinations. The mitral valve has important function in the regulation of blood flow from one chamber to another. Often, the mitral valve becomes abnormal with age, in Rheumatic fever or it is abnormal from birth (Congenital) or it can be destroyed by infection i.e. bacterial endocarditis and needs replacement. Myocardial function dep...

  19. Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas James Thestrup

    2014-01-01

    , we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC−MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples...... obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number...... of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included...

  20. Mitochondrial function in human skeletal muscle following high-altitude exposure

    DEFF Research Database (Denmark)

    Jacobs, Robert A; Boushel, Robert; Wright-Paradis, Cynthia

    2013-01-01

    Studies regarding mitochondrial modifications in human skeletal muscle following acclimatization to high altitude are conflicting, and these inconsistencies may be due to the prevalence of representing mitochondrial function through static and isolated measurements of specific mitochondrial...... characteristics. The aim of this study, therefore, was to investigate mitochondrial function in response to high-altitude acclimatization through measurements of respiratory control in the vastus lateralis muscle. Skeletal muscle biopsies were obtained from 10 lowland natives prior to and again after a total of 9......-11 days of exposure to 4559 m. High-resolution respirometry was performed on the muscle samples to compare respiratory chain function and respiratory capacities. Respirometric analysis revealed that mitochondrial function was largely unaffected, because high-altitude exposure did not affect the capacity...

  1. Transcriptional changes common to human cocaine, cannabis and phencyclidine abuse.

    Directory of Open Access Journals (Sweden)

    Elin Lehrmann

    2006-12-01

    Full Text Available A major goal of drug abuse research is to identify and understand drug-induced changes in brain function that are common to many or all drugs of abuse. As these may underlie drug dependence and addiction, the purpose of the present study was to examine if different drugs of abuse effect changes in gene expression that converge in common molecular pathways. Microarray analysis was employed to assay brain gene expression in postmortem anterior prefrontal cortex (aPFC from 42 human cocaine, cannabis and/or phencyclidine abuse cases and 30 control cases, which were characterized by toxicology and drug abuse history. Common transcriptional changes were demonstrated for a majority of drug abuse cases (N = 34, representing a number of consistently changed functional classes: Calmodulin-related transcripts (CALM1, CALM2, CAMK2B were decreased, while transcripts related to cholesterol biosynthesis and trafficking (FDFT1, APOL2, SCARB1, and Golgi/endoplasmic reticulum (ER functions (SEMA3B, GCC1 were all increased. Quantitative PCR validated decreases in calmodulin 2 (CALM2 mRNA and increases in apolipoprotein L, 2 (APOL2 and semaphorin 3B (SEMA3B mRNA for individual cases. A comparison between control cases with and without cardiovascular disease and elevated body mass index indicated that these changes were not due to general cellular and metabolic stress, but appeared specific to the use of drugs. Therefore, humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and Golgi/ER function. These changes represent common molecular features of drug abuse, which may underlie changes in synaptic function and plasticity that could have important ramifications for decision-making capabilities in drug abusers.

  2. Abnormal mitochondrial transport and morphology as early pathological changes in human models of spinal muscular atrophy

    Directory of Open Access Journals (Sweden)

    Chong-Chong Xu

    2016-01-01

    Full Text Available Spinal muscular atrophy (SMA, characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1 gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA.

  3. Human mitochondrial DNA (mtDNA) types in Malaysia

    International Nuclear Information System (INIS)

    Lian, L.H.; Koh, C.L.; Lim, M.E.

    2000-01-01

    Each human cell contains hundreds of mitochondria and thousands of double-stranded circular mtDNA. The delineation of human mtDNA variation and genetics over the past decade has provided unique and often startling insights into human evolution, degenerative diseases, and aging. Each mtDNA of 16,569 base pairs, encodes 13 polypeptides essential to the enzymes of the mitochondrial energy generating pathway, plus the necessary tRNAs and rRNAs. The highly polymorphic noncoding D-(displacement) loop region, also called the control region, is approximately 1.2 kb long. It contains two well-characterized hypervariable (HV-) regions, HV1 and HV2. MtDNA identification is usually based on these sequence differences. According to the TWTGDAM (Technical Working Group for DNA Analysis Methods), the minimum requirement for a mtDNA database for HV1 is from positions 16024 to 16365 and for HV2, from positions 00073 to 00340. The targeted Malaysian population subgroups for this study were mainly the Malays, Chinese, Indians, and indigenous Ibans, Bidayuhs, Kadazan-Dusuns, and Bajaus. Research methodologies undertaken included DNA extraction of samples from unrelated individuals, amplification of the specific regions via the polymerase chain reaction (PCR), and preparation of template DNA for sequencing by using an automated DNA sequencer. Sufficient nucleotide sequence data were generated from the mtDNA analysis. When the sequences were analyzed, sequence variations were found to be caused by nucleotide substitutions, insertions, and deletions. Of the three causes of the sequence variations, nucleotide substitutions (86.1%) accounted for the vast majority of polymorphism. It is noted that transitions (83.5%) were predominant when compared to the significantly lower frequencies of transversions (2.6%). Insertions (0.9%) and deletions (13.0%) were rather rare and found only in HV2. The data generated will also form the basis of a Malaysian DNA sequence database of mtDNA D

  4. Resveratrol induces mitochondrial biogenesis in endothelial cells.

    Science.gov (United States)

    Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

    2009-07-01

    Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

  5. Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus and humans

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    Zsurka Gábor

    2010-09-01

    Full Text Available Abstract Background We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee individuals to assess the detailed mitochondrial DNA (mtDNA phylogeny of this close relative of Homo sapiens. Results We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii A comparison of the ratios of non-synonymous to synonymous changes (dN/dS among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. Conclusions Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans.

  6. Copper and hypoxia modulate transcriptional and mitochondrial functional-biochemical responses in warm acclimated rainbow trout (Oncorhynchus mykiss)

    International Nuclear Information System (INIS)

    Sappal, Ravinder; Fast, Mark; Purcell, Sara; MacDonald, Nicole; Stevens, Don; Kibenge, Fred; Siah, Ahmed; Kamunde, Collins

    2016-01-01

    To survive in changing environments fish utilize a wide range of biological responses that require energy. We examined the effect of warm acclimation on the electron transport system (ETS) enzymes and transcriptional responses to hypoxia and copper (Cu) exposure in fish. Rainbow trout (Oncorhynchus mykiss) were acclimated to cold (11 °C; control) and warm (20 °C) temperatures for 3 weeks followed by exposure to Cu, hypoxia or both for 24 h. Activities of ETS enzyme complexes I-IV (CI–CIV) were measured in liver and gill mitochondria. Analyses of transcripts encoding for proteins involved in mitochondrial respiration (cytochrome c oxidase subunits 4-1 and 2: COX4-1 and COX4-2), metal detoxification/stress response (metallothioneins A and B: MT-A and MT-B) and energy sensing (AMP-activated protein kinase α1: AMPKα1) were done in liver mitochondria, and in whole liver and gill tissues by RT-qPCR. Warm acclimation inhibited activities of ETS enzymes while effects of Cu and hypoxia depended on the enzyme and thermal acclimation status. The genes encoding for COX4-1, COX4-2, MT-A, MT-B and AMPKα1 were strongly and tissue-dependently altered by warm acclimation. While Cu and hypoxia clearly increased MT-A and MT-B transcript levels in all tissues, their effects on COX4-1, COX4-2 and AMPKα1 mRNA levels were less pronounced. Importantly, warm acclimation differentially altered COX4-2/COX4-1 ratio in liver mitochondria and gill tissue. The three stressors showed both independent and joint actions on activities of ETS enzymes and transcription of genes involved in energy metabolism, stress response and metals homeostasis. Overall, we unveiled novel interactive effects that should not be overlooked in real world situations wherein fish normally encounter multiple stress factors. - Highlights: • Joint and individual effects of copper, hypoxia and warm acclimation differ quantitatively. • Energy metabolism genes are differentially altered by multiple stressors.

  7. Transcriptional profiling of putative human epithelial stem cells

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    Koçer Salih S

    2008-07-01

    Full Text Available Abstract Background Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells. Results Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells. Conclusion This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells

  8. DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

    Science.gov (United States)

    Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

    2014-01-01

    Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

  9. Stimulated human mast cells secrete mitochondrial components that have autocrine and paracrine inflammatory actions.

    Directory of Open Access Journals (Sweden)

    Bodi Zhang

    Full Text Available Mast cells are hematopoietically-derived tissue immune cells that participate in acquired and innate immunity, as well as in inflammation through release of many chemokines and cytokines, especially in response to the pro-inflammatory peptide substance P (SP. Inflammation is critical in the pathogenesis of many diseases, but the trigger(s is often unknown. We investigated if mast cell stimulation leads to secretion of mitochondrial components and whether these could elicit autocrine and/or paracrine inflammatory effects. Here we show that human LAD2 mast cells stimulated by IgE/anti-IgE or by the SP led to secretion of mitochondrial particles, mitochondrial (mt mtDNA and ATP without cell death. Mitochondria purified from LAD2 cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD(2, IL-8, TNF, and IL-1β. This stimulatory effect is partially inhibited by an ATP receptor antagonist and by DNAse. These results suggest that the mitochondrial protein fraction may also contribute. Purified mitochondria also stimulate IL-8 and vascular endothelial growth factor (VEGF release from cultured human keratinocytes, and VEGF release from primary human microvascular endothelial cells. In order to investigate if mitochondrial components could be secreted in vivo, we injected rats intraperiotoneally (ip with compound 48/80, which mimicks the action of SP. Peritoneal mast cells degranulated and mitochondrial particles were documented by transimission electron microscopy outside the cells. We also wished to investigate if mitochondrial components secreted locally could reach the systemic circulation. Administration ip of mtDNA isolated from LAD2 cells in rats was detected in their serum within 4 hr, indicating that extravascular mtDNA could enter the systemic circulation. Secretion of mitochondrial components from stimulated live mast cells may act as "autopathogens" contributing to the pathogenesis of inflammatory

  10. Significant associations of the mitochondrial transcription factor A promoter polymorphisms with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses

    International Nuclear Information System (INIS)

    Jiang, Zhihua; Kunej, Tanja; Michal, Jennifer J.; Gaskins, Charles T.; Reeves, Jerry J.; Busboom, Jan R.; Dovc, Peter; Wright, Raymond W.

    2005-01-01

    Mitochondrial transcription factor A (TFAM), a nucleus-encoded protein, regulates the initiation of transcription and replication of mitochondrial DNA (mtDNA). Decreased expression of nuclear-encoded mitochondrial genes has been associated with onset of obesity in mice. Therefore, we hypothesized genetic variants in TFAM gene influence mitochondrial biogenesis consequently affecting body fat deposition and energy metabolism. In the present study, both cDNA (2259 bp) and genomic DNA (16,666 bp) sequences were generated for the bovine TFAM gene using a combination of in silico cloning with targeted region PCR amplification. Alignment of both cDNA and genomic sequences led to the determination of genomic organization and characterization of the promoter region of the bovine TFAM gene. Two closely linked A/C and C/T single nucleotide polymorphisms (SNPs) were found in the bovine TFAM promoter and then genotyped on 237 Wagyu x Limousin F 2 animals with recorded phenotypes for marbling and subcutaneous fat depth (SFD). Statistical analysis demonstrated that both SNPs and their haplotypes were associated with marbling (P = 0.0153 for A/C, P = 0.0026 for C/T, and P = 0.0004 for haplotype) and SFD (P = 0.0200 for A/C, P = 0.0039 for C/T, and P = 0.0029 for haplotype), respectively. A search for transcriptional regulatory elements using MatInspector indicated that both SNPs lead to a gain/loss of six putative-binding sites for transcription factors relevant to fat deposition and energy metabolism. Our results suggest for the first time that TFAM gene plays an important role in lipid metabolism and may be a strong candidate gene for obesity in mammals

  11. Muscle biopsies from human muscle diseases with myopathic pathology reveal common alterations in mitochondrial function.

    Science.gov (United States)

    Sunitha, Balaraju; Gayathri, Narayanappa; Kumar, Manish; Keshava Prasad, Thottethodi Subrahmanya; Nalini, Atchayaram; Padmanabhan, Balasundaram; Srinivas Bharath, Muchukunte Mukunda

    2016-07-01

    Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX-deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX-SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated down-regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under-expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases. We investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies

  12. Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Naoya; Yamada, Shigeru [Division of Pharmacology, National Institute of Health Sciences (Japan); Asanagi, Miki [Division of Pharmacology, National Institute of Health Sciences (Japan); Faculty of Engineering, Department of Materials Science and Engineering, Yokohama National University (Japan); Sekino, Yuko [Division of Pharmacology, National Institute of Health Sciences (Japan); Kanda, Yasunari, E-mail: kanda@nihs.go.jp [Division of Pharmacology, National Institute of Health Sciences (Japan)

    2016-02-05

    Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 μM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 μM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.

  13. Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells

    International Nuclear Information System (INIS)

    Hirata, Naoya; Yamada, Shigeru; Asanagi, Miki; Sekino, Yuko; Kanda, Yasunari

    2016-01-01

    Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 μM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 μM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.

  14. Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

    Science.gov (United States)

    Ito, Hiroyuki; Uchida, Tokujiro; Makita, Koshi

    2015-01-01

    Purpose Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated. Methods Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD+ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy. Results Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P ketamine (100 μM) decreased the ATP level (22%, P ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation. Conclusions We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons. PMID:26020236

  15. Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis.

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    Dequina Nicholas

    Full Text Available Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96 analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

  16. Insights into the mutation-induced HHH syndrome from modeling human mitochondrial ornithine transporter-1.

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    Jing-Fang Wang

    Full Text Available Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109-113. Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and

  17. Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes.

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    Siamak Djafarzadeh

    Full Text Available During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α. Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB. The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis. Our data suggest that

  18. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

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    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  19. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  20. Mitochondrial DNA inheritance in the human fungal pathogen Cryptococcus gattii.

    Science.gov (United States)

    Wang, Zixuan; Wilson, Amanda; Xu, Jianping

    2015-02-01

    The inheritance of mitochondrial DNA (mtDNA) is predominantly uniparental in most sexual eukaryotes. In this study, we examined the mitochondrial inheritance pattern of Cryptococcus gattii, a basidiomycetous yeast responsible for the recent and ongoing outbreak of cryptococcal infections in the US Pacific Northwest and British Columbia (especially Vancouver Island) in Canada. Using molecular markers, we analyzed the inheritance of mtDNA in 14 crosses between strains within and between divergent lineages in C. gattii. Consistent with results from recent studies, our analyses identified significant variations in mtDNA inheritance patterns among strains and crosses, ranging from strictly uniparental to biparental. For two of the crosses that showed uniparental mitochondrial inheritance in standard laboratory conditions, we further investigated the effects of the following environmental variables on mtDNA inheritance: UV exposure, temperature, and treatments with the methylation inhibitor 5-aza-2'-deoxycytidine and with the ubiquitination inhibitor ammonium chloride. Interestingly, one of these crosses showed no response to these environmental variables while the other exhibited diverse patterns ranging from complete uniparental inheritance of the MATa parent mtDNA, to biparental inheritance, and to a significant bias toward inheritance of the MATα parental mtDNA. Our results indicate that mtDNA inheritance in C. gattii differs from that in its closely related species Cryptococcus neoformans. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. DNA methylation and transcriptional trajectories during human development and reprogramming of isogenic pluripotent stem cells

    NARCIS (Netherlands)

    Roost, Matthias S; Slieker, Roderick C; Bialecka, Monika; van Iperen, Liesbeth; Gomes Fernandes, Maria M; He, Nannan; Suchiman, H Eka D; Szuhai, Karoly; Carlotti, Françoise; de Koning, Eelco J P; Mummery, Christine L; Heijmans, Bastiaan T; Chuva de Sousa Lopes, Susana M

    2017-01-01

    Determining cell identity and maturation status of differentiated pluripotent stem cells (PSCs) requires knowledge of the transcriptional and epigenetic trajectory of organs during development. Here, we generate a transcriptional and DNA methylation atlas covering 21 organs during human fetal

  2. Complex forms of mitochondrial DNA in human B cells transformed by Epstein-Barr virus (EBV)

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Christiansen, C; Zeuthen, J

    1983-01-01

    Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed lymphoblast......Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed...

  3. Endangered species: mitochondrial DNA loss as a mechanism of human disease.

    Science.gov (United States)

    Herrera, Alan; Garcia, Iraselia; Gaytan, Norma; Jones, Edith; Maldonado, Alicia; Gilkerson, Robert

    2015-06-01

    Human mitochondrial DNA (mtDNA) is a small maternally inherited DNA, typically present in hundreds of copies in a single human cell. Thus, despite its small size, the mitochondrial genome plays a crucial role in the metabolic homeostasis of the cell. Our understanding of mtDNA genotype-phenotype relationships is derived largely from studies of the classical mitochondrial neuromuscular diseases, in which mutations of mtDNA lead to compromised mitochondrial bioenergetic function, with devastating pathological consequences. Emerging research suggests that loss, rather than mutation, of mtDNA plays a major role across a range of prevalent human diseases, including diabetes mellitus, cardiovascular disease, and aging. Here, we examine the 'rules' of mitochondrial genetics and function, the clinical settings in which loss of mtDNA is an emerging pathogenic mechanism, and explore mtDNA damage and its consequences for the organellar network and cell at large. As extranuclear genetic material arrayed throughout the cell to support metabolism, mtDNA is increasingly implicated in a host of disease conditions, opening a range of exciting questions regarding mtDNA and its role in cellular homeostasis.

  4. Regulatory motifs for CREB-binding protein and Nfe2l2 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene.

    Science.gov (United States)

    Rim, Jong S; Kozak, Leslie P

    2002-09-13

    Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.

  5. Tributyltin induces mitochondrial fission through NAD-IDH dependent mitofusin degradation in human embryonic carcinoma cells.

    Science.gov (United States)

    Yamada, Shigeru; Kotake, Yaichiro; Nakano, Mizuho; Sekino, Yuko; Kanda, Yasunari

    2015-08-01

    Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT acts at the nanomolar level through genomic pathways via the peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR). We recently reported that TBT inhibits cell growth and the ATP content in the human embryonic carcinoma cell line NT2/D1 via a non-genomic pathway involving NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which metabolizes isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial NAD-IDH and energy production. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusins 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess cytotoxicity associated with metal exposure.

  6. Changes in the human mitochondrial genome after treatment of malignant disease

    International Nuclear Information System (INIS)

    Wardell, Theresa M.; Ferguson, Elaine; Chinnery, Patrick F.; Borthwick, Gillian M.; Taylor, Robert W.; Jackson, Graham; Craft, Alan; Lightowlers, Robert N.; Howell, Neil; Turnbull, Douglass M.

    2003-01-01

    Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage

  7. Connective tissue growth factor decreases mitochondrial metabolism through ubiquitin-mediated degradation of mitochondrial transcription factor A in oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Wei-Ting Lai

    2018-03-01

    Conclusion: CTGF can decrease glycolysis, mitochondrial oxidative phosphorylation, ATP generation, and mtDNA copy number by increasing mtTFA protein degradation through ubiquitin proteasome pathway and in turn reduces migration and invasion of OSCC cells. Therefore, CTGF may be developed as a potential additive therapeutic drug for oral cancer in the near future.

  8. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf

    2010-10-21

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  9. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf; Schmeier, Sebastian; Bajic, Vladimir B.

    2010-01-01

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  10. Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus

    OpenAIRE

    El-Bacha , Tatiana; Midlej , Victor; Silva , Ana Paula Pereira Da; Costa , Leandro Silva Da; Benchimol , Marlene; Galina , Antonio; Poian , Andrea T. Da

    2007-01-01

    Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus correspondence: Corresponding author. Fax: +55 21 22708647. (El-Bacha, Tatiana) (El-Bacha, Tatiana) Laboratorio de Bioquimica de Virus, Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro - RJ-Brasil--> , Av. Bauhinia n? 400 ? CCS Bloco H 2? andar--> , sala 22. Ilha do Governador--> ...

  11. A revised timescale for human evolution based on ancient mitochondrial genomes

    Czech Academy of Sciences Publication Activity Database

    Fu, Q.; Mittnik, A.; Johnson, P. L. F.; Bos, K.; Lari, M.; Bollongino, R.; Sun, Ch.; Giemsch, L.; Schmitz, R.; Burger, J.; Ronchitelli, A. M.; Martini, F.; Cremonesi, R. G.; Svoboda, Jiří; Bauer, P.; Caramelli, D.; Castellano, S.; Reich, D.; Pääbo, S.; Krause, J.

    2013-01-01

    Roč. 23, April 8 (2013), s. 553-559 ISSN 0960-9822 Institutional support: RVO:68081758 Keywords : mitochondrial genome * human evolution * calibration Subject RIV: AC - Archeology, Anthropology, Ethnology OBOR OECD: Archaeology Impact factor: 9.916, year: 2013

  12. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Kazak, L; Wood, S R; Mao, C C; Fearnley, I M; Walker, J E; Holt, I J

    2012-07-01

    The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

  13. Distribution patterns of postmortem damage in human mitochondrial DNA

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Willerslev, Eske; Hansen, Anders J

    2002-01-01

    1 (HVR1) and cytochrome oxidase subunit III genes. A comparison of damaged sites within and between the regions reveals that damage hotspots exist and that, in the HVR1, these correlate with sites known to have high in vivo mutation rates. Conversely, HVR1 subregions with known structural function......, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed "low-diversity 1," through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions...... was significantly lower than in the HVR1, and, although hotspots were noted, these did not correlate with codon position. Finally, a simple method for the identification of incorrect archaeological haplogroup designations is introduced, on the basis of the observed spectrum of postmortem damage....

  14. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity

    Directory of Open Access Journals (Sweden)

    Peter W. Lindinger

    2015-09-01

    Full Text Available Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

  15. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

    Science.gov (United States)

    Lindinger, Peter W; Christe, Martine; Eberle, Alex N; Kern, Beatrice; Peterli, Ralph; Peters, Thomas; Jayawardene, Kamburapola J I; Fearnley, Ian M; Walker, John E

    2015-09-01

    Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

  16. Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Chen-Sung Lin

    2016-05-01

    Full Text Available We investigated the role of mitochondrial DNA (mtDNA copy number alteration in human renal cell carcinoma (RCC. The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR. An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM. Null target (NT and TFAM-knockdown (TFAM-KD represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1, ND6 and cytochrome c oxidase subunit 2 (COX-2; nuclear DNA (nDNA-encoded succinate dehydrogenase subunit A (SDHA; v-akt murine thymoma viral oncogene homolog 1 gene (AKT-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC-encoded MYC; glycolytic enzymes including hexokinase II (HK-II, glucose 6-phosphate isomerase (GPI, phosphofructokinase (PFK, and lactate dehydrogenase subunit A (LDHA; and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1, and pyruvate dehydrogenase E1 component α subunit (PDHA1 were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB and basal extracellular acidification rate (ECARB, were measured by a Seahorse XFe-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043. The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034, lower mRNA levels of TFAM (p = 0.008, ND1 (p = 0.007, and ND6 (p = 0.017, and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  17. Musa paradisiaca inflorescence induces human colon cancer cell death by modulating cascades of transcriptional events.

    Science.gov (United States)

    K B, Arun; Madhavan, Aravind; T R, Reshmitha; Thomas, Sithara; Nisha, P

    2018-01-24

    Colorectal cancer (CRC) is one of the leading causes of cancer death, and diet plays an important role in the etiology of CRC. Traditional medical practitioners in many South Asian countries use plantain inflorescence to treat various gastro-intestinal ailments. The aim of the present study was to investigate the anticancer effects of extracts of inflorescence of Musa paradisiaca against HT29 human colon cancer cells and elucidate the mechanism of these effects by studying the modulation of cascades of transcriptional events. In vitro assays depicted that methanol extract of Musa paradisiaca inflorescence (PIMET) was cytotoxic to HT29 cells. PIMET induced DNA damage and arrested the cell cycle at the G2/M phase. Expression studies showed that PIMET pretreatment upregulates pro-apoptotic Bcl2 and downregulates anti-apoptotic Bax proteins. Different assays showed that the deregulation of pro/antiapoptotic proteins reduces the mitochondrial membrane potential and ATP production; moreover, it enhances cytochrome c release, which triggers the apoptotic pathway, and further cleaves caspase 3 and PARP proteins, resulting in apoptosis. Changes in the protein expression profile of HT29 cells after PIMET treatment were analyzed using mass-spectrometry-based proteomics. PIMET treatment significantly altered the expression of HT29 protein; interestingly, X-linked inhibitor of apoptosis protein was also downregulated. Alteration in the expression of this protein has significant effects, leading to HT29 cell death.

  18. Cancer-specific SNPs originate from low-level heteroplasmic variants in human mitochondrial genomes of a matched cell line pair.

    Science.gov (United States)

    Hedberg, Annica; Knutsen, Erik; Løvhaugen, Anne Silje; Jørgensen, Tor Erik; Perander, Maria; Johansen, Steinar D

    2018-04-19

    Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

  19. Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2007-01-01

    In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects and exposed to mitochondrial inhibitors, either affecting the electron transport chain...... lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while......, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se...

  20. Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

    DEFF Research Database (Denmark)

    Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

    2002-01-01

    The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation...... procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit approximately 0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated...... as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore...

  1. Repeated static contractions increase mitochondrial vulnerability toward oxidative stress in human skeletal muscle

    DEFF Research Database (Denmark)

    Sahlin, Kent; Nielsen, Jens Steen; Mogensen, Martin

    2006-01-01

    Repeated static contractions (RSC) induce large fluctuations in tissue oxygen tension and increase the generation of reactive oxygen species (ROS). This study investigated the effect of RSC on muscle contractility, mitochondrial respiratory function, and in vitro sarcoplasmic reticulum (SR) Ca(2......+) kinetics in human muscle. Ten male subjects performed five bouts of static knee extension with 10-min rest in between. Each bout of RSC (target torque 66% of maximal voluntary contraction torque) was maintained to fatigue. Muscle biopsies were taken preexercise and 0.3 and 24 h postexercise from vastus...... lateralis. Mitochondria were isolated and respiratory function measured after incubation with H(2)O(2) (HPX) or control medium (Con). Mitochondrial function was not affected by RSC during Con. However, RSC exacerbated mitochondrial dysfunction during HPX, resulting in decreased respiratory control index...

  2. Tributyltin induces mitochondrial fission through Mfn1 degradation in human induced pluripotent stem cells.

    Science.gov (United States)

    Yamada, Shigeru; Asanagi, Miki; Hirata, Naoya; Itagaki, Hiroshi; Sekino, Yuko; Kanda, Yasunari

    2016-08-01

    Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity, including neurotoxicity and immunotoxicity. However, TBT toxicity has not been identified in normal stem cells. In the present study, we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production, which is a critical function of the mitochondria, we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1), and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5), suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus, mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

    2013-10-25

    Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia.

    Science.gov (United States)

    Lunetti, Paola; Damiano, Fabrizio; De Benedetto, Giuseppe; Siculella, Luisa; Pennetta, Antonio; Muto, Luigina; Paradies, Eleonora; Marobbio, Carlo Marya Thomas; Dolce, Vincenza; Capobianco, Loredana

    2016-09-16

    Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7).

    Science.gov (United States)

    Charizanis, C; Juhnke, H; Krems, B; Entian, K D

    1999-10-01

    In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.

  6. Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

    Directory of Open Access Journals (Sweden)

    Sameer Dixit

    2017-01-01

    Full Text Available A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei. Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1. Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome.

  7. DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA

    International Nuclear Information System (INIS)

    Pascucci, B.; Dogliotti, E.; Versteegh, A.; Hoffen, A. van; Zeeland, A.A. van; Mullenders, L.H.F.

    1997-01-01

    The induction and repair of DNA photolesions and mutations in the mitochondrial (mt) DNA of human cells in culture were analysed after cell exposure to UV-C light. The level of induction of cyclobutane pyrimidine dimers (CPD) in mitochondrial and nuclear DNA was comparable, while a higher frequency of pyrimidine (6-4) pyrimidone photoproducts (6-4 PP) was detected in mitochondrial than in nuclear DNA. Besides the known defect in CPD removal, mitochondria were shown to be deficient also in the excision of 6-4 PP. The effects of repair-defective conditions for the two major UV photolesions on mutagensis was assessed by analysing the frequency and spectrum of spontaneous and UV-induced mutations by restriction site mutation (RSM) method in a restriction endonuclease site, NciI (5'CCCGG3') located within the coding sequence of the mitochondrial gene for tRNA Leu . The spontaneous mutation frequency and spectrum at the NciI site of mitochondrial DNA was very similar to the RSM background mutation frequency (approximately 10 -5 ) and type (predominantly GC > AT transitions at GL 1 ) of the NciI site). Conversely, an approximately tenfold increase over background mutation frequency was recorded after cell exposure to 20 J/m 2 . In this case, the majority of mutations were C > T transitions preferentially located on the non-transcribed DNA strand at C 1 and C 2 of the NciI site. This mutation spectrum is expected by UV mutagenesis. This is the first evidence of induction of mutations in mitochondrial DNA by treatment of human cells with a carcinogen. (author)

  8. Mapping of Mitochondrial RNA-Protein Interactions by Digital RNase Footprinting

    Directory of Open Access Journals (Sweden)

    Ganqiang Liu

    2013-11-01

    Full Text Available Human mitochondrial DNA is transcribed as long polycistronic transcripts that encompass each strand of the genome and are processed subsequently into mature mRNAs, tRNAs, and rRNAs, necessitating widespread posttranscriptional regulation. Here, we establish methods for massively parallel sequencing and analyses of RNase-accessible regions of human mitochondrial RNA and thereby identify specific regions within mitochondrial transcripts that are bound by proteins. This approach provides a range of insights into the contribution of RNA-binding proteins to the regulation of mitochondrial gene expression.

  9. The Alu neurodegeneration hypothesis: A primate-specific mechanism for neuronal transcription noise, mitochondrial dysfunction, and manifestation of neurodegenerative disease.

    Science.gov (United States)

    Larsen, Peter A; Lutz, Michael W; Hunnicutt, Kelsie E; Mihovilovic, Mirta; Saunders, Ann M; Yoder, Anne D; Roses, Allen D

    2017-07-01

    It is hypothesized that retrotransposons have played a fundamental role in primate evolution and that enhanced neurologic retrotransposon activity in humans may underlie the origin of higher cognitive function. As a potential consequence of this enhanced activity, it is likely that neurons are susceptible to deleterious retrotransposon pathways that can disrupt mitochondrial function. An example is observed in the TOMM40 gene, encoding a β-barrel protein critical for mitochondrial preprotein transport. Primate-specific Alu retrotransposons have repeatedly inserted into TOMM40 introns, and at least one variant associated with late-onset Alzheimer's disease originated from an Alu insertion event. We provide evidence of enriched Alu content in mitochondrial genes and postulate that Alus can disrupt mitochondrial populations in neurons, thereby setting the stage for progressive neurologic dysfunction. This Alu neurodegeneration hypothesis is compatible with decades of research and offers a plausible mechanism for the disruption of neuronal mitochondrial homeostasis, ultimately cascading into neurodegenerative disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

    Science.gov (United States)

    In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

    1997-03-01

    Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

  11. Intergenic and repeat transcription in human, chimpanzee and macaque brains measured by RNA-Seq.

    Directory of Open Access Journals (Sweden)

    Augix Guohua Xu

    Full Text Available Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20-28% of non-ribosomal transcripts correspond to annotated exons and 20-23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40-48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20% represents 3'UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits.

  12. Disturbed mitochondrial function restricts glutamate uptake in the human Müller glia cell line, MIO-M1

    DEFF Research Database (Denmark)

    Vohra, Rupali; Gurubaran, Iswariyaraja Sridevi; Henriksen, Ulrik

    2017-01-01

    Using the human Müller cell line, MIO-M1, the aim was to study the impact of mitochondrial inhibition in Müller glia through antimycin A treatment. MIO-M1 cell survival, levels of released lactate, mitochondrial function, and glutamate uptake were studied in response to mitochondrial inhibition...... and glucose restriction. Lactate release decreased in response to glucose restriction. Combined glucose restriction and blocked mitochondrial activity decreased survival and caused collapse of the respiratory chain measured by oxygen consumption rate and extracellular acidification rate. Mitochondrial...... inhibition caused impaired glutamate uptake and decreased mRNA expression of the glutamate transporter, EAAT1. Over all, we show important roles of mitochondrial activity in MIO-M1 cell function and survival....

  13. Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

    International Nuclear Information System (INIS)

    Qi Xinming; Cai Yan; Gong Likun; Liu Linlin; Chen Fangping; Xiao Ying; Wu Xiongfei; Li Yan; Xue Xiang; Ren Jin

    2007-01-01

    Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca 2+ , AAI caused mitochondrial swelling, leakage of Ca 2+ , membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid

  14. Distribution of the DAZ gene transcripts in human testis.

    Directory of Open Access Journals (Sweden)

    J B Warchoł

    2004-07-01

    Full Text Available Involvement of variety of genes, especially located on Y chromosome, is critical for the regulation of spermatogenesis. In particular, fertility candidate genes such as deleted in azoospermia (DAZ are believed to have important function in sperm production, since DAZ is frequently deleted in azoospermic and severy oligozoospermic men. The role of the DAZ gene is supported by its exclusive expression in the testis and by its deletion in about 10% of azoospermic and severely oligozoospermic patients. The distribution of DAZ transcripts in seminiferous epithelium of human testis is reported in the present study. The use of Adobe Photoshop and Scion Image softwares allowed for semi-quantitative analysis of in situ RT-PCR (ISRT-PCR results. The intensity of ISRT-PCR product's fluorescence was different within individual seminiferous tubules. It was clearly shown by using the pseudocolour scale and transforming the intensity of the fluorescence into levels of greyscale images. The more intense fluorescence characterised single spermatogonia and those organized in small groups inside separate tubules. The most intense accumulation of DAZ mRNA was observed in spermatogonia.

  15. PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    Alison Wood-Kaczmar

    2008-06-01

    Full Text Available Parkinson's disease (PD is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.

  16. Mitochondrial nad2 gene is co-transcripted with CMS-associated orfB gene in cytoplasmic male-sterile stem mustard (Brassica juncea).

    Science.gov (United States)

    Yang, Jing-Hua; Zhang, Ming-Fang; Yu, Jing-Quan

    2009-02-01

    The transcriptional patterns of mitochondrial respiratory related genes were investigated in cytoplasmic male-sterile and fertile maintainer lines of stem mustard, Brassica juncea. There were numerous differences in nad2 (subunit 2 of NADH dehydrogenase) between stem mustard CMS and its maintainer line. One novel open reading frame, hereafter named orfB gene, was located at the downstream of mitochondrial nad2 gene in the CMS. The novel orfB gene had high similarity with YMF19 family protein, orfB in Raphanus sativus, Helianthus annuus, Nicotiana tabacum and Beta vulgaris, orfB-CMS in Daucus carota, atp8 gene in Arabidopsis thaliana, 5' flanking of orf224 in B. napus (nap CMS) and 5' flanking of orf220 gene in CMS Brassica juncea. Three copies probed by specific fragment (amplified by primers of nad2F and nad2R from CMS) were found in the CMS line following Southern blotting digested with HindIII, but only a single copy in its maintainer line. Meanwhile, two transcripts were shown in the CMS line following Northern blotting while only one transcript was detected in the maintainer line, which were probed by specific fragment (amplified by primers of nad2F and nad2R from CMS). Meanwhile, the expression of nad2 gene was reduced in CMS bud compared to that in its maintainer line. We thus suggested that nad2 gene may be co-transcripted with CMS-associated orfB gene in the CMS. In addition, the specific fragment that was amplified by primers of nad2F and nad2R just spanned partial sequences of nad2 gene and orfB gene. Such alterations in the nad2 gene would impact the activity of NADH dehydrogenase, and subsequently signaling, inducing the expression of nuclear genes involved in male sterility in this type of cytoplasmic male sterility.

  17. Genetically Determined Insulin Resistance is Characterized by Down-Regulation of Mitochondrial Oxidative Metabolism in Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Kristensen, Jonas M; Skov, Vibe; Wojtaszewski, Jørgen

    2010-01-01

    Transcriptional profiling of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated a co-ordinated down-regulation of oxidative phosphorylation (OxPhos) genes, suggesting a link between insulin resistance and mitochondrial dysfunction. However, whether...... mitochondrial dysfunction is a cause or consequence of insulin resistance remains to be clarified. In the present study, we tested the hypothesis that mitochondrial oxidative metabolism was down-regulated in skeletal muscle of patients with genetically determined insulin resistance. Skeletal muscle biopsies.......02), and complex V (ATP5B; p=0.005). Our data demonstrate that genetically determined insulin resistance is associated with a co-ordinated down-regulation of OxPhos components both at the transcriptional and translational level. These findings suggest that an impaired biological response to insulin in skeletal...

  18. Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Graham P. Holloway

    2018-03-01

    Full Text Available Summary: It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an in vitro method to simultaneously determine mitochondrial respiration and H2O2 emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H2O2 emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H2O2 and the fraction of electron leak to H2O2, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H2O2 emission rates in the presence of ADP, the fraction of electron leak to H2O2, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress. : Holloway et al. show that an inability of ADP to decrease mitochondrial reactive oxygen species emission contributes to redox stress in skeletal muscle tissue of older individuals and that this process is not recovered following prolonged resistance-type exercise training, despite the general benefits of resistance training for muscle health. Keywords: mitochondria, aging, muscle, ROS, H2O2, ADP, respiration, bioenergetics, exercise, resistance training

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

    Directory of Open Access Journals (Sweden)

    Andrew B J Prowse

    Full Text Available 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.

  20. Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus.

    Science.gov (United States)

    Ozga, Andrew T; Nieves-Colón, Maria A; Honap, Tanvi P; Sankaranarayanan, Krithivasan; Hofman, Courtney A; Milner, George R; Lewis, Cecil M; Stone, Anne C; Warinner, Christina

    2016-06-01

    Archaeological dental calculus is a rich source of host-associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. Extracted DNA from six individuals at the 700-year-old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in-solution capture techniques, followed by Illumina high-throughput sequencing. Full mitogenomes (7-34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92-100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220-228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. The Signal Transducer and Activator of Transcription 1 (STAT1 Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes.

    Directory of Open Access Journals (Sweden)

    Jennifer D Sisler

    Full Text Available The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT. Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.

  2. A transcript finishing initiative for closing gaps in the human transcriptome

    DEFF Research Database (Denmark)

    Sogayar, Mari Cleide; Camargo, Anamaria A; Bettoni, Fabiana

    2004-01-01

    We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected...

  3. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been

  4. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Kumer, Sean C.; Schmitt, Timothy M. [Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Ding, Wen-Xing [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2015-12-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.

  5. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    International Nuclear Information System (INIS)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-01-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.

  6. A functional test of Neandertal and modern human mitochondrial targeting sequences

    Energy Technology Data Exchange (ETDEWEB)

    Gralle, Matthias, E-mail: gralle@bioqmed.ufrj.br [Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, 21941-590 Rio de Janeiro (Brazil); Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig (Germany); Schaefer, Ingo; Seibel, Peter [Department of Molecular Cell Therapy, Leipzig University, Deutscher Platz 5, 04103 Leipzig (Germany); Paeaebo, Svante [Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig (Germany)

    2010-11-26

    Research highlights: {yields} Two mutations in mitochondrial targeting peptides occurred during human evolution, possibly after Neandertals split off from modern human lineage. {yields} The ancestral and modern human versions of these two targeting peptides were tested functionally for their effects on localization and cleavage rate. {yields} In spite of recent evolution, and to the contrary of other mutations in targeting peptides, these mutations had no visible effects. -- Abstract: Targeting of nuclear-encoded proteins to different organelles, such as mitochondria, is a process that can result in the redeployment of proteins to new intracellular destinations during evolution. With the sequencing of the Neandertal genome, it has become possible to identify amino acid substitutions that occurred on the modern human lineage since its separation from the Neandertal lineage. Here we analyze the function of two substitutions in mitochondrial targeting sequences that occurred and rose to high frequency recently during recent human evolution. The ancestral and modern versions of the two targeting sequences do not differ in the efficiency with which they direct a protein to the mitochondria, an observation compatible with the neutral theory of molecular evolution.

  7. A functional test of Neandertal and modern human mitochondrial targeting sequences

    International Nuclear Information System (INIS)

    Gralle, Matthias; Schaefer, Ingo; Seibel, Peter; Paeaebo, Svante

    2010-01-01

    Research highlights: → Two mutations in mitochondrial targeting peptides occurred during human evolution, possibly after Neandertals split off from modern human lineage. → The ancestral and modern human versions of these two targeting peptides were tested functionally for their effects on localization and cleavage rate. → In spite of recent evolution, and to the contrary of other mutations in targeting peptides, these mutations had no visible effects. -- Abstract: Targeting of nuclear-encoded proteins to different organelles, such as mitochondria, is a process that can result in the redeployment of proteins to new intracellular destinations during evolution. With the sequencing of the Neandertal genome, it has become possible to identify amino acid substitutions that occurred on the modern human lineage since its separation from the Neandertal lineage. Here we analyze the function of two substitutions in mitochondrial targeting sequences that occurred and rose to high frequency recently during recent human evolution. The ancestral and modern versions of the two targeting sequences do not differ in the efficiency with which they direct a protein to the mitochondria, an observation compatible with the neutral theory of molecular evolution.

  8. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

  9. Uncovering layers of human RNA polymerase II transcription

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In recent years DNA microarray and high-throughput sequencing technologies have challenged the “gene-centric” view that pre-mRNA is the only RNA species transcribed off protein-coding genes. Instead unorthodox transcription from within genic- and intergenic regions has been demonstrated to occur...

  10. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    Science.gov (United States)

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

  11. Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light.

    Science.gov (United States)

    Rascalou, Adeline; Lamartine, Jérôme; Poydenot, Pauline; Demarne, Frédéric; Bechetoille, Nicolas

    2018-05-05

    Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm 2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede

  12. Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

    Directory of Open Access Journals (Sweden)

    Seto Anita G

    2000-11-01

    Full Text Available Abstract Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

  13. A Role for the NF-kb/Rel Transcription Factors in Human Breast Cancer

    National Research Council Canada - National Science Library

    Baldwin, Albert

    1998-01-01

    Human breast cancer is characterized by the inappropriate expression of growth factors, kinases and possibly certain transcription factors Our project has focused on the regulation of the NF-kB family...

  14. E6-associated transcription patterns in human papilloma virus 16-positive cervical tissues.

    Science.gov (United States)

    Lin, Kezhi; Lu, Xulian; Chen, Jun; Zou, Ruanmin; Zhang, Lifang; Xue, Xiangyang

    2015-01-01

    The change in transcription pattern induced by post-transcriptional RNA splicing is an important mechanism in the regulation of the early gene expression of human papilloma virus (HPV). The present study was conducted to establish a method to specifically amplify HPV-16 E6-associated transcripts. The E6-related transcripts from 63 HPV-16-positive cervical tumor tissue samples were amplified, consisting of eight cases of low-risk intraepithelial lesions, 38 cases of high-risk intraepithelial lesions and 17 cases of cervical cancer (CxCa). The appropriate amplified segments were recovered following agarose gel electrophoresis, and subjected to further sequencing and sequence alignment analysis. Six groups of E6 transcription patterns were identified from HPV-16-positive cervical tumor tissue, including five newly-discovered transcripts. Different HPV-16 E6-associated transcription patterns were detected during the development of CxCa. Over the course of the progression of the low-grade squamous intraepithelial lesions to CxCa, the specific HPV-16 E6-associated transcription patterns and the dominant transcripts were all different. As indicated by this study, the transcription pattern of the E6 early gene of HPV-16 was closely associated with the stages of cervical carcinogenesis, and may also be involved in the development of CxCa.

  15. Cocaine- and amphetamine-regulated transcript peptide increases mitochondrial respiratory chain complex II activity and protects against oxygen-glucose deprivation in neurons.

    Science.gov (United States)

    Sha, Dujuan; Wang, Luna; Zhang, Jun; Qian, Lai; Li, Qiming; Li, Jin; Qian, Jian; Gu, Shuangshuang; Han, Ling; Xu, Peng; Xu, Yun

    2014-09-25

    The mechanisms of ischemic stroke, a main cause of disability and death, are complicated. Ischemic stroke results from the interaction of various factors including oxidative stress, a key pathological mechanism that plays an important role during the acute stage of ischemic brain injury. This study demonstrated that cocaine- and amphetamine-regulated transcript (CART) peptide, specifically CART55-102, increased the survival rate, but decreased the mortality of neurons exposed to oxygen-glucose deprivation (OGD), in a dose-dependent manner. The above-mentioned effects of CART55-102 were most significant at 0.4nM. These results indicated that CART55-102 suppressed neurotoxicity and enhanced neuronal survival after oxygen-glucose deprivation. CART55-102 (0.4nM) significantly diminished reactive oxygen species levels and markedly increased the activity of mitochondrial respiratory chain complex II in oxygen-glucose deprived neurons. In summary, CART55-102 suppressed oxidative stress in oxygen-glucose deprived neurons, possibly through elevating the activity of mitochondrial respiratory chain complex II. This result provides evidence for the development of CART55-102 as an antioxidant drug. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa).

    Science.gov (United States)

    Li, Xiao-Jie; Zhang, Ya-Feng; Hou, Mingming; Sun, Feng; Shen, Yun; Xiu, Zhi-Hui; Wang, Xiaomin; Chen, Zong-Liang; Sun, Samuel S M; Small, Ian; Tan, Bao-Cai

    2014-09-01

    RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, altering the amino acid specified by the DNA sequence. Here we report the identification of a critical editing factor of mitochondrial nad7 transcript via molecular characterization of a small kernel 1 (smk1) mutant in Zea mays (maize). Mutations in Smk1 arrest both the embryo and endosperm development. Cloning of Smk1 indicates that it encodes an E-subclass pentatricopeptide repeat (PPR) protein that is targeted to mitochondria. Loss of SMK1 function abolishes the C → U editing at the nad7-836 site, leading to the retention of a proline codon that is edited to encode leucine in the wild type. The smk1 mutant showed dramatically reduced complex-I assembly and NADH dehydrogenase activity, and abnormal biogenesis of the mitochondria. Analysis of the ortholog in Oryza sativa (rice) reveals that rice SMK1 has a conserved function in C → U editing of the mitochondrial nad7-836 site. T-DNA knock-out mutants showed abnormal embryo and endosperm development, resulting in embryo or seedling lethality. The leucine at NAD7-279 is highly conserved from bacteria to flowering plants, and analysis of genome sequences from many plants revealed a molecular coevolution between the requirement for C → U editing at this site and the existence of an SMK1 homolog. These results demonstrate that Smk1 encodes a PPR-E protein that is required for nad7-836 editing, and this editing is critical to NAD7 function in complex-I assembly in mitochondria, and hence to embryo and endosperm development in maize and rice. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  17. Resveratrol-Sensitized UVA Induced Apoptosis in Human Keratinocytes through Mitochondrial Oxidative Stress and Pore Opening

    Science.gov (United States)

    Boyer, Jean Z; Jandova, Jana; Janda, Jaroslav; Vleugels, Frank R; Elliott, David; Sligh, James E

    2012-01-01

    Resveratrol (3, 5, 4′-trihydroxy- trans- stilbene), a polyphenol compound, is derived from natural products such as the skin of red grapes, blueberries and cranberries. Resveratrol not only exhibits antioxidant, cardioprotection, and anti-aging properties, but can also inhibit cancer cell growth and induce apoptosis. It has been shown that resveratrol inhibits the activation of Nf-kB and subsequently down regulates the expression of Nf-kB regulated genes such as interleukin-2 and Bcl-2, leading to cell cycle arrest and increased apoptosis in multiple myeloma cells. In the skin, resveratrol has been reported to sensitize keratinocytes to UVA induced apoptosis. However, the effect of resveratrol on opening of the mitochondrial permeability transition pore has not been previously examined. Our data show that UVA (14J/cm2) along with resveratrol causes massive oxidative stress in mitochondria. As a consequence of oxidative stress, the mitochondrial membrane potential decreases which results in opening of the mitochondrial pores ultimately leading to apoptosis in human keratinocytes. These results may have clinical implications for development of future chemotherapeutic treatment for tumors of the skin. PMID:22673012

  18. Structure of the Human Mitochondrial Ribosome Studied In Situ by Cryoelectron Tomography.

    Science.gov (United States)

    Englmeier, Robert; Pfeffer, Stefan; Förster, Friedrich

    2017-10-03

    Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated mammalian mitoribosome, but its mode of membrane association remained hypothetical. In this study, we used cryoelectron tomography to visualize human mitoribosomes in isolated mitochondria. The subtomogram average of the membrane-associated human mitoribosome reveals a single major contact site with the inner membrane, mediated by the mitochondria-specific protein mL45. A second rRNA-mediated contact site that is present in yeast is absent in humans, resulting in a more variable association of the human mitoribosome with the inner membrane. Despite extensive structural differences of mammalian and fungal mitoribosomal structure, the principal organization of peptide exit tunnel and the mL45 homolog remains invariant, presumably to align the mitoribosome with the membrane-embedded insertion machinery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

    Science.gov (United States)

    Chaabane, Wiem; Cieślar-Pobuda, Artur; El-Gazzah, Mohamed; Jain, Mayur V; Rzeszowska-Wolny, Joanna; Rafat, Mehrdad; Stetefeld, Joerg; Ghavami, Saeid; Los, Marek J

    2014-09-01

    The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

    2009-06-09

    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.

  1. Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

    Science.gov (United States)

    Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

    2005-01-01

    The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

  2. Metabolic and transcriptional responses of gilthead sea bream (Sparus aurata L.) to environmental stress: New insights in fish mitochondrial phenotyping

    NARCIS (Netherlands)

    Bermejo-Nogales, A.; Nederlof, M.A.J.; Benedito-Palos, L.; Ballester-Lozano, G.F.; Folkedal, O.; Olsen, R.E.; Sitjà-Bobadilla, A.; Pérez-Sánchez, J.

    2014-01-01

    The aim of the current study was to phenotype fish metabolism and the transcriptionally-mediated response of hepatic mitochondria of gilthead sea bream to intermittent and repetitive environmental stressors: (i) changes in water temperature (T-ST), (ii) changes in water level and chasing (C-ST) and

  3. Mitochondrial cardiomyopathies

    Directory of Open Access Journals (Sweden)

    Ayman W. El-Hattab

    2016-07-01

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

  4. Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis

    International Nuclear Information System (INIS)

    Wu, C.-W.; Ping, Y.-H.; Yen, J.-C.; Chang, C.-Y.; Wang, S.-F.; Yeh, C.-L.; Chi, C.-W.; Lee, H.-C.

    2007-01-01

    Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in Brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects

  5. Mitochondrial Toxicity in Human Pregnancy: An Update on Clinical and Experimental Approaches in the Last 10 Years

    Directory of Open Access Journals (Sweden)

    Constanza Morén

    2014-09-01

    Full Text Available Mitochondrial toxicity can be one of the most dreadful consequences of exposure to a wide range of external agents including pathogens, therapeutic agents, abuse drugs, toxic gases and other harmful chemical substances. However, little is known about the effects of mitochondrial toxicity on pregnant women exposed to these agents that may exert transplacental activity and condition fetal remodeling. It has been hypothesized that mitochondrial toxicity may be involved in some adverse obstetric outcomes. In the present study, we investigated the association between exposure to mitochondrial toxic agents and pathologic conditions ranging from fertility defects, detrimental fetal development and impaired newborn health due to intra-uterine exposure. We have reviewed data from studies in human subjects to propose mechanisms of mitochondrial toxicity that could be associated with the symptoms present in both exposed pregnant and fetal patients. Since some therapeutic interventions or accidental exposure cannot be avoided, further research is needed to gain insight into the molecular pathways leading to mitochondrial toxicity during pregnancy. The ultimate objective of these studies should be to reduce the mitochondrial toxicity of these agents and establish biomarkers for gestational monitoring of harmful effects.

  6. A role for human mitochondrial complex II in the production of reactive oxygen species in human skin

    Directory of Open Access Journals (Sweden)

    Alasdair Anderson

    2014-01-01

    Full Text Available The mitochondrial respiratory chain is a major generator of cellular oxidative stress, thought to be an underlying cause of the carcinogenic and ageing process in many tissues including skin. Previous studies of the relative contributions of the respiratory chain (RC complexes I, II and III towards production of reactive oxygen species (ROS have focussed on rat tissues and certainly not on human skin which is surprising as this tissue is regularly exposed to UVA in sunlight, a potent generator of cellular oxidative stress. In a novel approach we have used an array of established specific metabolic inhibitors and DHR123 fluorescence to study the relative roles of the mitochondrial RC complexes in cellular ROS production in 2 types of human skin cells. These include additional enhancement of ROS production by exposure to physiological levels of UVA. The effects within epidermal and dermal derived skin cells are compared to other tissue cell types as well as those harbouring a compromised mitochondrial status (Rho-zero A549. The results show that the complex II inhibitor, TTFA, was the only RC inhibitor to significantly increase UVA-induced ROS production in both skin cell types (P<0.05 suggesting that the role of human skin complex II in terms of influencing ROS production is more important than previously thought particularly in comparison to liver cells. Interestingly, two-fold greater maximal activity of complex II enzyme was observed in both skin cell types compared to liver (P<0.001. The activities of RC enzymes appear to decrease with increasing age and telomere length is correlated with ageing. Our study showed that the level of maximal complex II activity was higher in the MRC5/hTERT (human lung fibroblasts transfected with telomerase cells than the corresponding wild type cells (P=0.0012 which can be considered (in terms of telomerase activity as models of younger and older cells respectively.

  7. Cocoa procyanidins and human cytokine transcription and secretion.

    Science.gov (United States)

    Mao, T; Van De Water, J; Keen, C L; Schmitz, H H; Gershwin, M E

    2000-08-01

    We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

  8. New insight into transcription of human endogenous retroviral elements

    Czech Academy of Sciences Publication Activity Database

    Pačes, Jan; Huang, Y.T.; Pačes, Václav; Rídl, Jakub; Chang, C.M.

    2013-01-01

    Roč. 30, č. 3 (2013), s. 314-318 ISSN 1871-6784 R&D Projects: GA ČR GCP305/10/J052 Grant - others:Chang Gung Memorial Hospital(TW) CMRPD170233; NSCNHRI Taiwan(TW) NSC 97-2314-B-182-001-MY2; NSCNHRI Taiwan(TW) NSC 99-2314-B-182-024-MY3; NSCNHRI Taiwan(TW) NSC 99-2923-B-182-001-MY3 Institutional support: RVO:68378050 Keywords : HERV * transcription * tissue-specific Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.106, year: 2013

  9. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: Dihydrolipoamide acetyltransferase

    International Nuclear Information System (INIS)

    Coppel, R.L.; McNeilage, L.J.; Surh, C.D.; Van De Water, J.; Spithill, T.W.; Whittingham, S.; Gershwin, M.E.

    1988-01-01

    Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency of autoantibodies to a M r 70,000 mitochondrial antigen a component of the M2 antigen complex. The authors have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects

  10. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  11. Influence of erythrocyte oxygenation and intravascular ATP on resting and exercising skeletal muscle blood flow in humans with mitochondrial myopathy

    DEFF Research Database (Denmark)

    Jeppesen, Tina D; Vissing, John; González-Alonso, José

    2012-01-01

    Oxygen (O(2)) extraction is impaired in exercising skeletal muscle of humans with mutations of mitochondrial DNA (mtDNA), but the muscle hemodynamic response to exercise has never been directly investigated. This study sought to examine the extent to which human skeletal muscle perfusion can incr...

  12. The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line

    DEFF Research Database (Denmark)

    Suzuki, Harukazu; Forrest, Alistair R R; van Nimwegen, Erik

    2009-01-01

    , we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks......Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites...... involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process....

  13. Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

    International Nuclear Information System (INIS)

    Zhang, H.; Wang, J.C.; Liu, L.F.

    1988-01-01

    Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

  14. Effects of Mitochondrial Uncoupling Protein 2 Inhibition by Genipin in Human Cumulus Cells

    Directory of Open Access Journals (Sweden)

    Hongshan Ge

    2015-01-01

    Full Text Available UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality.

  15. The Plant Decapeptide OSIP108 Can Alleviate Mitochondrial Dysfunction Induced by Cisplatin in Human Cells

    Directory of Open Access Journals (Sweden)

    Pieter Spincemaille

    2014-09-01

    Full Text Available We investigated the effect of the Arabidopsis thaliana-derived decapeptide OSIP108 on human cell tolerance to the chemotherapeutic agent cisplatin (Cp, which induces apoptosis and mitochondrial dysfunction. We found that OSIP108 increases the tolerance of HepG2 cells to Cp and prevents Cp-induced changes in basic cellular metabolism. More specifically, we demonstrate that OSIP108 reduces Cp-induced inhibition of respiration, decreases glycolysis and prevents Cp-uptake in HepG2 cells. Apart from its protective action against Cp in human cells, OSIP108 also increases the yeast Saccharomyces cerevisiae tolerance to Cp. A limited yeast-based study of OSIP108 analogs showed that cyclization does not severely affect its activity, which was further confirmed in HepG2 cells. Furthermore, the similarity in the activity of the D-stereoisomer (mirror image form of OSIP108 with the L-stereoisomer suggests that its mode of action does not involve binding to a stereospecific receptor. In addition, as OSIP108 decreases Cp uptake in HepG2 cells and the anti-Cp activity of OSIP108 analogs without free cysteine is reduced, OSIP108 seems to protect against Cp-induced toxicity only partly via complexation. Taken together, our data indicate that OSIP108 and its cyclic derivatives can protect against Cp-induced toxicity and, thus, show potential as treatment options for mitochondrial dysfunction- and apoptosis-related conditions.

  16. A transcriptional profile of aging in the human kidney.

    Directory of Open Access Journals (Sweden)

    Graham E J Rodwell

    2004-12-01

    Full Text Available In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.

  17. DOT/FAA Human Factors Workshop on Aviation (5th). Transcript.

    Science.gov (United States)

    1982-01-01

    This document is a verbatim transcript of the proceedings of the Fifth Human Factors Workshop held at the Mike Monroney Aeronautical Center in Oklahoma City, Oklahoma, on July 7-9, 1981. The Sixth Human Factors Workshop was held at the same facility ...

  18. Role of natural antisense transcripts pertaining to tumor suppressor genes in human carcinomas

    International Nuclear Information System (INIS)

    Pelicci, G.; Pierotti, M.

    2009-01-01

    Overlapping transcripts in opposite orientations can potentially form perfect sense-antisense duplex RNA. Recently, several studies have revealed the extent of natural antisense transcripts (NATs) and their role in important biological phenomena also in higher organisms. In order to test the hypothesis that the function of NATs in man might represent an essential element in the regulation of gene expression, especially at transcriptional level, in this study we planned to look for, systematically examine, and characterize NATs belonging in the human genome to the tumour suppressor class of genes, so to identify physiological (and potentially pathological) modulators in this gene class

  19. Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

    Science.gov (United States)

    Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

    2013-01-01

    Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, prespiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

  20. The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam; Palumbo, Elisa; Rampazzo, Chiara [Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova (Italy); Bianchi, Vera, E-mail: vbianchi@bio.unipd.it [Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova (Italy)

    2012-10-15

    In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [{sup 3}H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1{sup -} cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1{sup +} cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: Black-Right-Pointing-Pointer Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. Black-Right-Pointing-Pointer siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1{sup -} cells. Black-Right-Pointing-Pointer PNC1 overexpression accumulates dTTP in mitochondria of TK1{sup +} cells. Black-Right-Pointing-Pointer PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. Black-Right-Pointing-Pointer PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.

  1. The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

    International Nuclear Information System (INIS)

    Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam; Palumbo, Elisa; Rampazzo, Chiara; Bianchi, Vera

    2012-01-01

    In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [ 3 H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1 − cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1 + cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: ► Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. ► siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1 − cells. ► PNC1 overexpression accumulates dTTP in mitochondria of TK1 + cells. ► PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. ► PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.

  2. Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

    Science.gov (United States)

    André, P; Kim, A; Khrapko, K; Thilly, W G

    1997-08-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

  3. Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation

    International Nuclear Information System (INIS)

    Chen Xiaosui; Zhou Lijun; Wang Yuxiao; Qu Jia; Feng Jiangbing; Lu Xue; Chen Deqing; Liu Qingjie

    2006-01-01

    Objective: To construct a stable plasmid that spanning deleted human mitochondrial DNA (mtDNA) 4977 bp induced by ionizing radiation and another one for control DNA fragment, in order to use in the human mitochondrial genome study in the future. Methods: The peripheral blood, which had no mtDNA 4977 bp deletion found in previous study, was exposed to 10 Gy 60 Co γ-rays in vitro. The total cell DNA was extracted and PCR was carried out: a nest-PCR of three-round PCR was used for the mtDNA 4977 bp deletion and one- round regular PCR was used for the control ND1 gene. The PCR products were used for transfection by electroporation and the positive clones were obtained after screening. The plasmid DNA was isolated and sequenced after enzymatic digestion and purification. The sequence result was BLASTed with the human mitochondrial genome. Results: The sizes of PCR products for the flanked 4977 bp deletion and the ND1 gene were similar with those predicted according to GeneBank. The sequences for the positive clones were above 99 per cent homologous with the human mitochondrial genome after BLASTed. Conclusion: The plasmids for deleted human mtDNA 4977 bp and control DNA fragment have been constructed successfully, and they could be used in the quality and quantity studies on human mtDNA 4977 bp deletion. (authors)

  4. GAD2 Alternative Transcripts in the Human Prefrontal Cortex, and in Schizophrenia and Affective Disorders.

    Directory of Open Access Journals (Sweden)

    Kasey N Davis

    Full Text Available Genetic variation and early adverse environmental events work together to increase risk for schizophrenia. γ-aminobutyric acid (GABA, the major inhibitory neurotransmitter in adult mammalian brain, plays a major role in normal brain development, and has been strongly implicated in the pathobiology of schizophrenia. GABA synthesis is controlled by two glutamic acid decarboxylase (GAD genes, GAD1 and GAD2, both of which produce a number of alternative transcripts. Genetic variants in the GAD1 gene are associated with increased risk for schizophrenia, and reduced expression of its major transcript in the human dorsolateral prefrontal cortex (DLPFC. No consistent changes in GAD2 expression have been found in brains from patients with schizophrenia. In this work, with the use of RNA sequencing and PCR technologies, we confirmed and tracked the expression of an alternative truncated transcript of GAD2 (ENST00000428517 in human control DLPFC homogenates across lifespan besides the well-known full length transcript of GAD2. In addition, using quantitative RT-PCR, expression of GAD2 full length and truncated transcripts were measured in the DLPFC of patients with schizophrenia, bipolar disorder and major depression. The expression of GAD2 full length transcript is decreased in the DLPFC of schizophrenia and bipolar disorder patients, while GAD2 truncated transcript is increased in bipolar disorder patients but decreased in schizophrenia patients. Moreover, the patients with schizophrenia with completed suicide or positive nicotine exposure showed significantly higher expression of GAD2 full length transcript. Alternative transcripts of GAD2 may be important in the growth and development of GABA-synthesizing neurons as well as abnormal GABA signaling in the DLPFC of patients with schizophrenia and affective disorders.

  5. Human macrophages support persistent transcription from unintegrated HIV-1 DNA

    International Nuclear Information System (INIS)

    Kelly, Jeremy; Beddall, Margaret H.; Yu Dongyang; Iyer, Subashini R.; Marsh, Jon W.; Wu Yuntao

    2008-01-01

    Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines

  6. Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation.

    Science.gov (United States)

    Emelyanova, Larisa; Ashary, Zain; Cosic, Milanka; Negmadjanov, Ulugbek; Ross, Gracious; Rizvi, Farhan; Olet, Susan; Kress, David; Sra, Jasbir; Tajik, A Jamil; Holmuhamedov, Ekhson L; Shi, Yang; Jahangir, Arshad

    2016-07-01

    Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF. Copyright © 2016 the American Physiological Society.

  7. Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate.

    Science.gov (United States)

    Adomako-Ankomah, Yaw; English, Elizabeth D; Danielson, Jeffrey J; Pernas, Lena F; Parker, Michelle L; Boulanger, Martin J; Dubey, Jitender P; Boyle, Jon P

    2016-05-01

    In Toxoplasma gondii, an intracellular parasite of humans and other animals, host mitochondrial association (HMA) is driven by a gene family that encodes multiple mitochondrial association factor 1 (MAF1) proteins. However, the importance of MAF1 gene duplication in the evolution of HMA is not understood, nor is the impact of HMA on parasite biology. Here we used within- and between-species comparative analysis to determine that the MAF1 locus is duplicated in T. gondii and its nearest extant relative Hammondia hammondi, but not another close relative, Neospora caninum Using cross-species complementation, we determined that the MAF1 locus harbors multiple distinct paralogs that differ in their ability to mediate HMA, and that only T. gondii and H. hammondi harbor HMA(+) paralogs. Additionally, we found that exogenous expression of an HMA(+) paralog in T. gondii strains that do not normally exhibit HMA provides a competitive advantage over their wild-type counterparts during a mouse infection. These data indicate that HMA likely evolved by neofunctionalization of a duplicate MAF1 copy in the common ancestor of T. gondii and H. hammondi, and that the neofunctionalized gene duplicate is selectively advantageous. Copyright © 2016 by the Genetics Society of America.

  8. Laminarin Induces Apoptosis of Human Colon Cancer LOVO Cells through a Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    He Zhang

    2012-08-01

    Full Text Available Many scientific studies have shown that laminarin has anti-tumor effects, but the anti-tumor mechanism was unclear. The purpose of this study was to investigate the effect of laminarin on the induction of apoptosis in human colon cancer LOVO cells and the molecular mechanism involved. LOVO cells were treated with different concentrations of laminarin at different times. Morphology observations were performed to determine the effects of laminarin on apoptosis of LOVO cells. Flow cytometry (FCM was used to detect the level of intracellular reactive oxygen species (ROS and pH. Laser scanning confocal microscope (LSCM was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP and mitochondrial membrane potential (MMP. Western blotd were performed to analyze the expressions of Cyt-C, Caspase-9 and -3. The results showed the apoptosis morphology, which showed cell protuberance, concentrated cytoplasm and apoptotic bodies, was obvious after 72 h treatment. Laminarin treatment for 24 h increased the intracellular level of ROS and Ca2+; decreased pH value; activated intracellular MPTP and decreased MMP in dose-dependent manners. It also induced the release of Cyt-C and the activation of Caspase-9 and -3. In conclusion, laminarin induces LOVO cell apoptosis through a mitochondrial pathway, suggesting that it could be a potent agent for cancer prevention and treatment.

  9. Loneliness, eudaimonia, and the human conserved transcriptional response to adversity.

    Science.gov (United States)

    Cole, Steven W; Levine, Morgan E; Arevalo, Jesusa M G; Ma, Jeffrey; Weir, David R; Crimmins, Eileen M

    2015-12-01

    Chronic social adversity activates a conserved transcriptional response to adversity (CTRA) marked by increased expression of pro-inflammatory genes and decreased expression of antiviral- and antibody-related genes. Recent findings suggest that some psychological resilience factors may help buffer CTRA activation, but the relative impact of resilience and adversity factors remains poorly understood. Here we examined the relative strength of CTRA association for the two best-established psychological correlates of CTRA gene expression-the risk factor of perceived social isolation (loneliness) and the resilience factor of eudaimonic well-being (purpose and meaning in life). Peripheral blood samples and validated measures of loneliness and eudaimonic well-being were analyzed in 108 community-dwelling older adults participating in the longitudinal US Health and Retirement Study (56% female, mean age 73). Mixed effect linear model analyses quantified the strength of association between CTRA gene expression and measures of loneliness and eudaimonic well-being in separate and joint analyses. As in previous studies, separate analyses found CTRA gene expression to be up-regulated in association with loneliness and down-regulated in association with eudaimonic well-being. In joint analyses, effects of loneliness were completely abrogated whereas eudaimonic well-being continued to associate with CTRA down-regulation. Similar eudaimonia-dominant effects were observed for positive and negative affect, optimism and pessimism, and anxiety symptoms. All results were independent of demographic and behavioral health risk factors. Eudaimonic well-being may have the potential to compensate for the adverse impact of loneliness on CTRA gene expression. Findings suggest a novel approach to targeting the health risks associated with social isolation by promoting purpose and meaning in life. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Codon usage and expression level of human mitochondrial 13 protein coding genes across six continents.

    Science.gov (United States)

    Chakraborty, Supriyo; Uddin, Arif; Mazumder, Tarikul Huda; Choudhury, Monisha Nath; Malakar, Arup Kumar; Paul, Prosenjit; Halder, Binata; Deka, Himangshu; Mazumder, Gulshana Akthar; Barbhuiya, Riazul Ahmed; Barbhuiya, Masuk Ahmed; Devi, Warepam Jesmi

    2017-12-02

    The study of codon usage coupled with phylogenetic analysis is an important tool to understand the genetic and evolutionary relationship of a gene. The 13 protein coding genes of human mitochondria are involved in electron transport chain for the generation of energy currency (ATP). However, no work has yet been reported on the codon usage of the mitochondrial protein coding genes across six continents. To understand the patterns of codon usage in mitochondrial genes across six different continents, we used bioinformatic analyses to analyze the protein coding genes. The codon usage bias was low as revealed from high ENC value. Correlation between codon usage and GC3 suggested that all the codons ending with G/C were positively correlated with GC3 but vice versa for A/T ending codons with the exception of ND4L and ND5 genes. Neutrality plot revealed that for the genes ATP6, COI, COIII, CYB, ND4 and ND4L, natural selection might have played a major role while mutation pressure might have played a dominant role in the codon usage bias of ATP8, COII, ND1, ND2, ND3, ND5 and ND6 genes. Phylogenetic analysis indicated that evolutionary relationships in each of 13 protein coding genes of human mitochondria were different across six continents and further suggested that geographical distance was an important factor for the origin and evolution of 13 protein coding genes of human mitochondria. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  11. Oxygen consumption rate and mitochondrial density in human melanoma monolayer cultures and multicellular spheroids.

    Science.gov (United States)

    Hystad, M E; Rofstad, E K

    1994-05-15

    Rate of oxygen consumption per cell has been shown in previous studies to decrease with increasing depth in the viable rim of multicellular spheroids initiated from rodent cells, human colon-carcinoma cells, and human glioma cells, due to progressive accumulation of quiescent cells during spheroid growth. The purpose of our work was to determine oxygen-consumption profiles in human melanoma spheroids. Monolayer cultures of 4 lines (BEX-c, COX-c, SAX-c, and WIX-c) and spheroid cultures of 2 lines (BEX-c and WIX-c) were subjected to investigation. Spheroids were initiated from monolayer cell cultures and grown in spinner flasks. Rate of oxygen consumption was measured with a Clarke-type electrode. Mitochondrial density was determined by stereological analysis of transmission electron micrographs. Thickness of viable rim and cell packing density were assessed by light microscopy of central spheroid sections. Cell-cycle distribution was determined by analysis of DNA histograms measured by flow cytometry. Cell volume was measured by an electronic particle counter. Rate of oxygen consumption per cell differed by a factor of approximately 1.8 between the 4 cell lines and was positively correlated to total volume of mitochondria per cell. Rate of oxygen consumption per cell and total volume of mitochondria per cell were equal for monolayer cell cultures, 600-microns spheroids and 1,200-microns spheroids of the same line. Mitochondrial density and location in the cell did not differ between cells at the spheroid surface, in the middle of the viable rim and adjacent to the central necrosis. Cell-cycle distribution, cell volume, and cell-packing density in the outer and inner halves of the viable rim were not significantly different. Consequently, the rate of oxygen consumption per cell in inner regions of the viable rim was probably equal to that at the spheroid surface, suggesting that oxygen diffusion distances may be shorter in some melanomas than in many other tumor

  12. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

    Science.gov (United States)

    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  13. Nucleotide sequences of cDNAs for human papillomavirus type 18 transcripts in HeLa cells

    International Nuclear Information System (INIS)

    Inagaki, Yutaka; Tsunokawa, Youko; Takebe, Naoko; Terada, Masaaki; Sugimura, Takashi; Nawa, Hiroyuki; Nakanishi, Shigetada

    1988-01-01

    HeLa cells expressed 3.4- and 1.6-kilobase (kb) transcripts of the integrated human papillomavirus (HPV) type 18 genome. Two types of cDNA clones representing each size of HPV type 18 transcript were isolated. Sequence analysis of these two types of cDNA clones revealed that the 3.4-kb transcript contained E6, E7, the 5' portion of E1, and human sequence and that the 1.6-kb transcript contained spliced and frameshifted E6 (E6 * ), E7, and human sequence. There was a common human sequence containing a poly(A) addition signal in the 3' end portions of both transcripts, indicating that they were transcribed from the HPV genome at the same integration site with different splicing. Furthermore, the 1.6-kb transcript contained both of the two viral TATA boxes upstream of E6, strongly indicating that a cellular promoter was used for its transcription

  14. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation

    International Nuclear Information System (INIS)

    Sahijdak, W.M.; Yang, Chin-Rang; Zuckerman, J.S.; Meyers, M.; Boothman, D.A.

    1994-01-01

    We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation. Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kB and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3, or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. 22 refs., 2 figs

  15. Detection of mitochondrial DNA deletions in human cells induced by ionizing radiation

    International Nuclear Information System (INIS)

    Liu, Qing-Jie; Feng, Jiang-Bin; Lu, Xue; Li, Yu-Wen; Chen, De-Qing

    2008-01-01

    Full text: Purpose: To screen the novel mitochondrial DNA (mt DNA) deletions induced by ionizing radiation, and analyze the several kinds of mt DNA deletions, known as 3895 bp, 889 bp, 7436 bp or 4934 bp deletions. Methods: Long-range PCR with two pairs of primers, which could amplify the whole human mitochondrial genome, was used to analyze the lymphoblastoid cell line before and after exposed to 10 Gy 60 Co γ-rays. The limited condition PCR was used to certify the possible mt DNA deletion showed by long-range PCR. The PCR products were purified, cloned, sequenced and the sequence result were BLASTed. Regular PCR or nest-PCR were used to analyze the 3895 bp, 889 bp, 7436 bp or 4934 bp deletions before and after radiation exposure. The final PCR products were purified, sequenced and BALSTed on standard human mitochondrial genome sequence database. Results: (1) The predicted bands of mt DNA were observed on the control cell lines, and the possible mt DNA deletions were also detected on the irradiated cell lines. The deletions were certified by the limited condition PCR. The sequence BLAST results of the cloned PCR products showed that two kinds of deletions, 7455 bp deletion (nt 475-7929 in heavy strand) and 9225 bp deletion (nt 7714-369 in heavy strand), which were between two 8 bp direct repeats. Further bioinformatics analysis showed that the two deletions were novel deletions. (2) The 889 bp and 3895 bp deletion were not detected for the cell line samples not exposed to 60 Co γ-rays. The 889 bp and 3895 bp deletions were detected on samples exposed to 10 Gy 60 Co γ-rays. The BALST results showed that the 889 bp and 3895 deletions flanked nt 11688 bp-12576, nt 548 bp-4443, respectively. The 7436 bp deletion levels were not changed much before and after irradiation. (3) The 4934 bp deletions had the same pattern as 7436 bp deletion, but it could induced by radiation. Conclusions: Ionizing radiation could induce the human lymphoblastoid two novel mt DNA

  16. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  17. Relationship between human cytomegalovirus transcription and symptomatic apical periodontitis in Iran.

    Science.gov (United States)

    Yazdi, K A; Sabeti, M; Jabalameli, F; Eman eini, M; Kolahdouzan, S A; Slots, J

    2008-12-01

    Apical periodontitis of endodontic origin may develop as a result of cooperative interactions among herpesviruses, specific pathogenic bacteria and tissue-destructive inflammatory mediators. This study sought to identify the presence of Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) transcripts in symptomatic and asymptomatic periapical lesions of individuals living in Iran. Fifty endodontic patients (28 with symptomatic periapical lesions and 22 with asymptomatic periapical lesions) were included in the study. In each study subject, a microbiological periapical sample was collected using a curette in conjunction with periapical surgery. A reverse transcription-polymerase chain reaction assay was used to identify transcripts of EBV and HCMV. Human cytomegalovirus transcript was detected in 15 of the 28 (53.6%) symptomatic and in six of the 22 (27.3%) asymptomatic periapical study lesions (significant difference between symptomatic and asymptomatic lesions; P = 0.03, chi-square test). Epstein-Barr virus transcript was identified in one symptomatic and in two asymptomatic periapical lesions. This study establishes that HCMV transcription is common in apical periodontitis and is most frequent in symptomatic lesions. The high frequency of active herpesvirus infections in severe apical periodontitis changes the pathogenic paradigm of the disease and may also have preventive and therapeutic implications.

  18. Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

    Science.gov (United States)

    Genga, Ryan M; Kearns, Nicola A; Maehr, René

    2016-05-15

    The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts

    DEFF Research Database (Denmark)

    Guillery, O.; Malka, F.; Frachon, P.

    2008-01-01

    induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under...... basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked...... to mitochondrial oxidative phosphorylation. Its alteration by glycolysis, inhibition points to a severe oxidative phosphorylation defect. (C) 2008 Elsevier B.V. All rights reserved Udgivelsesdato: 2008/4...

  20. Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

    Science.gov (United States)

    Philippeos, Christina; Telerman, Stephanie B; Oulès, Bénédicte; Pisco, Angela O; Shaw, Tanya J; Elgueta, Raul; Lombardi, Giovanna; Driskell, Ryan R; Soldin, Mark; Lynch, Magnus D; Watt, Fiona M

    2018-04-01

    Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  1. O-GlcNAc transferase regulates transcriptional activity of human Oct4.

    Science.gov (United States)

    Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

    2017-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  3. PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

    Science.gov (United States)

    Miranda, Dante; Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra; Montoya, Margarita

    2016-01-01

    Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

  4. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    International Nuclear Information System (INIS)

    Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  5. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

    2013-10-15

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  6. Radiosensitivity evaluation of Human tumor cell lines by detecting 4977bp deletion in mitochondrial DNA

    International Nuclear Information System (INIS)

    Zhang Yipei

    2009-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using the assay of mtDNA4977bp deletion. Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction(SF), the ratio of mtDNA4977bp deletion and DNA damage were detected by MTT assay and nested PCR technique respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4and 8Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. PCR method:The differences on mtDNA 4977bp deletion in mitochondrial DNA among HepG 2 , EC-9706 and MCF-7 were not significant after 1Gy and 4Gy γ-ray irradiation. The ratio of 4977bp deletion in mitochondrial DNA of HepG 2 and EC-9706 increased while that of MCF-7 decreased after 8Gy irradiation. The ratio of mtDNA 4977bp deletion of HepG 2 and EC-9706 was higher significantly than that of MCF-7, which implies that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF -7. Conclusion: As a new biological marker, mtDNA4977bp deletion may be hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  7. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

  8. Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression

    Directory of Open Access Journals (Sweden)

    Carlos R. Morales

    2014-01-01

    Full Text Available Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.

  9. High-confidence assessment of functional impact of human mitochondrial non-synonymous genome variations by APOGEE.

    Directory of Open Access Journals (Sweden)

    Stefano Castellana

    2017-06-01

    Full Text Available 24,189 are all the possible non-synonymous amino acid changes potentially affecting the human mitochondrial DNA. Only a tiny subset was functionally evaluated with certainty so far, while the pathogenicity of the vast majority was only assessed in-silico by software predictors. Since these tools proved to be rather incongruent, we have designed and implemented APOGEE, a machine-learning algorithm that outperforms all existing prediction methods in estimating the harmfulness of mitochondrial non-synonymous genome variations. We provide a detailed description of the underlying algorithm, of the selected and manually curated training and test sets of variants, as well as of its classification ability.

  10. Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

    Science.gov (United States)

    Wan, Xianxiu; Gupta, Shivali; Zago, Maria P.; Davidson, Mercy M.; Dousset, Pierre; Amoroso, Alejandro; Garg, Nisha Jain

    2012-01-01

    Background Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. Methods and Results We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. Conclusions The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for

  11. Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics.

    Science.gov (United States)

    Zampieri, Sandra; Mammucari, Cristina; Romanello, Vanina; Barberi, Laura; Pietrangelo, Laura; Fusella, Aurora; Mosole, Simone; Gherardi, Gaia; Höfer, Christian; Löfler, Stefan; Sarabon, Nejc; Cvecka, Jan; Krenn, Matthias; Carraro, Ugo; Kern, Helmut; Protasi, Feliciano; Musarò, Antonio; Sandri, Marco; Rizzuto, Rosario

    2016-12-01

    Age-related sarcopenia is characterized by a progressive loss of muscle mass with decline in specific force, having dramatic consequences on mobility and quality of life in seniors. The etiology of sarcopenia is multifactorial and underlying mechanisms are currently not fully elucidated. Physical exercise is known to have beneficial effects on muscle trophism and force production. Alterations of mitochondrial Ca 2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) have been recently shown to affect muscle trophism in vivo in mice. To understand the relevance of MCU-dependent mitochondrial Ca 2+ uptake in aging and to investigate the effect of physical exercise on MCU expression and mitochondria dynamics, we analyzed skeletal muscle biopsies from 70-year-old subjects 9 weeks trained with either neuromuscular electrical stimulation (ES) or leg press. Here, we demonstrate that improved muscle function and structure induced by both trainings are linked to increased protein levels of MCU Ultrastructural analyses by electron microscopy showed remodeling of mitochondrial apparatus in ES-trained muscles that is consistent with an adaptation to physical exercise, a response likely mediated by an increased expression of mitochondrial fusion protein OPA1. Altogether these results indicate that the ES-dependent physiological effects on skeletal muscle size and force are associated with changes in mitochondrial-related proteins involved in Ca 2+ homeostasis and mitochondrial shape. These original findings in aging human skeletal muscle confirm the data obtained in mice and propose MCU and mitochondria-related proteins as potential pharmacological targets to counteract age-related muscle loss. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  12. Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells.

    Science.gov (United States)

    Swalwell, Helen; Latimer, Jennifer; Haywood, Rachel M; Birch-Machin, Mark A

    2012-02-01

    Skin cancer incidence is dramatically increasing worldwide, with exposure to ultraviolet radiation (UVR) a predominant factor. The UVA component initiates oxidative stress in human skin, although its exact role in the initiation of skin cancer, particularly malignant melanoma, remains unclear and is controversial because there is evidence for a melanin-dependent mechanism in UVA-linked melanoma studies. Nonpigmented (CHL-1, A375), moderately pigmented (FM55, SKmel23), and highly pigmented (FM94, hyperpigmented FM55) human melanoma cell lines have been used to investigate UVA-induced production of reactive oxygen species using FACS analysis, at both the cellular (dihydrorhodamine-123) and the mitochondrial (MitoSOX) level, where most cellular stress is generated. For the first time, downstream mtDNA damage (utilizing a quantitative long-PCR assay) has been investigated. Using UVA, UVB, and H(2)O(2) as cellular stressors, we have explored the dual roles of melanin as a photoprotector and photosensitizer. The presence of melanin has no influence over cellular oxidative stress generation, whereas, in contrast, melanin protects against mitochondrial superoxide generation and mtDNA damage (one-way ANOVA with post hoc Tukey's analysis, Pmelanin binds directly to DNA, it acts as a direct photosensitizer of mtDNA damage during UVA irradiation (Pmelanin. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Transcriptional dynamics during human adipogenesis and its link to adipose morphology and distribution

    DEFF Research Database (Denmark)

    Ehrlund, Anna; Mejhert, Niklas; Björk, Christel

    2017-01-01

    White adipose tissue (WAT) can develop into several phenotypes with different pathophysiological impact on type 2 diabetes. To better understand the adipogenic process, the transcriptional events that occur during in vitro differentiation of human adipocytes were investigated and the findings lin...

  14. DOT/FAA Human Factors Workshop on Aviation (6th). Transcript.

    Science.gov (United States)

    1982-05-01

    This document is a verbatim transcript of the proceedings of the DOT/FAA Sixth Human Factors Workshop on Aviation held at the Mike Monroney Aeronautical Center, Oklahoma City, Oklahoma on July 7-8, 1981. The subject of the workshop was aviation maint...

  15. Associating transcription factors and conserved RNA structures with gene regulation in the human brain

    DEFF Research Database (Denmark)

    Hecker, Nikolai; Seemann, Stefan E.; Silahtaroglu, Asli

    2017-01-01

    Anatomical subdivisions of the human brain can be associated with different neuronal functions. This functional diversification is reflected by differences in gene expression. By analyzing post-mortem gene expression data from the Allen Brain Atlas, we investigated the impact of transcription fac...

  16. [Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937].

    Science.gov (United States)

    Fujii, Satoshi; Muraoka, Sanae; Miyamoto, Atsushi; Sakurai, Koichi

    2018-01-01

     Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

  17. Transcriptional regulation of human RANK ligand gene expression by E2F1

    International Nuclear Information System (INIS)

    Hu Yan; Sun Meng; Nadiminty, Nagalakshmi; Lou Wei; Pinder, Elaine; Gao, Allen C.

    2008-01-01

    Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890 bp 5'-flanking sequence of human RANKL gene (-1782 bp to +108 bp relative to the transcription start site). Using a series of deletion mutations of the 1890 bp RANKL promoter, we identified a 72 bp region (-172 to -100 bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72 bp region in the human RANKL promoter. Overexpression of E2F1 increased RANKL promoter activity, while down-regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site

  18. The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids*

    Science.gov (United States)

    Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

    2014-01-01

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation. PMID:24652292

  19. The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

    Science.gov (United States)

    Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

    2014-05-09

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

  20. HmtDB 2016: data update, a better performing query system and human mitochondrial DNA haplogroup predictor.

    Science.gov (United States)

    Clima, Rosanna; Preste, Roberto; Calabrese, Claudia; Diroma, Maria Angela; Santorsola, Mariangela; Scioscia, Gaetano; Simone, Domenico; Shen, Lishuang; Gasparre, Giuseppe; Attimonelli, Marcella

    2017-01-04

    The HmtDB resource hosts a database of human mitochondrial genome sequences from individuals with healthy and disease phenotypes. The database is intended to support both population geneticists as well as clinicians undertaking the task to assess the pathogenicity of specific mtDNA mutations. The wide application of next-generation sequencing (NGS) has provided an enormous volume of high-resolution data at a low price, increasing the availability of human mitochondrial sequencing data, which called for a cogent and significant expansion of HmtDB data content that has more than tripled in the current release. We here describe additional novel features, including: (i) a complete, user-friendly restyling of the web interface, (ii) links to the command-line stand-alone and web versions of the MToolBox package, an up-to-date tool to reconstruct and analyze human mitochondrial DNA from NGS data and (iii) the implementation of the Reconstructed Sapiens Reference Sequence (RSRS) as mitochondrial reference sequence. The overall update renders HmtDB an even more handy and useful resource as it enables a more rapid data access, processing and analysis. HmtDB is accessible at http://www.hmtdb.uniba.it/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. 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

    Science.gov (United States)

    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

  2. A role for mitochondrial oxidants in stress-induced premature senescence of human vascular smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Yogita Mistry

    2013-01-01

    Full Text Available Mitochondria are a major source of cellular oxidants and have been implicated in aging and associated pathologies, notably cardiovascular diseases. Vascular cell senescence is observed in experimental and human cardiovascular pathologies. Our previous data highlighted a role for angiotensin II in the induction of telomere-dependent and -independent premature senescence of human vascular smooth muscle cells and suggested this was due to production of superoxide by NADPH oxidase. However, since a role for mitochondrial oxidants was not ruled out we hypothesise that angiotensin II mediates senescence by mitochondrial superoxide generation and suggest that inhibition of superoxide may prevent vascular smooth muscle cell aging in vitro. Cellular senescence was induced using a stress-induced premature senescence protocol consisting of three successive once-daily exposure of cells to 1×10−8 mol/L angiotensin II and was dependent upon the type-1 angiotensin II receptor. Angiotensin stimulated NADPH-dependent superoxide production as estimated using lucigenin chemiluminescence in cell lysates and this was attenuated by the mitochondrial electron transport chain inhibitor, rotenone. Angiotensin also resulted in an increase in mitoSOX fluorescence indicating stimulation of mitochondrial superoxide. Significantly, the induction of senescence by angiotensin II was abrogated by rotenone and by the mitochondria-targeted superoxide dismutase mimetic, mitoTEMPO. These data suggest that mitochondrial superoxide is necessary for the induction of stress-induced premature senescence by angiotensin II and taken together with other data suggest that mitochondrial cross-talk with NADPH oxidases, via as yet unidentified signalling pathways, is likely to play a key role.

  3. Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

    Science.gov (United States)

    André, Paulo; Kim, Andrea; Khrapko, Konstantin; Thilly, William G.

    1997-01-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be

  4. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

    Directory of Open Access Journals (Sweden)

    Yiwen Li

    2012-11-01

    Full Text Available Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

  5. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yiwen; Xu, Yuqing [Department of Oncology,Second Affiliated Hospital, Harbin Medical University, Nangang District, Harbin, Heilongjiang (China); Lei, Bo [Department of Breast Surgery, Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang (China); Wang, Wenxiu [Department of Oncology,Second Affiliated Hospital, Harbin Medical University, Nangang District, Harbin, Heilongjiang (China); Ge, Xin; Li, Jingrui [Department of General Surgery, Heilongjiang Province Hospital, Harbin, Heilongjiang (China)

    2012-08-03

    Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM) for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

  6. Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration

    Science.gov (United States)

    Alves, Chrystian J.; Dariolli, Rafael; Jorge, Frederico M.; Monteiro, Matheus R.; Maximino, Jessica R.; Martins, Roberto S.; Strauss, Bryan E.; Krieger, José E.; Callegaro, Dagoberto; Chadi, Gerson

    2015-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that leads to widespread motor neuron death, general palsy and respiratory failure. The most prevalent sporadic ALS form is not genetically inherited. Attempts to translate therapeutic strategies have failed because the described mechanisms of disease are based on animal models carrying specific gene mutations and thus do not address sporadic ALS. In order to achieve a better approach to study the human disease, human induced pluripotent stem cell (hiPSC)-differentiated motor neurons were obtained from motor nerve fibroblasts of sporadic ALS and non-ALS subjects using the STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus system and submitted to microarray analyses using a whole human genome platform. DAVID analyses of differentially expressed genes identified molecular function and biological process-related genes through Gene Ontology. REVIGO highlighted the related functions mRNA and DNA binding, GTP binding, transcription (co)-repressor activity, lipoprotein receptor binding, synapse organization, intracellular transport, mitotic cell cycle and cell death. KEGG showed pathways associated with Parkinson's disease and oxidative phosphorylation, highlighting iron homeostasis, neurotrophic functions, endosomal trafficking and ERK signaling. The analysis of most dysregulated genes and those representative of the majority of categorized genes indicates a strong association between mitochondrial function and cellular processes possibly related to motor neuron degeneration. In conclusion, iPSC-derived motor neurons from motor nerve fibroblasts of sporadic ALS patients may recapitulate key mechanisms of neurodegeneration and may offer an opportunity for translational investigation of sporadic ALS. Large gene profiling of differentiated motor neurons from sporadic ALS patients highlights mitochondrial participation in the establishment of autonomous mechanisms associated with sporadic ALS

  7. Two distinct promoters drive transcription of the human D1A dopamine receptor gene.

    Science.gov (United States)

    Lee, S H; Minowa, M T; Mouradian, M M

    1996-10-11

    The human D1A dopamine receptor gene has a GC-rich, TATA-less promoter located upstream of a small, noncoding exon 1, which is separated from the coding exon 2 by a 116-base pair (bp)-long intron. Serial 3'-deletions of the 5'-noncoding region of this gene, including the intron and 5'-end of exon 2, resulted in 80 and 40% decrease in transcriptional activity of the upstream promoter in two D1A-expressing neuroblastoma cell lines, SK-N-MC and NS20Y, respectively. To investigate the function of this region, the intron and 245 bp at the 5'-end of exon 2 were investigated. Transient expression analyses using various chloramphenicol acetyltransferase constructs showed that the transcriptional activity of the intron is higher than that of the upstream promoter by 12-fold in SK-N-MC cells and by 5.5-fold in NS20Y cells in an orientation-dependent manner, indicating that the D1A intron is a strong promoter. Primer extension and ribonuclease protection assays revealed that transcription driven by the intron promoter is initiated at the junction of intron and exon 2 and at a cluster of nucleotides located 50 bp downstream from this junction. The same transcription start sites are utilized by the chloramphenicol acetyltransferase constructs employed in transfections as well as by the D1A gene expressed within the human caudate. The relative abundance of D1A transcripts originating from the upstream promoter compared with those transcribed from the intron promoter is 1.5-2.9 times in SK-N-MC cells and 2 times in the human caudate. Transcript stability studies in SK-N-MC cells revealed that longer D1A mRNA molecules containing exon 1 are degraded 1.8 times faster than shorter transcripts lacking exon 1. Although gel mobility shift assay could not detect DNA-protein interaction at the D1A intron, competitive co-transfection using the intron as competitor confirmed the presence of trans-acting factors at the intron. These data taken together indicate that the human D1A gene has

  8. 3,3'-Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells.

    Science.gov (United States)

    Gong, Yixuan; Sohn, Heesook; Xue, Ling; Firestone, Gary L; Bjeldanes, Leonard F

    2006-05-01

    Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.

  9. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  10. Discovery of a Regulatory Motif for Human Satellite DNA Transcription in Response to BATF2 Overexpression.

    Science.gov (United States)

    Bai, Xuejia; Huang, Wenqiu; Zhang, Chenguang; Niu, Jing; Ding, Wei

    2016-03-01

    One of the basic leucine zipper transcription factors, BATF2, has been found to suppress cancer growth and migration. However, little is known about the genes downstream of BATF2. HeLa cells were stably transfected with BATF2, then chromatin immunoprecipitation-sequencing was employed to identify the DNA motifs responsive to BATF2. Comprehensive bioinformatics analyses indicated that the most significant motif discovered as TTCCATT[CT]GATTCCATTC[AG]AT was primarily distributed among the chromosome centromere regions and mostly within human type II satellite DNA. Such motifs were able to prime the transcription of type II satellite DNA in a directional and asymmetrical manner. Consistently, satellite II transcription was up-regulated in BATF2-overexpressing cells. The present study provides insight into understanding the role of BATF2 in tumours and the importance of satellite DNA in the maintenance of genomic stability. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  11. Assessing quality and completeness of human transcriptional regulatory pathways on a genome-wide scale

    Directory of Open Access Journals (Sweden)

    Aifantis Iannis

    2011-02-01

    Full Text Available Abstract Background Pathway databases are becoming increasingly important and almost omnipresent in most types of biological and translational research. However, little is known about the quality and completeness of pathways stored in these databases. The present study conducts a comprehensive assessment of transcriptional regulatory pathways in humans for seven well-studied transcription factors: MYC, NOTCH1, BCL6, TP53, AR, STAT1, and RELA. The employed benchmarking methodology first involves integrating genome-wide binding with functional gene expression data to derive direct targets of transcription factors. Then the lists of experimentally obtained direct targets are compared with relevant lists of transcriptional targets from 10 commonly used pathway databases. Results The results of this study show that for the majority of pathway databases, the overlap between experimentally obtained target genes and targets reported in transcriptional regulatory pathway databases is surprisingly small and often is not statistically significant. The only exception is MetaCore pathway database which yields statistically significant intersection with experimental results in 84% cases. Additionally, we suggest that the lists of experimentally derived direct targets obtained in this study can be used to reveal new biological insight in transcriptional regulation and suggest novel putative therapeutic targets in cancer. Conclusions Our study opens a debate on validity of using many popular pathway databases to obtain transcriptional regulatory targets. We conclude that the choice of pathway databases should be informed by solid scientific evidence and rigorous empirical evaluation. Reviewers This article was reviewed by Prof. Wing Hung Wong, Dr. Thiago Motta Venancio (nominated by Dr. L Aravind, and Prof. Geoff J McLachlan.

  12. The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney

    Science.gov (United States)

    O’Brown, Zach K.; Van Nostrand, Eric L.; Higgins, John P.; Kim, Stuart K.

    2015-01-01

    Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney. PMID:26678048

  13. The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pan, Li; Zhang, Yuming; Zhao, Wanlu; Zhou, Xia; Wang, Chunxia; Deng, Fan

    2017-07-01

    Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca 2+ was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02 µM, whereas for NCM460 cells 0.56 µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca 2+ concentration, but decreased GSH concentration in the cells. The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

  14. Distinguishing the Transcription Regulation Patterns in Promoters of Human Genes with Different Function or Evolutionary Age

    KAUST Repository

    Alam, Tanvir

    2012-07-01

    Distinguishing transcription regulatory patterns of different gene groups is a common problem in various bioinformatics studies. In this work we developed a methodology to deal with such a problem based on machine learning techniques. We applied our method to two biologically important problems related to detecting a difference in transcription regulation of: a/ protein-coding and long non-coding RNAs (lncRNAs) in human, as well as b/ a difference between primate-specific and non-primate-specific long non-coding RNAs. Our method is capable to classify RNAs using various regulatory features of genes that transcribe into these RNAs, such as nucleotide frequencies, transcription factor binding sites, de novo sequence motifs, CpG islands, repetitive elements, histone modification marks, and others. Ten-fold cross-validation tests suggest that our model can distinguish protein-coding and non-coding RNAs with accuracy above 80%. Twenty-fold cross-validation tests suggest that our model can distinguish primate-specific from non-primate-specific promoters of lncRNAs with accuracy above 80%. Consequently, we can hypothesize that transcription of the groups of genes mentioned above are regulated by different mechanisms. Feature selection techniques allowed us to reduce the number of features significantly while keeping the accuracy around 80%. Consequently, we can conclude that selected features play significant role in transcription regulation of coding and non-coding genes, as well as primate-specific and non-primate-specific lncRNA genes.

  15. Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

    Science.gov (United States)

    Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

    2017-08-15

    In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. A network of paralogous stress response transcription factors in the human pathogen Candida glabrata.

    Directory of Open Access Journals (Sweden)

    Jawad eMerhej

    2016-05-01

    Full Text Available The yeast Candida glabrata has become the second cause of systemic candidemia in humans. However, relatively few genome-wide studies have been conducted in this organism and our knowledge of its transcriptional regulatory network is quite limited. In the present work, we combined genome-wide chromatin immunoprecipitation (ChIP-seq, transcriptome analyses and DNA binding motif predictions to describe the regulatory interactions of the seven Yap (Yeast AP1 transcription factors of C. glabrata. We described a transcriptional network containing 255 regulatory interactions and 309 potential target genes. We predicted with high confidence the preferred DNA binding sites for 5 of the 7 CgYaps and showed a strong conservation of the Yap DNA binding properties between S. cerevisiae and C. glabrata. We provided reliable functional annotation for 3 of the 7 Yaps and identified for Yap1 and Yap5 a core regulon which is conserved in S. cerevisiae, C. glabrata and C. albicans. We uncovered new roles for CgYap7 in the regulation of iron-sulfur cluster biogenesis, for CgYap1 in the regulation of heme biosynthesis and for CgYap5 in the repression of GRX4 in response to iron starvation. These transcription factors define an interconnected transcriptional network at the cross-roads between redox homeostasis, oxygen consumption and iron metabolism.

  17. Transcriptional profiling of five isolated size-matched stages of human preantral follicles

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Ebbesen, Pernille; Andersen, Claus Yding

    2015-01-01

    Little is known of the early stages of human follicular development and the complex processes that regulate follicular growth. To identify genes of potential importance, we analysed follicle-related transcripts in five populations of isolated size-matched human preantral follicles by microarray...... factors of NOTCH signalling, IGF2, orphan nuclear receptor LRH-1, and homeobox gene HOXA7, indicating potentially important regulatory roles for these genes during early human folliculogenesis. We also found that FSHR mRNA and protein were present in the earliest stages of preantral follicles, whereas LHR...

  18. The mitochondrial membrane potential in human platelets: a sensitive parameter for platelet quality

    NARCIS (Netherlands)

    Verhoeven, Arthur J.; Verhaar, Robin; Gouwerok, Eric G. W.; de Korte, Dirk

    2005-01-01

    BACKGROUND: Deterioration of platelet (PLT) quality during storage is accompanied by an increase in lactate production, indicating a decrease in mitochondrial function. In this study, the optimal conditions under which the fluorescent dye JC-1 can be used to detect changes in mitochondrial function

  19. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

    KAUST Repository

    Schmeier, Sebastian

    2016-10-17

    Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

  20. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

    KAUST Repository

    Schmeier, Sebastian; Alam, Tanvir; Essack, Magbubah; Bajic, Vladimir B.

    2016-01-01

    Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

  1. Transcriptional correlates of disease outcome in anticoagulant-treated non-human primates infected with ebolavirus.

    Directory of Open Access Journals (Sweden)

    Sara Garamszegi

    Full Text Available Ebola virus (EBOV infection in humans and non-human primates (NHPs is highly lethal, and there is limited understanding of the mechanisms associated with pathogenesis and survival. Here, we describe a transcriptomic analysis of NHPs that survived lethal EBOV infection, compared to NHPs that did not survive. It has been previously demonstrated that anticoagulant therapeutics increase the survival rate in EBOV-infected NHPs, and that the characteristic transcriptional profile of immune response changes in anticoagulant-treated NHPs. In order to identify transcriptional signatures that correlate with survival following EBOV infection, we compared the mRNA expression profile in peripheral blood mononuclear cells from EBOV-infected NHPs that received anticoagulant treatment, to those that did not receive treatment. We identified a small set of 20 genes that are highly confident predictors and can accurately distinguish between surviving and non-surviving animals. In addition, we identified a larger predictive signature of 238 genes that correlated with disease outcome and treatment; this latter signature was associated with a variety of host responses, such as the inflammatory response, T cell death, and inhibition of viral replication. Notably, among survival-associated genes were subsets of genes that are transcriptionally regulated by (1 CCAAT/enhancer-binding protein alpha, (2 tumor protein 53, and (3 megakaryoblastic leukemia 1 and myocardin-like protein 2. These pathways merit further investigation as potential transcriptional signatures of host immune response to EBOV infection.

  2. Sp1 and CREB regulate basal transcription of the human SNF2L gene

    International Nuclear Information System (INIS)

    Xia Yu; Jiang Baichun; Zou Yongxin; Gao Guimin; Shang Linshan; Chen Bingxi; Liu Qiji; Gong Yaoqin

    2008-01-01

    Imitation Switch (ISWI) is a member of the SWI2/SNF2 superfamily of ATP-dependent chromatin remodelers, which are involved in multiple nuclear functions, including transcriptional regulation, replication, and chromatin assembly. Mammalian genomes encode two ISWI orthologs, SNF2H and SNF2L. In order to clarify the molecular mechanisms governing the expression of human SNF2L gene, we functionally examined the transcriptional regulation of human SNF2L promoter. Reporter gene assays demonstrated that the minimal SNF2L promoter was located between positions -152 to -86 relative to the transcription start site. In this region we have identified a cAMP-response element (CRE) located at -99 to -92 and a Sp1-binding site at -145 to -135 that play a critical role in regulating basal activity of human SNF2L gene, which were proven by deletion and mutation of specific binding sites, EMSA, and down-regulating Sp1 and CREB via RNAi. This study provides the first insight into the mechanisms that control basal expression of human SNF2L gene

  3. Bisphenol A and Bisphenol S Induce Distinct Transcriptional Profiles in Differentiating Human Primary Preadipocytes.

    Directory of Open Access Journals (Sweden)

    Jonathan G Boucher

    Full Text Available Bisphenol S (BPS is increasingly used as a replacement plasticizer for bisphenol A (BPA but its effects on human health have not been thoroughly examined. Recent evidence indicates that both BPA and BPS induce adipogenesis, although the mechanisms leading to this effect are unclear. In an effort to identify common and distinct mechanisms of action in inducing adipogenesis, transcriptional profiles of differentiating human preadipocytes exposed to BPA or BPS were compared. Human subcutaneous primary preadipocytes were differentiated in the presence of either 25 μM BPA or BPS for 2 and 4 days. Poly-A RNA-sequencing was used to identify differentially expressed genes (DEGs. Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis. BPA-treatment resulted in 472 and 176 DEGs on days 2 and 4, respectively, affecting pathways such as liver X receptor (LXR/retinoid X receptor (RXR activation, hepatic fibrosis and cholestasis. BPS-treatment resulted in 195 and 51 DEGs on days 2 and 4, respectively, revealing enrichment of genes associated with adipogenesis and lipid metabolism including the adipogenesis pathway and cholesterol biosynthesis. Interestingly, the transcription repressor N-CoR was identified as a negative upstream regulator in both BPA- and BPS-treated cells. This study presents the first comparison of BPA- and BPS-induced transcriptional profiles in human differentiating preadipocytes. While we previously showed that BPA and BPS both induce adipogenesis, the results from this study show that BPS affects adipose specific transcriptional changes earlier than BPA, and alters the expression of genes specifically related to adipogenesis and lipid metabolism. The findings provide insight into potential BPS and BPA-mediated mechanisms of action in inducing adipogenesis in human primary preadipocytes.

  4. Human, donkey and cow milk differently affects energy efficiency and inflammatory state by modulating mitochondrial function and gut microbiota.

    Science.gov (United States)

    Trinchese, Giovanna; Cavaliere, Gina; Canani, Roberto Berni; Matamoros, Sebastien; Bergamo, Paolo; De Filippo, Chiara; Aceto, Serena; Gaita, Marcello; Cerino, Pellegrino; Negri, Rossella; Greco, Luigi; Cani, Patrice D; Mollica, Maria Pina

    2015-11-01

    Different nutritional components are able, by modulating mitochondrial function and gut microbiota composition, to influence body composition, metabolic homeostasis and inflammatory state. In this study, we aimed to evaluate the effects produced by the supplementation of different milks on energy balance, inflammatory state, oxidative stress and antioxidant/detoxifying enzyme activities and to investigate the role of the mitochondrial efficiency and the gut microbiota in the regulation of metabolic functions in an animal model. We compared the intake of human milk, gold standard for infant nutrition, with equicaloric supplementation of donkey milk, the best substitute for newborns due to its nutritional properties, and cow milk, the primary marketed product. The results showed a hypolipidemic effect produced by donkey and human milk intake in parallel with enhanced mitochondrial activity/proton leakage. Reduced mitochondrial energy efficiency and proinflammatory signals (tumor necrosis factor α, interleukin-1 and lipopolysaccharide levels) were associated with a significant increase of antioxidants (total thiols) and detoxifying enzyme activities (glutathione-S-transferase, NADH quinone oxidoreductase) in donkey- and human milk-treated animals. The beneficial effects were attributable, at least in part, to the activation of the nuclear factor erythroid-2-related factor-2 pathway. Moreover, the metabolic benefits induced by human and donkey milk may be related to the modulation of gut microbiota. In fact, milk treatments uniquely affected the proportions of bacterial phyla and genera, and we hypothesized that the increased concentration of fecal butyrate in human and donkey milk-treated rats was related to the improved lipid and glucose metabolism and detoxifying activities. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies.

    Science.gov (United States)

    Thaler, David S; Stoeckle, Mark Y

    2016-10-01

    DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648-bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein-encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most - possibly all - synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well-curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.

  6. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

    Science.gov (United States)

    He, Jiuya; Ford, Holly C; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-03-28

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1 , ATP5G2 , and ATP5G3 , encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1 , ATP5G2 , and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

  7. Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate.

    Science.gov (United States)

    Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro

    2013-03-08

    Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

  8. Activation of IGF-1 and insulin signaling pathways ameliorate mitochondrial function and energy metabolism in Huntington's Disease human lymphoblasts.

    Science.gov (United States)

    Naia, Luana; Ferreira, I Luísa; Cunha-Oliveira, Teresa; Duarte, Ana I; Ribeiro, Márcio; Rosenstock, Tatiana R; Laço, Mário N; Ribeiro, Maria J; Oliveira, Catarina R; Saudou, Frédéric; Humbert, Sandrine; Rego, A Cristina

    2015-02-01

    Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Mitochondrial dysfunction associated with energy failure plays an important role in this untreated pathology. In the present work, we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin (at low nM) on signaling and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt, and ERK was largely restored by IGF-1 and, at a less extent, by insulin in HD human lymphoblasts. Importantly, both neurotrophic factors stimulated huntingtin phosphorylation at Ser421 in HD cells. IGF-1 and insulin also rescued energy levels in HD peripheral cells, as evaluated by increased ATP and phosphocreatine, and decreased lactate levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential (Δψm) in HD lymphoblasts, which occurred concomitantly with increased levels of cytochrome c. Indeed, constitutive phosphorylation of huntingtin was able to restore the Δψm in lymphoblasts expressing an abnormal expansion of polyglutamines. HD lymphoblasts further exhibited increased intracellular Ca(2+) levels before and after exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca(2+) accumulation, being the later recovered by IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, the data support an important role for IR/IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts.

  9. Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

    Science.gov (United States)

    Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

    1991-01-01

    Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

  10. Elevated expression and potential roles of human Sp5, a member of Sp transcription factor family, in human cancers

    International Nuclear Information System (INIS)

    Chen Yongxin; Guo Yingqiu; Ge Xijin; Itoh, Hirotaka; Watanabe, Akira; Fujiwara, Takeshi; Kodama, Tatsuhiko; Aburatani, Hiroyuki

    2006-01-01

    In this report, we describe the expression and function of human Sp5, a member of the Sp family of zinc finger transcription factors. Like other family members, the Sp5 protein contains a Cys2His2 zinc finger DNA binding domain at the C-terminus. Our experiments employing Gal4-Sp5 fusion proteins reveal multiple transcriptional domains, including a N-terminal activity domain, an intrinsic repressive element, and a C-terminal synergistic domain. Elevated expression of Sp5 was noted in several human tumors including hepatocellular carcinoma, gastric cancer, and colon cancer. To study the effects of the Sp5 protein on growth properties of human cancer cells and facilitate the identification of its downstream genes, we combined an inducible gene expression system with microarray analysis to screen for its transcriptional targets. Transfer of Sp5 into MCF-7 cells that expressed no detectable endogenous Sp5 protein elicited significant growth promotion activity. Several of the constitutively deregulated genes have been associated with tumorigenesis (CDC25C, CEACAM6, TMPRSS2, XBP1, MYBL1, ABHD2, and CXCL12) and Wnt/β-Catenin signaling pathways (BAMBI, SIX1, IGFBP5, AES, and p21 WAF1 ). This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against human cancers

  11. Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin (ISO) on human bladder cancer cells.

    Science.gov (United States)

    Jiang, Guosong; Huang, Chao; Li, Jingxia; Huang, Haishan; Wang, Jingjing; Li, Yawei; Xie, Fei; Jin, Honglei; Zhu, Junlan; Huang, Chuanshu

    2018-03-08

    There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.

  12. Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli

    OpenAIRE

    Sullivan, Gareth J.; Bridger, Joanna M.; Cuthbert, Andrew P.; Newbold, Robert F.; Bickmore, Wendy A.; McStay, Brian

    2001-01-01

    Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of in...

  13. Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

    Directory of Open Access Journals (Sweden)

    Mueller Nancy

    2005-10-01

    Full Text Available Abstract Background Human T-cell leukemia virus type I (HTLV-I causes adult T-cell leukemia (ATL after a long latent period. Among accessory genes encoded by HTLV-I, the tax gene is thought to play a central role in oncogenesis. However, Tax expression is disrupted by several mechanims including genetic changes of the tax gene, deletion/hypermethylation of 5'-LTR. To clarify the role of epigenetic changes, we analyzed DNA methylation and histone modification in the whole HTLV-I provirus genome. Results The gag, pol and env genes of HTLV-I provirus were more methylated than pX region, whereas methylation of 5'-LTR was variable and 3'-LTR was not methylated at all. In ATL cell lines, complete DNA methylation of 5'-LTR was associated with transcriptional silencing of viral genes. HTLV-I provirus was more methylated in primary ATL cells than in carrier state, indicating the association with disease progression. In seroconvertors, DNA methylation was already observed in internal sequences of provirus just after seroconversion. Taken together, it is speculated that DNA methylation first occurs in the gag, pol and env regions and then extends in the 5' and 3' directions in vivo, and when 5'-LTR becomes methylated, viral transcription is silenced. Analysis of histone modification in the HTLV-I provirus showed that the methylated provirus was associated with hypoacetylation. However, the tax gene transcript could not be detected in fresh ATL cells regardless of hyperacetylated histone H3 in 5'-LTR. The transcription rapidly recovered after in vitro culture in such ATL cells. Conclusion These results showed that epigenetic changes of provirus facilitated ATL cells to evade host immune system by suppressing viral gene transcription. In addition, this study shows the presence of another reversible mechanism that suppresses the tax gene transcription without DNA methylation and hypoacetylated histone.

  14. Effect of DHA and CoenzymeQ10 Against Aβ- and Zinc-Induced Mitochondrial Dysfunction in Human Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Nadia Sadli

    2013-07-01

    Full Text Available Background: Beta-amyloid (Aβ protein is a key factor in the pathogenesis of Alzheimer's disease (AD and it has been reported that mitochondria is involved in the biochemical pathway by which Aβ can lead to neuronal dysfunction. Coenzyme Q10 (CoQ10 is an essential cofactor involved in the mitochondrial electron transport chain and has been suggested as a potential therapeutic agent in AD. Zinc toxicity also affects cellular energy production by decreasing oxygen consumption rate (OCR and ATP turnover in human neuronal cells, which can be restored by the neuroprotective effect of docosahexaenoic acid (DHA. Method: In the present study, using Seahorse XF-24 Metabolic Flux Analysis we investigated the effect of DHA and CoQ10 alone and in combination against Aβ- and zinc-mediated changes in the mitochondrial function of M17 neuroblastoma cell line. Results: Here, we observed that DHA is specifically neuroprotective against zinc-triggered mitochondrial dysfunction, but does not directly affect Aβ neurotoxicity. CoQ10 has shown to be protective against both Aβ- and zinc-induced alterations in mitochondrial function. Conclusion: Our results indicate that DHA and CoQ10 may be useful for the prevention, treatment and management of neurodegenerative diseases such as AD.

  15. Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Dehai Yu

    2017-01-01

    Full Text Available Background. Fetal heart can regenerate to restore its normal anatomy and function in response to injury, but this regenerative capacity is lost within the first week of postnatal life. Although the specific molecular mechanisms remain to be defined, it is presumed that aging of cardiac stem or progenitor cells may contribute to the loss of regenerative potential. Methods. To study this aging-related dysfunction, we cultured mesenchymal stem cells (MSCs from human fetal heart tissues. Senescence was induced by exposing cells to chronic oxidative stress/low serum. Mitochondrial DNA methylation was examined during the period of senescence. Results. Senescent MSCs exhibited flattened and enlarged morphology and were positive for the senescence-associated beta-galactosidase (SA-β-Gal. By scanning the entire mitochondrial genome, we found that four CpG islands were hypomethylated in close association with senescence in MSCs. The mitochondrial COX1 gene, which encodes the main subunit of the cytochrome c oxidase complex and contains the differentially methylated CpG island 4, was upregulated in MSCs in parallel with the onset of senescence. Knockdown of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3B also upregulated COX1 expression and induced cellular senescence in MSCs. Conclusions. This study demonstrates that mitochondrial CpG hypomethylation may serve as a critical biomarker associated with cellular senescence induced by chronic oxidative stress.

  16. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

    Science.gov (United States)

    Nisr, Raid B; Affourtit, Charles

    2014-02-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

  17. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation☆

    Science.gov (United States)

    Nisr, Raid B.; Affourtit, Charles

    2014-01-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. PMID:24212054

  18. Jaceosidin Induces Apoptosis in Human Ovary Cancer Cells through Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Wen Lv

    2008-01-01

    Full Text Available We examined the antiproliferation effect of Jaceosidin (4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV-3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose polymerase (PARP, while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells. Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.

  19. Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

    Science.gov (United States)

    Nagle, Nano; Ballantyne, Kaye N; van Oven, Mannis; Tyler-Smith, Chris; Xue, Yali; Wilcox, Stephen; Wilcox, Leah; Turkalov, Rust; van Oorschot, Roland A H; van Holst Pellekaan, Sheila; Schurr, Theodore G; McAllister, Peter; Williams, Lesley; Kayser, Manfred; Mitchell, R John

    2017-03-01

    Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.

  20. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

    International Nuclear Information System (INIS)

    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee

    2009-01-01

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with 137 Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H 2 O 2 -treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H 2 O 2 -treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells

  1. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2009-06-15

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with {sup 137}Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H{sub 2}O{sub 2}-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H{sub 2}O{sub 2}-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

  2. Dissecting interferon-induced transcriptional programs in human peripheral blood cells.

    Directory of Open Access Journals (Sweden)

    Simon J Waddell

    2010-03-01

    Full Text Available Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles.We used human cDNA microarrays to (1 compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs elicited by 6 major mediators of the immune response: interferons alpha, beta, omega and gamma, IL12 and TNFalpha; and (2 characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings.

  3. Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

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    Groscurth Peter

    2007-06-01

    Full Text Available Abstract Background Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522 were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. Results In this study we investigated generation of granulysin in lymphokine activated killer (LAK cells and antigen (Listeria specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation. Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. Conclusion This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.

  4. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Benoît Couvigny

    Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  5. The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.

    Directory of Open Access Journals (Sweden)

    Maléne E Lindholm

    2016-09-01

    Full Text Available Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

  6. Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

    Science.gov (United States)

    Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth; Dutch, Rebecca Ellis

    2017-12-15

    Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to

  7. Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors | Office of Cancer Genomics

    Science.gov (United States)

    Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells.

  8. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  9. High DNA melting temperature predicts transcription start site location in human and mouse.

    LENUS (Irish Health Repository)

    Dineen, David G

    2009-12-01

    The accurate computational prediction of transcription start sites (TSS) in vertebrate genomes is a difficult problem. The physicochemical properties of DNA can be computed in various ways and a many combinations of DNA features have been tested in the past for use as predictors of transcription. We looked in detail at melting temperature, which measures the temperature, at which two strands of DNA separate, considering the cooperative nature of this process. We find that peaks in melting temperature correspond closely to experimentally determined transcription start sites in human and mouse chromosomes. Using melting temperature alone, and with simple thresholding, we can predict TSS with accuracy that is competitive with the most accurate state-of-the-art TSS prediction methods. Accuracy is measured using both experimentally and manually determined TSS. The method works especially well with CpG island containing promoters, but also works when CpG islands are absent. This result is clear evidence of the important role of the physical properties of DNA in the process of transcription. It also points to the importance for TSS prediction methods to include melting temperature as prior information.

  10. Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

    Science.gov (United States)

    Vadrot, Nathalie; Ghanem, Sarita; Braut, Françoise; Gavrilescu, Laura; Pilard, Nathalie; Mansouri, Abdellah; Moreau, Richard; Reyl-Desmars, Florence

    2012-01-01

    During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

  11. Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

    Directory of Open Access Journals (Sweden)

    Nathalie Vadrot

    Full Text Available During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α targets hepatocytes and induces abnormal reactive oxygen species (ROS production responsible for mitochondrial DNA (mtDNA alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC was used to measure the rapid (10 min and transient TNF-α induced increase in ROS production (168±15%. A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM. In addition, mitochondrial D-loop immunoprecipitation (mtDIP revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

  12. Low nucleosome occupancy is encoded around functional human transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Daenen Floris

    2008-07-01

    Full Text Available Abstract Background Transcriptional regulation of genes in eukaryotes is achieved by the interactions of multiple transcription factors with arrays of transcription factor binding sites (TFBSs on DNA and with each other. Identification of these TFBSs is an essential step in our understanding of gene regulatory networks, but computational prediction of TFBSs with either consensus or commonly used stochastic models such as Position-Specific Scoring Matrices (PSSMs results in an unacceptably high number of hits consisting of a few true functional binding sites and numerous false non-functional binding sites. This is due to the inability of the models to incorporate higher order properties of sequences including sequences surrounding TFBSs and influencing the positioning of nucleosomes and/or the interactions that might occur between transcription factors. Results Significant improvement can be expected through the development of a new framework for the modeling and prediction of TFBSs that considers explicitly these higher order sequence properties. It would be particularly interesting to include in the new modeling framework the information present in the nucleosome positioning sequences (NPSs surrounding TFBSs, as it can be hypothesized that genomes use this information to encode the formation of stable nucleosomes over non-functional sites, while functional sites have a more open chromatin configuration. In this report we evaluate the usefulness of the latter feature by comparing the nucleosome occupancy probabilities around experimentally verified human TFBSs with the nucleosome occupancy probabilities around false positive TFBSs and in random sequences. Conclusion We present evidence that nucleosome occupancy is remarkably lower around true functional human TFBSs as compared to non-functional human TFBSs, which supports the use of this feature to improve current TFBS prediction approaches in higher eukaryotes.

  13. Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway.

    Science.gov (United States)

    Koshikawa, Nobuko; Hayashi, Jun-Ichi; Nakagawara, Akira; Takenaga, Keizo

    2009-11-27

    Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

  14. Selective toxicity of persian gulf sea cucumber holothuria parva on human chronic lymphocytic leukemia b lymphocytes by direct mitochondrial targeting.

    Science.gov (United States)

    Salimi, Ahmad; Motallebi, Abbasali; Ayatollahi, Maryam; Seydi, Enayatollah; Mohseni, Ali Reza; Nazemi, Melika; Pourahmad, Jalal

    2017-04-01

    Natural products isolated from marine environment are well known for their pharmacodynamic potential in diversity of disease treatments such as cancer or inflammatory conditions. Sea cucumbers are one of the marine animals of the phylum Echinoderm. Many studies have shown that the sea cucumber contains antioxidants and anti-cancer compounds. Chronic lymphocytic leukemia (CLL) is a disease characterized by the relentless accumulation of CD5 + B lymphocytes. CLL is the most common leukemia in adults, about 25-30% of all leukemias. In this study B lymphocytes and their mitochondria (cancerous and non-cancerous) were obtained from peripheral blood of human subjects and B lymphocyte cytotoxicity assay, and caspase 3 activation along with mitochondrial upstream events of apoptosis signaling including reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential (MMP) and mitochondrial swelling were determined following the addition of Holothuria parva extract to both cancerous and non-cancerous B lymphocytes and their mitochondria. Our in vitro finding showed that mitochondrial ROS formation, MMP collapse, and mitochondrial swelling and cytochrome c release were significantly (P < 0.05) increased after addition of different concentrations of H. parva only in cancerous BUT NOT normal non-cancerous mitochondria. Consistently, different concentrations of H. parva significantly (P < 0.05) increased cytotoxicity and caspase 3 activation only in cancerous BUT NOT normal non-cancerous B lymphocytes. These results showed that H. parva methanolic extract has a selective mitochondria mediated apoptotic effect on chronic lymphocytic leukemia B lymphocytes hence may be promising in the future anticancer drug development for treatment of CLL. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1158-1169, 2017. © 2016 Wiley Periodicals, Inc.

  15. Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Arwa S Kathiria

    Full Text Available Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα, both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB, which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells

  16. Nutritional Ketosis and Mitohormesis: Potential Implications for Mitochondrial Function and Human Health

    Science.gov (United States)

    Villamena, Frederick A.

    2018-01-01

    Impaired mitochondrial function often results in excessive production of reactive oxygen species (ROS) and is involved in the etiology of many chronic diseases, including cardiovascular disease, diabetes, neurodegenerative disorders, and cancer. Moderate levels of mitochondrial ROS, however, can protect against chronic disease by inducing upregulation of mitochondrial capacity and endogenous antioxidant defense. This phenomenon, referred to as mitohormesis, is induced through increased reliance on mitochondrial respiration, which can occur through diet or exercise. Nutritional ketosis is a safe and physiological metabolic state induced through a ketogenic diet low in carbohydrate and moderate in protein. Such a diet increases reliance on mitochondrial respiration and may, therefore, induce mitohormesis. Furthermore, the ketone β-hydroxybutyrate (BHB), which is elevated during nutritional ketosis to levels no greater than those resulting from fasting, acts as a signaling molecule in addition to its traditionally known role as an energy substrate. BHB signaling induces adaptations similar to mitohormesis, thereby expanding the potential benefit of nutritional ketosis beyond carbohydrate restriction. This review describes the evidence supporting enhancement of mitochondrial function and endogenous antioxidant defense in response to nutritional ketosis, as well as the potential mechanisms leading to these adaptations. PMID:29607218

  17. Luffa echinata Roxb. Induces Human Colon Cancer Cell (HT-29 Death by Triggering the Mitochondrial Apoptosis Pathway

    Directory of Open Access Journals (Sweden)

    Yan Yu

    2012-05-01

    Full Text Available The antiproliferative properties and cell death mechanism induced by the extract of the fruits of Luffa echinata Roxb. (LER were investigated. The methanolic extract of LER inhibited the proliferation of human colon cancer cells (HT-29 in both dose-dependent and time-dependent manners and caused a significant increase in the population of apoptotic cells. In addition, obvious shrinkage and destruction of the monolayer were observed in LER-treated cells, but not in untreated cells. Analysis of the cell cycle after treatment of HT-29 cells with various concentrations indicated that LER extracts inhibited the cellular proliferation of HT-29 cells via G2/M phase arrest of the cell cycle. The Reactive oxygen species (ROS level determination revealed that LER extracts induced apoptotic cell death via ROS generation. In addition, LER treatment led to a rapid drop in mitochondrial membrane potential (MMP as a decrease in fluorescence. The transcripts of several apoptosis-related genes were investigated by RT-PCR analysis. The caspase-3 transcripts of HT-29 cells significantly accumulated and the level of Bcl-XL mRNA was decreased after treatment with LER extract. Furthermore, the ratio of mitochondria-dependent apoptosis genes (Bax and Bcl-2 was sharply increased from 1.6 to 54.1. These experiments suggest that LER has anticancer properties via inducing the apoptosis in colon cancer cells, which provided the impetus for further studies on the therapeutic potential of LER against human colon carcinoma.

  18. Variations in mitochondrial DNA and gene transcription in freezing-tolerant larvae of Eurosta solidaginis (Diptera: Tephritidae) and Gynaephora groenlandica (Lepidoptera: Lymantriidae).

    Science.gov (United States)

    Levin, D B; Danks, H V; Barber, S A

    2003-06-01

    Respiration, mitochondrial (mt)DNA content, and mitochondrial-specific RNA expression in fat body cells from active and cold-adapted larvae of the goldenrod gall fly, Eurosta solidaginis, and the Arctic woolly bear caterpillar, Gynaephora groenlandica, were compared. Reduced amounts of mtDNA were observed in cold-adapted larvae of both E. solidaginis and G. groenlandica collected in fall or winter, compared with summer-collected larvae. mtDNA increased to levels similar to those of summer-collected larvae after incubation at 10 degrees C or 15 degrees C for 5 h. Mitochondrial-specific RNAs (COI and 16S) were observed in fat body cells of both active and cold-adapted E. solidaginis larvae. Our results suggest that mitochondrial proteins required for respiration may be restored rapidly from stable RNAs present in overwintering larvae.

  19. Human BLCAP transcript: new editing events in normal and cancerous tissues.

    Science.gov (United States)

    Galeano, Federica; Leroy, Anne; Rossetti, Claudia; Gromova, Irina; Gautier, Philippe; Keegan, Liam P; Massimi, Luca; Di Rocco, Concezio; O'Connell, Mary A; Gallo, Angela

    2010-07-01

    Bladder cancer-associated protein (BLCAP) is a highly conserved protein among species, and it is considered a novel candidate tumor suppressor gene originally identified from human bladder carcinoma. However, little is known about the regulation or the function of this protein. Here, we show that the human BLCAP transcript undergoes multiple A-to-I editing events. Some of the new editing events alter the highly conserved amino terminus of the protein creating alternative protein isoforms by changing the genetically coded amino acids. We found that both ADAR1 and ADAR2-editing enzymes cooperate to edit this transcript and that different tissues displayed distinctive ratios of edited and unedited BLCAP transcripts. Moreover, we observed a general decrease in BLCAP-editing level in astrocytomas, bladder cancer and colorectal cancer when compared with the related normal tissues. The newly identified editing events, found to be downregulated in cancers, could be useful for future studies as a diagnostic tool to distinguish malignancies or epigenetic changes in different tumors.

  20. Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-12-15

    Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

  1. Mutations in the Caenorhabditis elegans orthologs of human genes required for mitochondrial tRNA modification cause similar electron transport chain defects but different nuclear responses.

    Science.gov (United States)

    Navarro-González, Carmen; Moukadiri, Ismaïl; Villarroya, Magda; López-Pascual, Ernesto; Tuck, Simon; Armengod, M-Eugenia

    2017-07-01

    Several oxidative phosphorylation (OXPHOS) diseases are caused by defects in the post-transcriptional modification of mitochondrial tRNAs (mt-tRNAs). Mutations in MTO1 or GTPBP3 impair the modification of the wobble uridine at position 5 of the pyrimidine ring and cause heart failure. Mutations in TRMU affect modification at position 2 and cause liver disease. Presently, the molecular basis of the diseases and why mutations in the different genes lead to such different clinical symptoms is poorly understood. Here we use Caenorhabditis elegans as a model organism to investigate how defects in the TRMU, GTPBP3 and MTO1 orthologues (designated as mttu-1, mtcu-1, and mtcu-2, respectively) exert their effects. We found that whereas the inactivation of each C. elegans gene is associated with a mild OXPHOS dysfunction, mutations in mtcu-1 or mtcu-2 cause changes in the expression of metabolic and mitochondrial stress response genes that are quite different from those caused by mttu-1 mutations. Our data suggest that retrograde signaling promotes defect-specific metabolic reprogramming, which is able to rescue the OXPHOS dysfunction in the single mutants by stimulating the oxidative tricarboxylic acid cycle flux through complex II. This adaptive response, however, appears to be associated with a biological cost since the single mutant worms exhibit thermosensitivity and decreased fertility and, in the case of mttu-1, longer reproductive cycle. Notably, mttu-1 worms also exhibit increased lifespan. We further show that mtcu-1; mttu-1 and mtcu-2; mttu-1 double mutants display severe growth defects and sterility. The animal models presented here support the idea that the pathological states in humans may initially develop not as a direct consequence of a bioenergetic defect, but from the cell's maladaptive response to the hypomodification status of mt-tRNAs. Our work highlights the important association of the defect-specific metabolic rewiring with the pathological phenotype

  2. High Glucose-Induced Oxidative Stress Increases the Copy Number of Mitochondrial DNA in Human Mesangial Cells

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    Ghada Al-Kafaji

    2013-01-01

    Full Text Available Oxidative damage to mitochondrial DNA (mtDNA has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells cultured in 5 mM glucose, 25 mM glucose, and mannitol (osmotic control, as well as in cells cultured in 25 mM glucose in the presence and absence of 200 μM MnTBAP. Intracellular ROS was assessed by confocal microscopy and flow cytometry in human mesangial cells. The copy number of mtDNA was significantly increased when human mesangial cells were incubated with 25 mM glucose compared to 5 mM glucose and mannitol. In addition, 25 mM glucose rapidly generated ROS in the cells, which was not detected in 5 mM glucose. Furthermore, mtDNA copy number was significantly decreased and maintained to normal following treatment of cells with 25 mM glucose and MnTBAP compared to 25 mM glucose alone. Inclusion of MnTBAP during 25 mM glucose incubation inhibited mitochondrial superoxide in human mesangial cells. Increased mtDNA copy number in human mesangial cells by high glucose could contribute to increased mitochondrial superoxide, and prevention of mtDNA copy number could have potential in retarding the development of diabetic nephropathy.

  3. Transcriptional profiles of supragranular-enriched genes associate with corticocortical network architecture in the human brain.

    Science.gov (United States)

    Krienen, Fenna M; Yeo, B T Thomas; Ge, Tian; Buckner, Randy L; Sherwood, Chet C

    2016-01-26

    The human brain is patterned with disproportionately large, distributed cerebral networks that connect multiple association zones in the frontal, temporal, and parietal lobes. The expansion of the cortical surface, along with the emergence of long-range connectivity networks, may be reflected in changes to the underlying molecular architecture. Using the Allen Institute's human brain transcriptional atlas, we demonstrate that genes particularly enriched in supragranular layers of the human cerebral cortex relative to mouse distinguish major cortical classes. The topography of transcriptional expression reflects large-scale brain network organization consistent with estimates from functional connectivity MRI and anatomical tracing in nonhuman primates. Microarray expression data for genes preferentially expressed in human upper layers (II/III), but enriched only in lower layers (V/VI) of mouse, were cross-correlated to identify molecular profiles across the cerebral cortex of postmortem human brains (n = 6). Unimodal sensory and motor zones have similar molecular profiles, despite being distributed across the cortical mantle. Sensory/motor profiles were anticorrelated with paralimbic and certain distributed association network profiles. Tests of alternative gene sets did not consistently distinguish sensory and motor regions from paralimbic and association regions: (i) genes enriched in supragranular layers in both humans and mice, (ii) genes cortically enriched in humans relative to nonhuman primates, (iii) genes related to connectivity in rodents, (iv) genes associated with human and mouse connectivity, and (v) 1,454 gene sets curated from known gene ontologies. Molecular innovations of upper cortical layers may be an important component in the evolution of long-range corticocortical projections.

  4. Metabolites from invasive pests inhibit mitochondrial complex II: A potential strategy for the treatment of human ovarian carcinoma?

    Energy Technology Data Exchange (ETDEWEB)

    Ferramosca, Alessandra, E-mail: alessandra.ferramosca@unisalento.it [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce (Italy); Conte, Annalea; Guerra, Flora; Felline, Serena [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce (Italy); Rimoli, Maria Grazia [Dipartimento di Farmacia, Università di Napoli Federico II, Napoli (Italy); Mollo, Ernesto [Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli (Italy); Zara, Vincenzo [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce (Italy); Terlizzi, Antonio [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce (Italy); Stazione Zoologica Anton Dohrn, Napoli (Italy)

    2016-05-13

    The red pigment caulerpin, a secondary metabolite from the marine invasive green algae Caulerpa cylindracea can be accumulated and transferred along the trophic chain, with detrimental consequences on biodiversity and ecosystem functioning. Despite increasing research efforts to understand how caulerpin modifies fish physiology, little is known on the effects of algal metabolites on mammalian cells. Here we report for the first time the mitochondrial targeting activity of both caulerpin, and its closely related derivative caulerpinic acid, by using as experimental model rat liver mitochondria, a system in which bioenergetics mechanisms are not altered. Mitochondrial function was tested by polarographic and spectrophotometric methods. Both compounds were found to selectively inhibit respiratory complex II activity, while complexes I, III, and IV remained functional. These results led us to hypothesize that both algal metabolites could be used as antitumor agents in cell lines with defects in mitochondrial complex I. Ovarian cancer cisplatin-resistant cells are a good example of cell lines with a defective complex I function on which these molecules seem to have a toxic effect on proliferation. This provided novel insight toward the potential use of metabolites from invasive Caulerpa species for the treatment of human ovarian carcinoma cisplatin-resistant cells. -- Highlights: •Novel insight toward the potential use of the algal metabolites for the treatment of human diseases. •Caulerpin and caulerpinic acid inhibit respiratory complex II activity. •Both algal metabolites could be used as antitumor agents in ovarian cancer cisplatin-resistant cells.

  5. Metabolites from invasive pests inhibit mitochondrial complex II: A potential strategy for the treatment of human ovarian carcinoma?

    International Nuclear Information System (INIS)

    Ferramosca, Alessandra; Conte, Annalea; Guerra, Flora; Felline, Serena; Rimoli, Maria Grazia; Mollo, Ernesto; Zara, Vincenzo; Terlizzi, Antonio

    2016-01-01

    The red pigment caulerpin, a secondary metabolite from the marine invasive green algae Caulerpa cylindracea can be accumulated and transferred along the trophic chain, with detrimental consequences on biodiversity and ecosystem functioning. Despite increasing research efforts to understand how caulerpin modifies fish physiology, little is known on the effects of algal metabolites on mammalian cells. Here we report for the first time the mitochondrial targeting activity of both caulerpin, and its closely related derivative caulerpinic acid, by using as experimental model rat liver mitochondria, a system in which bioenergetics mechanisms are not altered. Mitochondrial function was tested by polarographic and spectrophotometric methods. Both compounds were found to selectively inhibit respiratory complex II activity, while complexes I, III, and IV remained functional. These results led us to hypothesize that both algal metabolites could be used as antitumor agents in cell lines with defects in mitochondrial complex I. Ovarian cancer cisplatin-resistant cells are a good example of cell lines with a defective complex I function on which these molecules seem to have a toxic effect on proliferation. This provided novel insight toward the potential use of metabolites from invasive Caulerpa species for the treatment of human ovarian carcinoma cisplatin-resistant cells. -- Highlights: •Novel insight toward the potential use of the algal metabolites for the treatment of human diseases. •Caulerpin and caulerpinic acid inhibit respiratory complex II activity. •Both algal metabolites could be used as antitumor agents in ovarian cancer cisplatin-resistant cells.

  6. Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

    OpenAIRE

    In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A

    1997-01-01

    Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, delet...

  7. Substrate availability and transcriptional regulation of metabolic genes in human skeletal muscle during recovery from exercise

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Osada, Takuya; Andersen, Lisbeth Tingsted

    2005-01-01

    before exercise and 2, 5, 8, and 24 hours after exercise. Muscle glycogen was restored to near resting levels within 5 hours in the HC trial, but remained depressed through 24 hours in the LC trial. During the 2- to 8-hour recovery period, leg glucose uptake was 5- to 15-fold higher with HC ingestion......In skeletal muscle of humans, transcription of several metabolic genes is transiently induced during recovery from exercise when no food is consumed. To determine the potential influence of substrate availability on the transcriptional regulation of metabolic genes during recovery from exercise, 9...... male subjects (aged 22-27) completed 75 minutes of cycling exercise at 75% V¿o2max on 2 occasions, consuming either a high-carbohydrate (HC) or low-carbohydrate (LC) diet during the subsequent 24 hours of recovery. Nuclei were isolated and tissue frozen from vastus lateralis muscle biopsies obtained...

  8. Genome-wide transcriptional profiling of human glioblastoma cells in response to ITE treatment.

    Science.gov (United States)

    Kang, Bo; Zhou, Yanwen; Zheng, Min; Wang, Ying-Jie

    2015-09-01

    A ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is recently revealed to play a key role in embryogenesis and tumorigenesis (Feng et al. [1], Safe et al. [2]) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Song et al. [3]) is an endogenous AhR ligand that possesses anti-tumor activity. In order to gain insights into how ITE acts via the AhR in embryogenesis and tumorigenesis, we analyzed the genome-wide transcriptional profiles of the following three groups of cells: the human glioblastoma U87 parental cells, U87 tumor sphere cells treated with vehicle (DMSO) and U87 tumor sphere cells treated with ITE. Here, we provide the details of the sample gathering strategy and show the quality controls and the analyses associated with our gene array data deposited into the Gene Expression Omnibus (GEO) under the accession code of GSE67986.

  9. Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

    International Nuclear Information System (INIS)

    Reed, Jason; Hsueh, Carlin; Gimzewski, James K; Mishra, Bud

    2008-01-01

    We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing

  10. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  11. Expression of the Transcription Factor E4BP4 in Human Basophils

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Gohr, Maria; Poulsen, Lars Kærgaard

    2014-01-01

    Rationale The cytokine IL-3 plays an important role for human basophil development, function and survival. IL-3 is also reported to induce the expression of the transcription factor E4BP4, but it is not known whether E4BP4 is expressed in basophils and influences basophil responsiveness. The aim...... by Alcian blue. RNA was extracted (0.005-0.02 µg RNA from 0.5 - 1 x 106 cells), and the corresponding cDNA analyzed by real-time PCR where E4BP4 expression was calculated as 2-(CT(E4BP4) - CT(β-actin)). E4BP4 protein expression was visualized in basophil lysates (107 cells/ml) by Western blot followed...... the transcription factor E4BP4 which might have an impact on basophil histamine release....

  12. Plastic downregulation of the transcriptional repressor BCL6 during maturation of human dendritic cells

    International Nuclear Information System (INIS)

    Pantano, Serafino; Jarrossay, David; Saccani, Simona; Bosisio, Daniela; Natoli, Gioacchino

    2006-01-01

    Dendritic cell (DC) maturation links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. Here, we describe an as yet unrecognized modulator of human DC maturation, the transcriptional repressor BCL6. We found that both myeloid and plasmacytoid DCs constitutively express BCL6, which is rapidly downregulated following maturation triggered by selected stimuli. Both in unstimulated and maturing DCs, control of BCL6 protein levels reflects the convergence of several mechanisms regulating BCL6 stability, mRNA transcription and nuclear export. By regulating the induction of several genes implicated in the immune response, including inflammatory cytokines, chemokines and survival genes, BCL6 may represent a pivotal modulator of the afferent branch of the immune response

  13. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major

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    Rodrigo Lombraña

    2016-08-01

    Full Text Available Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs. Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

  14. The complete mitochondrial genomes for three Toxocara species of human and animal health significance

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    Wu Xiang-Yun

    2008-05-01

    Full Text Available Abstract Background Studying mitochondrial (mt genomics has important implications for various fundamental areas, including mt biochemistry, physiology and molecular biology. In addition, mt genome sequences have provided useful markers for investigating population genetic structures, systematics and phylogenetics of organisms. Toxocara canis, Toxocara cati and Toxocara malaysiensis cause significant health problems in animals and humans. Although they are of importance in human and animal health, no information on the mt genomes for any of Toxocara species is available. Results The sizes of the entire mt genome are 14,322 bp for T. canis, 14029 bp for T. cati and 14266 bp for T. malaysiensis, respectively. These circular genomes are amongst the largest reported to date for all secernentean nematodes. Their relatively large sizes relate mainly to an increased length in the AT-rich region. The mt genomes of the three Toxocara species all encode 12 proteins, two ribosomal RNAs and 22 transfer RNA genes, but lack the ATP synthetase subunit 8 gene, which is consistent with all other species of Nematode studied to date, with the exception of Trichinella spiralis. All genes are transcribed in the same direction and have a nucleotide composition high in A and T, but low in G and C. The contents of A+T of the complete genomes are 68.57% for T. canis, 69.95% for T. cati and 68.86% for T. malaysiensis, among which the A+T for T. canis is the lowest among all nematodes studied to date. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. The mt genome structures for three Toxocara species, including genes and non-coding regions, are in the same order as for Ascaris suum and Anisakis simplex, but differ from Ancylostoma duodenale, Necator americanus and Caenorhabditis elegans only in the location of the AT-rich region, whereas there are substantial differences when compared with Onchocerca volvulus

  15. Analysis of transcript and protein overlap in a human osteosarcoma cell line

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    Emanuelsson Olof

    2010-12-01

    Full Text Available Abstract Background An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC and immunofluorescence microscopy (IF. Results A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2% were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies. Conclusion This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.

  16. Telmisartan enhances mitochondrial activity and alters cellular functions in human coronary artery endothelial cells via AMP-activated protein kinase pathway.

    Science.gov (United States)

    Kurokawa, Hirofumi; Sugiyama, Seigo; Nozaki, Toshimitsu; Sugamura, Koichi; Toyama, Kensuke; Matsubara, Junichi; Fujisue, Koichiro; Ohba, Keisuke; Maeda, Hirofumi; Konishi, Masaaki; Akiyama, Eiichi; Sumida, Hitoshi; Izumiya, Yasuhiro; Yasuda, Osamu; Kim-Mitsuyama, Shokei; Ogawa, Hisao

    2015-04-01

    Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. Telmisartan is a unique angiotensin II type I receptor blocker that has been shown to prevent cardiovascular events in high risk patients. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. This study assessed whether telmisartan enhances mitochondrial function and alters cellular functions via AMPK in human coronary artery endothelial cells (HCAECs). In cultured HCAECs, telmisartan significantly enhanced mitochondrial activity assessed by mitochondrial reductase activity and intracellular ATP production and increased the expression of mitochondria related genes. Telmisartan prevented cellular senescence and exhibited the anti-apoptotic and pro-angiogenic properties. The expression of genes related anti-oxidant and pro-angiogenic properties were increased by telmisartan. Telmisartan increased endothelial NO synthase and AMPK phosphorylation. Peroxisome proliferator-activated receptor gamma signaling was not involved in telmisartan-induced improvement of mitochondrial function. All of these effects were abolished by inhibition of AMPK. Telmisartan enhanced mitochondrial activity and exhibited anti-senescence effects and improving endothelial function through AMPK in HCAECs. Telmisartan could provide beneficial effects on vascular diseases via enhancement of mitochondrial activity and modulating endothelial function through AMPK activation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Mitochondrial lineage M1 traces an early human backflow to Africa.

    Science.gov (United States)

    González, Ana M; Larruga, José M; Abu-Amero, Khaled K; Shi, Yufei; Pestano, José; Cabrera, Vicente M

    2007-07-09

    The out of Africa hypothesis has gained generalized consensus. However, many specific questions remain unsettled. To know whether the two M and N macrohaplogroups that colonized Eurasia were already present in Africa before the exit is puzzling. It has been proposed that the east African clade M1 supports a single origin of haplogroup M in Africa. To test the validity of that hypothesis, the phylogeographic analysis of 13 complete mitochondrial DNA (mtDNA) sequences and 261 partial sequences belonging to haplogroup M1 was carried out. The coalescence age of the African haplogroup M1 is younger than those for other M Asiatic clades. In contradiction to the hypothesis of an eastern Africa origin for modern human expansions out of Africa, the most ancestral M1 lineages have been found in Northwest Africa and in the Near East, instead of in East Africa. The M1 geographic distribution and the relative ages of its different subclades clearly correlate with those of haplogroup U6, for which an Eurasian ancestor has been demonstrated. This study provides evidence that M1, or its ancestor, had an Asiatic origin. The earliest M1 expansion into Africa occurred in northwestern instead of eastern areas; this early spread reached the Iberian Peninsula even affecting the Basques. The majority of the M1a lineages found outside and inside Africa had a more recent eastern Africa origin. Both western and eastern M1 lineages participated in the Neolithic colonization of the Sahara. The striking parallelism between subclade ages and geographic distribution of M1 and its North African U6 counterpart strongly reinforces this scenario. Finally, a relevant fraction of M1a lineages present today in the European Continent and nearby islands possibly had a Jewish instead of the commonly proposed Arab/Berber maternal ascendance.

  18. Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

    Science.gov (United States)

    Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

    2017-10-15

    Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

  19. Mitochondrial lineage M1 traces an early human backflow to Africa

    Directory of Open Access Journals (Sweden)

    Pestano José

    2007-07-01

    Full Text Available Abstract Background The out of Africa hypothesis has gained generalized consensus. However, many specific questions remain unsettled. To know whether the two M and N macrohaplogroups that colonized Eurasia were already present in Africa before the exit is puzzling. It has been proposed that the east African clade M1 supports a single origin of haplogroup M in Africa. To test the validity of that hypothesis, the phylogeographic analysis of 13 complete mitochondrial DNA (mtDNA sequences and 261 partial sequences belonging to haplogroup M1 was carried out. Results The coalescence age of the African haplogroup M1 is younger than those for other M Asiatic clades. In contradiction to the hypothesis of an eastern Africa origin for modern human expansions out of Africa, the most ancestral M1 lineages have been found in Northwest Africa and in the Near East, instead of in East Africa. The M1 geographic distribution and the relative ages of its different subclades clearly correlate with those of haplogroup U6, for which an Eurasian ancestor has been demonstrated. Conclusion This study provides evidence that M1, or its ancestor, had an Asiatic origin. The earliest M1 expansion into Africa occurred in northwestern instead of eastern areas; this early spread reached the Iberian Peninsula even affecting the Basques. The majority of the M1a lineages found outside and inside Africa had a more recent eastern Africa origin. Both western and eastern M1 lineages participated in the Neolithic colonization of the Sahara. The striking parallelism between subclade ages and geographic distribution of M1 and its North African U6 counterpart strongly reinforces this scenario. Finally, a relevant fraction of M1a lineages present today in the European Continent and nearby islands possibly had a Jewish instead of the commonly proposed Arab/Berber maternal ascendance.

  20. Transcriptional and epigenetic regulation of KIAA1199 gene expression in human breast cancer.

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    Cem Kuscu

    Full Text Available Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension study with 5'RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1, Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.

  1. Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy

    DEFF Research Database (Denmark)

    Suetta, Charlotte Arneboe; Frandsen, Ulrik; Jensen, Line

    2012-01-01

    Important insights concerning the molecular basis of skeletal muscle disuse-atrophy and aging related muscle loss have been obtained in cell culture and animal models, but these regulatory signaling pathways have not previously been studied in aging human muscle. In the present study, muscle...... atrophy was induced by immobilization in healthy old and young individuals to study the time-course and transcriptional factors underlying human skeletal muscle atrophy. The results reveal that irrespectively of age, mRNA expression levels of MuRF-1 and Atrogin-1 increased in the very initial phase (2......-4 days) of human disuse-muscle atrophy along with a marked reduction in PGC-1α and PGC-1β (1-4 days) and a ∼10% decrease in myofiber size (4 days). Further, an age-specific decrease in Akt and S6 phosphorylation was observed in young muscle within the first days (1-4 days) of immobilization. In contrast...

  2. Is human blood a good surrogate for brain tissue in transcriptional studies?

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    van den Berg Leonard H

    2010-10-01

    Full Text Available Abstract Background Since human brain tissue is often unavailable for transcriptional profiling studies, blood expression data is frequently used as a substitute. The underlying hypothesis in such studies is that genes expressed in brain tissue leave a transcriptional footprint in blood. We tested this hypothesis by relating three human brain expression data sets (from cortex, cerebellum and caudate nucleus to two large human blood expression data sets (comprised of 1463 individuals. Results We found mean expression levels were weakly correlated between the brain and blood data (r range: [0.24,0.32]. Further, we tested whether co-expression relationships were preserved between the three brain regions and blood. Only a handful of brain co-expression modules showed strong evidence of preservation and these modules could be combined into a single large blood module. We also identified highly connected intramodular "hub" genes inside preserved modules. These preserved intramodular hub genes had the following properties: first, their expression levels tended to be significantly more heritable than those from non-preserved intramodular hub genes (p -90; second, they had highly significant positive correlations with the following cluster of differentiation genes: CD58, CD47, CD48, CD53 and CD164; third, a significant number of them were known to be involved in infection mechanisms, post-transcriptional and post-translational modification and other basic processes. Conclusions Overall, we find transcriptome organization is poorly preserved between brain and blood. However, the subset of preserved co-expression relationships characterized here may aid future efforts to identify blood biomarkers for neurological and neuropsychiatric diseases when brain tissue samples are unavailable.

  3. Preclinical evaluation of transcriptional targeting strategy for human hepatocellular carcinoma in an orthotopic xenograft mouse model.

    Science.gov (United States)

    Sia, Kian Chuan; Huynh, Hung; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Hui, Kam Man; Lam, Paula Yeng Po

    2013-08-01

    Gene regulation of many key cell-cycle players in S-, G(2) phase, and mitosis results from transcriptional repression in their respective promoter regions during the G(0) and G(1) phases of cell cycle. Within these promoter regions are phylogenetically conserved sequences known as the cell-cycle-dependent element (CDE) and cell-cycle genes homology regions (CHR) sites. Thus, we hypothesize that transcriptional regulation of cell-cycle regulation via the CDE/CHR region together with liver-specific apolipoprotein E (apoE)-hAAT promoter could bring about a selective transgene expression in proliferating human hepatocellular carcinoma. We show that the newly generated vector AH-6CC-L2C could mediate hepatocyte-targeted luciferase gene expression in tumor cells and freshly isolated short-term hepatocellular carcinoma cultures from patient biopsy. In contrast, normal murine and human hepatocytes infected with AH-6CC-L2C expressed minimal or low luciferase activities. In the presence of prodrug 5-fluorocytosine (5-FC), AH-6CC-L2C effectively suppressed the growth of orthotopic hepatocellular carcinoma patient-derived xenograft mouse model via the expression of yeast cytosine deaminase (yCD) that converts 5-FC to anticancer metabolite 5-fluoruracil. More importantly, we show that combination treatment of AH-6CC-L2C with an EZH2 inhibitor, DZNep, that targets EpCAM-positive hepatocellular carcinoma, can bring about a greater therapeutic efficacy compared with a single treatment of virus or inhibitor. Our study showed that targeting proliferating human hepatocellular carcinoma cells through the transcriptional control of therapeutic gene could represent a feasible approach against hepatocellular carcinoma.

  4. Transcriptional factor PU.1 regulates decidual C1q expression in early pregnancy in human

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    Priyaa Madhukaran Raj

    2015-02-01

    Full Text Available C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells. Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue- specific. Recently, PU.1 has been shown to regulate C1q gene expression in dendritic cells and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation.

  5. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

    Science.gov (United States)

    Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

    2013-10-01

    Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

  6. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    Science.gov (United States)

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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. © 2015 The Authors. BioEssays Published by WILEY Periodicals, Inc.

  8. Effects of acute and chronic endurance exercise on mitochondrial uncoupling in human skeletal muscle

    DEFF Research Database (Denmark)

    Fernström, Maria; Tonkonogi, Michail; Sahlin, Kent

    2004-01-01

    Mitochondrial proteins such as uncoupling protein 3 (UCP3) and adenine nucleotide translocase (ANT) may mediate back-leakage of protons and serve as uncouplers of oxidative phosphorylation. We hypothesized that UCP3 and ANT increase after prolonged exercise and/or endurance training, resulting...... respiration or state 3). Protein expression of UCP3 and ANT was measured with Western blotting. After endurance training, .VO2peak, citrate synthase activity (CS), state 3 respiration and ANT increased by 24, 47, 40 and 95%, respectively (all P ... mitochondrial resistance to Ca2+ overload but does not influence UCR or protein expression of UCP3 and ANT. The increased Ca2+ resistance may prevent mitochondrial degradation and the mechanism needs to be further explored....

  9. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene

    International Nuclear Information System (INIS)

    Heilbronn, T.; Jahn, G.; Buerkle, A.; Freese, U.K.; Fleckenstein, B.; Zur Hausen, H.

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSF-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at T/sub m/ - 25/degrees/C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Esptein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein

  10. Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

    Science.gov (United States)

    Bhosale, Priyanka G; Pandey, Manishkumar; Desai, Rajiv S; Patil, Asawari; Kane, Shubhada; Prabhash, Kumar; Mahimkar, Manoj B

    2016-11-01

    In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Mycobacterium leprae induces NF-κB-dependent transcription repression in human Schwann cells

    International Nuclear Information System (INIS)

    Pereira, Renata M.S.; Calegari-Silva, Teresa Cristina; Hernandez, Maristela O.; Saliba, Alessandra M.; Redner, Paulo; Pessolani, Maria Cristina V.; Sarno, Euzenir N.; Sampaio, Elizabeth P.; Lopes, Ulisses G.

    2005-01-01

    Mycobacterium leprae, the causative agent of leprosy, invades peripheral nerve Schwann cells, resulting in deformities associated with this disease. NF-κB is an important transcription factor involved in the regulation of host immune antimicrobial responses. We aimed in this work to investigate NF-κB signaling pathways in the human ST88-14 Schwannoma cell line infected with M. leprae. Gel shift and supershift assays indicate that two NF-κB dimers, p65/p50 and p50/p50, translocate to the nucleus in Schwann cells treated with lethally irradiated M. leprae. Consistent with p65/p50 and p50/p50 activation, we observed IκB-α degradation and reduction of p105 levels. The nuclear translocation of p50/p50 complex due to M. leprae treatment correlated with repression of NF-κB-driven transcription induced by TNF-α. Moreover, thalidomide inhibited p50 homodimer nuclear translocation induced by M. leprae and consequently rescues Schwann cells from NF-κB-dependent transcriptional repression. Here, we report for the first time that M. leprae induces NF-κB activation in Schwann cells and thalidomide is able to modulate this activation

  12. Human mitochondrial haplogroup H: the highest VO2max consumer--is it a paradox?

    Science.gov (United States)

    Martínez-Redondo, Diana; Marcuello, Ana; Casajús, José A; Ara, Ignacio; Dahmani, Yahya; Montoya, Julio; Ruiz-Pesini, Eduardo; López-Pérez, Manuel J; Díez-Sánchez, Carmen

    2010-03-01

    Mitochondrial background has been demonstrated to influence maximal oxygen uptake (VO(2max), in mLkg(-1)min(-1)), but this genetic influence can be compensated for by regular exercise. A positive correlation among electron transport chain (ETC) coupling, ATP and reactive oxygen species (ROS) production has been established, and mitochondrial variants have been reported to show differences in their ETC performance. In this study, we examined in detail the VO(2max) differences found among mitochondrial haplogroups. We recruited 81 healthy male Spanish Caucasian individuals and determined their mitochondrial haplogroup. Their VO(2max) was determined using incremental cycling exercise (ICE). VO(2max) was lower in J than in non-J haplogroup individuals (P=0.04). The H haplogroup was responsible for this difference (VO(2max); J vs. H; P=0.008) and this group also had significantly higher mitochondrial oxidative damage (mtOD) than the J haplogroup (P=0.04). In agreement with these results, VO(2max) and mtOD were positively correlated (P=0.01). Given that ROS production is the major contributor to mtOD and consumes four times more oxygen per electron than the ETC, our results strongly suggest that ROS production is responsible for the higher VO(2max) found in the H variant. These findings not only contribute to a better understanding of the mechanisms underneath VO(2max), but also help to explain some reported associations between mitochondrial haplogroups and mtOD with longevity, sperm motility, premature aging and susceptibility to different pathologies.

  13. Transcriptional interactions suggest niche segregation among microorganisms in the human gut

    DEFF Research Database (Denmark)

    Plichta, Damian Rafal; Juncker, Agnieszka; dos Santos, Marcelo Bertalan Quintanilha

    2016-01-01

    The human gastrointestinal (GI) tract is the habitat for hundreds of microbial species, of which many cannot be cultivated readily, presumably because of the dependencies between species 1. Studies of microbial co-occurrence in the gut have indicated community substructures that may reflect...... functional and metabolic interactions between cohabiting species 2,3. To move beyond species co-occurrence networks, we systematically identified transcriptional interactions between pairs of coexisting gut microbes using metagenomics and microarray-based metatranscriptomics data from 233 stool samples from...

  14. Mitochondrial Disease

    OpenAIRE

    Bulent Kurt; Turgut Topal

    2013-01-01

    Mitochondria are the major energy source of cells. Mitochondrial disease occurs due to a defect in mitochondrial energy production. A valuable energy production in mitochondria depend a healthy interconnection between nuclear and mitochondrial DNA. A mutation in nuclear or mitochondrial DNA may cause abnormalities in ATP production and single or multiple organ dysfunctions, secondarily. In this review, we summarize mitochondrial physiology, mitochondrial genetics, and clinical expression and ...

  15. 2-ethylpyridine, a cigarette smoke component, causes mitochondrial damage in human retinal pigment epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    S Mansoor

    2014-01-01

    Full Text Available Purpose: Our goal was to identify the cellular and molecular effects of 2-ethylpyridine (2-EP, a component of cigarette smoke on human retinal pigment epithelial cells (ARPE-19 in vitro. Materials and Methods: ARPE-19 cells were exposed to varying concentrations of 2-EP. Cell viability (CV was measured by a trypan blue dye exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS was detected with a 2′,7′-dichlorodihydrofluorescein diacetate dye assay. The JC-1 assay was used to measure mitochondrial membrane potential (ΔΨm. Mitochondrial redox potential was measured using a RedoxSensor Red kit and mitochondria were evaluated with Mitotracker dye. Results: After 2-EP exposure, ARPE-19 cells showed significantly decreased CV, increased caspase-3/7 and caspase-9 activities, elevated ROS/RNS levels, decreased ΔΨm value and decreased redox fluorescence when compared with control samples. Conclusions: These results show that 2-EP treatment induced cell death by caspase-dependent apoptosis associated with an oxidative stress and mitochondrial dysfunction. These data represent a possible mechanism by which smoking contributes to age-related macular degeneration and other retinal diseases and identify mitochondria as a target for future therapeutic interventions.

  16. Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways.

    Directory of Open Access Journals (Sweden)

    Yao Shen

    Full Text Available Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.

  17. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Levin, Inna; Kessler, Naama [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Moor, Nina [Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk (Russian Federation); Klipcan, Liron [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Koc, Emine [Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 (United States); Templeton, Paul [Department Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215 (United States); Spremulli, Linda [Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290 (United States); Safro, Mark, E-mail: mark.safro@weizmann.ac.il [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2007-09-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

  18. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    International Nuclear Information System (INIS)

    Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

    2007-01-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 55, b = 90, c = 96 Å

  19. Accurate Gene Expression-Based Biodosimetry Using a Minimal Set of Human Gene Transcripts

    Energy Technology Data Exchange (ETDEWEB)

    Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, Michigan (United States); Joiner, Michael C. [Department of Radiation Oncology, Wayne State University, Detroit, Michigan (United States); Thomas, Robert A.; Grever, William E.; Bakhmutsky, Marina V. [Department of Biological Sciences, Wayne State University, Detroit, Michigan (United States); Chinkhota, Chantelle N.; Smolinski, Joseph M. [Department of Electrical and Computer Engineering, Wayne State University, Detroit, Michigan (United States); Divine, George W. [Department of Public Health Sciences, Henry Ford Hospital, Detroit, Michigan (United States); Auner, Gregory W. [Department of Electrical and Computer Engineering, Wayne State University, Detroit, Michigan (United States)

    2014-03-15

    Purpose: Rapid and reliable methods for conducting biological dosimetry are a necessity in the event of a large-scale nuclear event. Conventional biodosimetry methods lack the speed, portability, ease of use, and low cost required for triaging numerous victims. Here we address this need by showing that polymerase chain reaction (PCR) on a small number of gene transcripts can provide accurate and rapid dosimetry. The low cost and relative ease of PCR compared with existing dosimetry methods suggest that this approach may be useful in mass-casualty triage situations. Methods and Materials: Human peripheral blood from 60 adult donors was acutely exposed to cobalt-60 gamma rays at doses of 0 (control) to 10 Gy. mRNA expression levels of 121 selected genes were obtained 0.5, 1, and 2 days after exposure by reverse-transcriptase real-time PCR. Optimal dosimetry at each time point was obtained by stepwise regression of dose received against individual gene transcript expression levels. Results: Only 3 to 4 different gene transcripts, ASTN2, CDKN1A, GDF15, and ATM, are needed to explain ≥0.87 of the variance (R{sup 2}). Receiver-operator characteristics, a measure of sensitivity and specificity, of 0.98 for these statistical models were achieved at each time point. Conclusions: The actual and predicted radiation doses agree very closely up to 6 Gy. Dosimetry at 8 and 10 Gy shows some effect of saturation, thereby slightly diminishing the ability to quantify higher exposures. Analyses of these gene transcripts may be advantageous for use in a field-portable device designed to assess exposures in mass casualty situations or in clinical radiation emergencies.

  20. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    Science.gov (United States)

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  1. Photosensitized 2-amino-3-hydroxypyridine-induced mitochondrial apoptosis via Smac/DIABLO in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Goyal, Shruti; Amar, Saroj Kumar [Photobiology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR — Indian Institute of Toxicology Research (CSIR-IITR), M.G, Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research (AcSIR), CSIR — IITR, Lucknow 226001 (India); Dwivedi, Ashish; Mujtaba, Syed Faiz; Kushwaha, Hari Narayan; Chopra, Deepti; Pal, Manish Kumar; Singh, Dhirendra [Photobiology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR — Indian Institute of Toxicology Research (CSIR-IITR), M.G, Marg, Lucknow 226001, Uttar Pradesh (India); Chaturvedi, Rajnish Kumar [Developmental Toxicology Division, CSIR — Indian Institute of Toxicology Research, P. O. Box 80, M.G. Marg, Lucknow 226001 (India); Ray, Ratan Singh, E-mail: ratanray.2011@rediffmail.com [Photobiology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR — Indian Institute of Toxicology Research (CSIR-IITR), M.G, Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research (AcSIR), CSIR — IITR, Lucknow 226001 (India)

    2016-04-15

    The popularity of hair dyes use has been increasing regularly throughout the world as per the demand of hair coloring fashion trends and other cosmetic products. 2-Amino-3-hydroxypyridine (A132) is widely used as a hair dye ingredient around the world. We are reporting first time the phototoxicity mechanism of A132 under ambient environmental UV-B radiation. It showed maximum absorption in UV-B region (317 nm) and forms a photoproduct within an hour exposure of UV-B irradiation. Photocytotoxicity of A132 in human keratinocytes (HaCaT) was measured by mitochondrial (MTT), lysosomal (NRU) and LDH assays which illustrated the significant reduction in cell viability. The role of reactive oxygen species (ROS) generation for A132 phototoxicity was established photo- chemically as well as intracellularly. Noteworthy, formation of tail DNA (comet assay), micronuclei and cyclobutane pyrimidine dimers (CPDs) (immunocytochemistry) formation confirmed the photogenotoxic potential of dye. Cell cycle study (sub-G1peak) and staining with EB/AO revealed the cell cycle arrest and apoptosis. Further, mitochondrial mediated apoptosis was corroborated by reduced MMP, release of cytochrome c and upregulation of caspase-3. Release of mitochondrial Smac/DIABLO in cytoplasm demonstrated the caspase dependent apoptotic cell death by photolabile A132 dye. In-addition increased Bax/Bcl2 ratio again proved the apoptosis. Thus, study suggests that A132 induces photogenotoxicity, phototoxicity and apoptotic cell death through the involvement of Smac/DIABLO in mitochondrial apoptosis via caspase dependent manner. Therefore, the long term use of A132 dye and sunlight exposure jointly increased the oxidative stress in skin which causes premature hair loss, damage to progenitor cells of hair follicles. - Highlights: • Photodegradation of A132 and formation of novel photoproduct • Involvement of ROS in A132 phototoxicity • Role of ROS in DNA damage, CPD and micronuclei formation • Release of

  2. Photosensitized 2-amino-3-hydroxypyridine-induced mitochondrial apoptosis via Smac/DIABLO in human skin cells

    International Nuclear Information System (INIS)

    Goyal, Shruti; Amar, Saroj Kumar; Dwivedi, Ashish; Mujtaba, Syed Faiz; Kushwaha, Hari Narayan; Chopra, Deepti; Pal, Manish Kumar; Singh, Dhirendra; Chaturvedi, Rajnish Kumar; Ray, Ratan Singh

    2016-01-01

    The popularity of hair dyes use has been increasing regularly throughout the world as per the demand of hair coloring fashion trends and other cosmetic products. 2-Amino-3-hydroxypyridine (A132) is widely used as a hair dye ingredient around the world. We are reporting first time the phototoxicity mechanism of A132 under ambient environmental UV-B radiation. It showed maximum absorption in UV-B region (317 nm) and forms a photoproduct within an hour exposure of UV-B irradiation. Photocytotoxicity of A132 in human keratinocytes (HaCaT) was measured by mitochondrial (MTT), lysosomal (NRU) and LDH assays which illustrated the significant reduction in cell viability. The role of reactive oxygen species (ROS) generation for A132 phototoxicity was established photo- chemically as well as intracellularly. Noteworthy, formation of tail DNA (comet assay), micronuclei and cyclobutane pyrimidine dimers (CPDs) (immunocytochemistry) formation confirmed the photogenotoxic potential of dye. Cell cycle study (sub-G1peak) and staining with EB/AO revealed the cell cycle arrest and apoptosis. Further, mitochondrial mediated apoptosis was corroborated by reduced MMP, release of cytochrome c and upregulation of caspase-3. Release of mitochondrial Smac/DIABLO in cytoplasm demonstrated the caspase dependent apoptotic cell death by photolabile A132 dye. In-addition increased Bax/Bcl2 ratio again proved the apoptosis. Thus, study suggests that A132 induces photogenotoxicity, phototoxicity and apoptotic cell death through the involvement of Smac/DIABLO in mitochondrial apoptosis via caspase dependent manner. Therefore, the long term use of A132 dye and sunlight exposure jointly increased the oxidative stress in skin which causes premature hair loss, damage to progenitor cells of hair follicles. - Highlights: • Photodegradation of A132 and formation of novel photoproduct • Involvement of ROS in A132 phototoxicity • Role of ROS in DNA damage, CPD and micronuclei formation • Release of

  3. DDX11L: a novel transcript family emerging from human subtelomeric regions

    Directory of Open Access Journals (Sweden)

    D'Urso Michele

    2009-05-01

    Full Text Available Abstract Background The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members. Results During a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin. Conclusion Our data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.

  4. Whole genome transcription profiling of Anaplasma phagocytophilum in human and tick host cells by tiling array analysis

    Directory of Open Access Journals (Sweden)

    Chavez Adela

    2008-07-01

    Full Text Available Abstract Background Anaplasma phagocytophilum (Ap is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6 and pathogenesis (human; HL-60 and HMEC-1. Results Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6 and the human (HL-60 and HMEC-1 cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins paralogs (of 114 total, through elevated signal produced to the unique hypervariable region of each – 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6. Conclusion Using these methods, whole genome transcription profiles can likely be generated for Ap, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.

  5. Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H+-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    Pelin, Marco; Ponti, Cristina; Sosa, Silvio; Gibellini, Davide; Florio, Chiara; Tubaro, Aurelia

    2013-01-01

    In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na + influx due to the transformation of Na + /K + ATPase in a cationic channel. Recently, we have demonstrated that Na + overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na + intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O 2 − production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na + -mediated H + -imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O 2 − production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O 2 − production induced by PLTX-mediated ionic imbalance. Indeed, the H + intracellular overload that follows PLTX-induced intracellular Na + accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O 2 − production by reversing mitochondrial electron transport. Highlights: ► PLTX induces superoxide (O 2 − ) production by reversing mitochondrial transport chain. ► The mechanism of O 2 − production is dependent on PLTX-induced ionic imbalance. ► The results led to the

  6. Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H{sup +}-dependent mitochondrial pathway

    Energy Technology Data Exchange (ETDEWEB)

    Pelin, Marco, E-mail: marco.pelin@phd.units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Ponti, Cristina, E-mail: cponti@units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Sosa, Silvio, E-mail: silvio.sosa@econ.units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Gibellini, Davide, E-mail: davide.gibellini@unibo.it [Department of Haematology and Oncological Sciences, University of Bologna, Via Massarenti 9, 40138 Bologna (Italy); Florio, Chiara, E-mail: florioc@units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Tubaro, Aurelia, E-mail: tubaro@units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy)

    2013-01-01

    In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na{sup +} influx due to the transformation of Na{sup +}/K{sup +} ATPase in a cationic channel. Recently, we have demonstrated that Na{sup +} overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na{sup +} intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O{sub 2}{sup −} production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na{sup +}-mediated H{sup +}-imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O{sub 2}{sup −} production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O{sub 2}{sup −} production induced by PLTX-mediated ionic imbalance. Indeed, the H{sup +} intracellular overload that follows PLTX-induced intracellular Na{sup +} accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O{sub 2}{sup −} production by reversing mitochondrial electron transport. Highlights: ► PLTX induces superoxide (O{sub 2}{sup −}) production by reversing mitochondrial transport chain. ► The mechanism of

  7. Caffeic Acid Induces Apoptosis in Human Cervical Cancer Cells Through the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Chun Chang

    2010-12-01

    Conclusion: Caffeic acid induces apoptosis by inhibiting Bcl-2 activity, leading to release of cytochrome c and subsequent activation of caspase-3, indicating that caffeic acid induces apoptosis via the mitochondrial apoptotic pathway. This also suggests that caffeic acid has a strong anti-tumor effect and may be a promising chemopreventive or chemotherapeutic agent.

  8. Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen

    Czech Academy of Sciences Publication Activity Database

    Lin, R.-H.; Lai, D.-H.; Zheng, L.-L.; Wu, J.; Lukeš, Julius; Hide, G.; Lun, Z.-R.

    2015-01-01

    Roč. 8, 30 December 2015 (2015), s. 665 ISSN 1756-3305 Institutional support: RVO:60077344 Keywords : Trypanosoma lewisi * Kinetoplast maxicircle * Mitochondrial DNA * RNA editing * Palindrome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2015

  9. Decreased mitochondrial oxidative phosphorylation capacity in the human heart with left ventricular systolic dysfunction

    DEFF Research Database (Denmark)

    Stride, Nis; Larsen, Steen; Hey-Mogensen, Martin

    2013-01-01

    Heart failure (HF) with left ventricular systolic dysfunction (LVSD) is associated with a shift in substrate utilization and a compromised energetic state. Whether these changes are connected with mitochondrial dysfunction is not known. We hypothesized that the cardiac phenotype in LVSD could...

  10. Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

    Science.gov (United States)

    Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

    2010-01-01

    This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

  11. A novel method for determining human ex vivo submaximal skeletal muscle mitochondrial function

    DEFF Research Database (Denmark)

    Hey-Mogensen, Martin; Gram, Martin; Jensen, Martin Borch

    2015-01-01

    previously used. Muscle biopsies were taken from 64 old or young male subjects (60-70 or 20-30 years old). Aged subjects were recruited as trained or untrained. Muscle biopsies were used for isolation of mitochondria and subsequent measurements of DNA repair, antioxidant capacity and mitochondrial protein...

  12. Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy

    Directory of Open Access Journals (Sweden)

    Weihong Zeng

    2017-06-01

    Full Text Available Decidual CD4+ T (dCD4 T cells are crucial for the maternal-fetal immune tolerance required for a healthy pregnancy outcome. However, their molecular and functional characteristics are not well elucidated. In this study, we performed the first analysis of transcriptional and alternative splicing (AS landscapes for paired decidual and peripheral blood CD4+ T (pCD4 T cells in human early pregnancy using high throughput mRNA sequencing. Our data showed that dCD4 T cells are endowed with a unique transcriptional signature when compared to pCD4 T cells: dCD4 T cells upregulate 1,695 genes enriched in immune system process whereas downregulate 1,011 genes mainly related to mRNA catabolic process and the ribosome. Moreover, dCD4 T cells were observed to be at M phase, and show increased activation, proliferation, and cytokine production, as well as display an effector-memory phenotype and a heterogenous nature containing Th1, Th17, and Treg cell subsets. However, dCD4 T cells undergo a comparable number of upregulated and downregulated AS events, both of which are enriched in the genes related to cellular metabolic process. And the changes at the AS event level do not reflect measurable differences at the gene expression level in dCD4 T cells. Collectively, our findings provide a comprehensive portrait of the unique transcriptional signature and AS profile of CD4+ T cells in human decidua and help us gain more understanding of the functional characteristic of these cells during early pregnancy.

  13. Oxidative damage of mitochondrial and nuclear DNA induced by ionizing radiation in human hepatoblastoma cells

    International Nuclear Information System (INIS)

    Morales, Albert; Miranda, Merce; Sanchez-Reyes, Alberto; Biete, Alberto; Fernandez-Checa, Jose C.

    1998-01-01

    Purpose: Since reactive oxygen species (ROS) act as mediators of radiation-induced cellular damage, the aim of our studies was to determine the effects of ionizing radiation on the regulation of hepatocellular reduced glutathione (GSH), survival and integrity of nuclear and mitochondrial DNA (mtDNA) in human hepatoblastoma cells (Hep G2) depleted of GSH prior to radiation. Methods and Materials: GSH, oxidized glutathione (GSSG), and generation of ROS were determined in irradiated (50-500 cGy) Hep G2 cells. Clonogenic survival, nuclear DNA fragmentation, and integrity of mtDNA were assessed in cells depleted of GSH prior to radiation. Results: Radiation of Hep G2 cells (50-400 cGy) resulted in a dose-dependent generation of ROS, an effect accompanied by a decrease of reduced GSH, ranging from a 15% decrease for 50 cGy to a 25% decrease for 400 cGy and decreased GSH/GSSG from a ratio of 17 to a ratio of 7 for controls and from 16 to 6 for diethyl maleate (DEM)-treated cells. Depletion of GSH prior to radiation accentuated the increase of ROS by 40-50%. The depletion of GSH by radiation was apparent in different subcellular sites, being particularly significant in mitochondria. Furthermore, depletion of nuclear GSH to 50-60% of initial values prior to irradiation (400 cGy) resulted in DNA fragmentation and apoptosis. Consequently, the survival of Hep G2 to radiation was reduced from 25% of cells not depleted of GSH to 10% of GSH-depleted cells. Fitting the survival rate of cells as a function of GSH using a theoretical model confirmed cellular GSH as a key factor in determining intrinsic sensitivity of Hep G2 cells to radiation. mtDNA displayed an increased susceptibility to the radiation-induced loss of integrity compared to nuclear DNA, an effect that was potentiated by GSH depletion in mitochondria (10-15% intact mtDNA in GSH-depleted cells vs. 25-30% of repleted cells). Conclusion: GSH plays a critical protective role in maintaining nuclear and mtDNA functional

  14. Influence of heavy ions on cell survival, cytogenetic damage and mitochondrial function of human endothelial cells

    Science.gov (United States)

    Ritter, Sylvia; Helm, Alexander; Lee, Ryonfa; Pollet, Dieter; Durante, Marco

    There is increasing evidence that there is an elevated risk of cardiovascular disease among atomic bomb survivors and radiotherapy patients, typically developing with a long latency. However, essentially no information is available on the potential cardiovascular risks associated with space radiation, in particular heavy ions. To address this issue, we have chosen human umbilical vein endothelial cells (HUVEC) as a model system. Cells at an early passage number were irradiated with 0.1 to 4 Gy of either 9.8 MeV/u C-ions (LET=170 keV/µm), 91 MeV/u C-ions (LET=29 keV/µm) or 250 kV X-rays. Cells were regularly subcultured up to 40 days (20 population doublings) post-irradiation. Immediately after exposure cell inactivation was deter-mined by the colony forming assay. Furthermore, at selected time-points cytogenetic damage (formation of micronuclei in binucleated cells) and the mitochondrial membrane potential ΨM (flow cytometric analysis following JC-1 staining) were assessed. Measurement of the directly induced radiation damage showed that 9.8 MeV/u and 91 MeV/u C-ions were more effective than X-rays (i.e. about 3 and 2 times, respectively) with respect to cell inactivation or the in-duction of cytogenetic damage. At the subsequent days in the irradiated cultures the number of cells with micronuclei declined to the control level (3-5Altogether our data indicate that under the applied radiation conditions the integrity of mitochondria which play a significant role in the regulation of cardiovascular cell function is not impaired. With respect to directly induced genetic damage C-ions are more effective than X-rays as observed in other cell systems. If the effectiveness of charged particles for the occurrence of late chromosomal damage in endothelial cells is higher than that of sparsely ionizing radiation needs further clarification. The data obtained up to now indicate that sophisticated cytogenetic techniques have to be applied in order to draw any firm

  15. Transcriptional Inhibition of the Human Papilloma Virus Reactivates Tumor Suppressor p53 in Cervical Carcinoma Cells

    Science.gov (United States)

    Kochetkov, D. V.; Ilyinskaya, G. V.; Komarov, P. G.; Strom, E.; Agapova, L. S.; Ivanov, A. V.; Budanov, A. V.; Frolova, E. I.; Chumakov, P. M.

    2009-01-01

    Inactivation of tumor suppressor p53 accompanies the majority of human malignancies. Restoration of p53 function causes death of tumor cells and is potentially suitable for gene therapy of cancer. In cervical carcinoma, human papilloma virus (HPV) E6 facilitates proteasomal degradation of p53. Hence, a possible approach to p53 reactivation is the use of small molecules suppressing the function of viral proteins. HeLa cervical carcinoma cells (HPV-18) with a reporter construct containing the b-galactosidase gene under the control of a p53-responsive promoter were used as a test system to screen a library of small molecules for restoration of the transcriptional activity of p53. The effect of the two most active compounds was studied with cell lines differing in the state of p53-dependent signaling pathways. The compounds each specifically activated p53 in cells expressing HPV-18 and, to a lesser extent, HPV-16 and exerted no effect on control p53-negative cells or cells with the intact p53-dependent pathways. Activation of p53 in cervical carcinoma cells was accompanied by induction of p53-dependent CDKN1 (p21), inhibition of cell proliferation, and induction of apoptosis. In addition, the two compounds dramatically decreased transcription of the HPV genome, which was assumed to cause p53 reactivation. The compounds were low-toxic for normal cells and can be considered as prototypes of new anticancer drugs. PMID:17685229

  16. Distinct Contributions of Replication and Transcription to Mutation Rate Variation of Human Genomes

    KAUST Repository

    Cui, Peng; Ding, Feng; Lin, Qiang; Zhang, Lingfang; Li, Ang; Zhang, Zhang; Hu, Songnian; Yu, Jun

    2012-01-01

    Here, we evaluate the contribution of two major biological processes—DNA replication and transcription—to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes.

  17. Transcription factors and stress response gene alterations in human keratinocytes following Solar Simulated Ultra Violet Radiation.

    Science.gov (United States)

    Marais, Thomas L Des; Kluz, Thomas; Xu, Dazhong; Zhang, Xiaoru; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

    2017-10-19

    Ultraviolet radiation (UVR) from sunlight is the major effector for skin aging and carcinogenesis. However, genes and pathways altered by solar-simulated UVR (ssUVR), a mixture of UVA and UVB, are not well characterized. Here we report global changes in gene expression as well as associated pathways and upstream transcription factors in human keratinocytes exposed to ssUVR. Human HaCaT keratinocytes were exposed to either a single dose or 5 repetitive doses of ssUVR. Comprehensive analyses of gene expression profiles as well as functional annotation were performed at 24 hours post irradiation. Our results revealed that ssUVR modulated genes with diverse cellular functions changed in a dose-dependent manner. Gene expression in cells exposed to a single dose of ssUVR differed significantly from those that underwent repetitive exposures. While single ssUVR caused a significant inhibition in genes involved in cell cycle progression, especially G2/M checkpoint and mitotic regulation, repetitive ssUVR led to extensive changes in genes related to cell signaling and metabolism. We have also identified a panel of ssUVR target genes that exhibited persistent changes in gene expression even at 1 week after irradiation. These results revealed a complex network of transcriptional regulators and pathways that orchestrate the cellular response to ssUVR.

  18. Distinct Contributions of Replication and Transcription to Mutation Rate Variation of Human Genomes

    KAUST Repository

    Cui, Peng

    2012-03-23

    Here, we evaluate the contribution of two major biological processes—DNA replication and transcription—to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes.

  19. UVB induces IL-12 transcription in human keratinocytes in vivo and in vitro

    International Nuclear Information System (INIS)

    Enk, C.D.; Blauvet, A.; Katz, S.I.; Mahanty, S.

    1996-01-01

    Human epidermal cells produce a wide range of cytokines, including those characteristic of Th2-like responses such as interleukin (IL)-4 and IL-10. As well, keratinocytes have recently been shown to produce Th1-like cytokines such as IL-12. Exposure to UVB has profound effects on the skin and systemic immune system, which is in part mediated by secretion of tumor necrosis factor (TNF)-α by epidermal cells. Because IL-12 induces production of TNF-α by certain cells of the immune system, we sought to determine whether UVB is an inducer of IL-12 gene expression in epidermal cells. Human epidermal cells were exposed to UVB radiation in vivo, isolated by suction blister technique and trypsinization and transcription of the IL-12 p35 and p40 chains was examined by RT-PCR. (Author)

  20. Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

    Science.gov (United States)

    Moqtaderi, Zarmik; Wang, Jie; Raha, Debasish; White, Robert J; Snyder, Michael; Weng, Zhiping; Struhl, Kevin

    2010-05-01

    Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

  1. Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

    LENUS (Irish Health Repository)

    Anoopkumar-Dukie, S

    2009-10-01

    Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax\\/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

  2. Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

    Science.gov (United States)

    Doersen, C J; Stanbridge, E J

    1981-04-01

    HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

  3. Friends-enemies: endogenous retroviruses are major transcriptional regulators of human DNA

    Science.gov (United States)

    Buzdin, Anton A.; Prassolov, Vladimir; Garazha, Andrew V.

    2017-06-01

    Endogenous retroviruses are mobile genetic elements hardly distinguishable from infectious, or “exogenous”, retroviruses at the time of insertion in the host DNA. Human endogenous retroviruses (HERVs) are not rare. They gave rise to multiple families of closely related mobile elements that occupy 8% of the human genome. Together, they shape genomic regulatory landscape by providing at least 320,000 human transcription factor binding sites (TFBS) located on 110,000 individual HERV elements. The HERVs host as many as 155,000 mapped DNaseI hypersensitivity sites, which denote loci active in the regulation of gene expression or chromatin structure. The contemporary view of the HERVs evolutionary dynamics suggests that at the early stages after insertion, the HERV is treated by the host cells as a foreign genetic element, and is likely to be suppressed by the targeted methylation and mutations. However, at the later stages, when significant number of mutations has been already accumulated and when the retroviral genes are broken, the regulatory potential of a HERV may be released and recruited to modify the genomic balance of transcription factor binding sites. This process goes together with further accumulation and selection of mutations, which reshape the regulatory landscape of the human DNA. However, developmental reprogramming, stress or pathological conditions like cancer, inflammation and infectious diseases, can remove the blocks limiting expression and HERV-mediated host gene regulation. This, in turn, can dramatically alter the gene expression equilibrium and shift it to a newer state, thus further amplifying instability and exacerbating the stressful situation.

  4. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

    International Nuclear Information System (INIS)

    Dykens, James A.; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A.; Will, Yvonne

    2008-01-01

    As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction

  5. Nuclear localization of the mitochondrial ncRNAs in normal and cancer cells.

    Science.gov (United States)

    Landerer, Eduardo; Villegas, Jaime; Burzio, Veronica A; Oliveira, Luciana; Villota, Claudio; Lopez, Constanza; Restovic, Franko; Martinez, Ronny; Castillo, Octavio; Burzio, Luis O

    2011-08-01

    We have previously shown a differential expression of a family of mitochondrial ncRNAs in normal and cancer cells. Normal proliferating cells and cancer cells express the sense mitochondrial ncRNA (SncmtRNA). In addition, while normal proliferating cells express two antisense mitochondrial ncRNAs (ASncmtRNAs-1 and -2), these transcripts seem to be universally down-regulated in cancer cells. In situ hybridization (ISH) of some normal and cancer tissues reveals nuclear localization of these transcripts suggesting that they are exported from mitochondria. FISH and confocal microscopy, in situ digestion with RNase previous to ISH and electron microscopy ISH was employed to confirm the extra-mitochondrial localization of the SncmtRNA and the ASncmtRNAs in normal proliferating and cancer cells of human and mouse. In normal human kidney and mouse testis the SncmtRNA and the ASncmtRNAs were found outside the organelle and especially localized in the nucleus associated to heterochromatin. In cancer cells, only the SncmtRNA was expressed and was found associated to heterochromatin and nucleoli. The ubiquitous localization of these mitochondrial transcripts in the nucleus suggests that they are new players in the mitochondrial-nuclear communication pathway or retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step on neoplastic transformation and cancer progression.

  6. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian; MacPherson, Cameron R; Essack, Magbubah; Kaur, Mandeep; Schaefer, Ulf; Suzuki, Harukazu; Hayashizaki, Yoshihide; Bajic, Vladimir B.

    2009-01-01

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  7. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian

    2009-12-10

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  8. Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdel-Mohsen

    2016-06-01

    Full Text Available Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART, and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9 potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002, more potently than vorinostat (p = 0.02. rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05. rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006 and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02 and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009, suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies.

  9. Transient Genome-Wide Transcriptional Response to Low-Dose Ionizing Radiation In Vivo in Humans

    International Nuclear Information System (INIS)

    Berglund, Susanne R.; Rocke, David M.; Dai Jian; Schwietert, Chad W.; Santana, Alison; Stern, Robin L.; Lehmann, Joerg; Hartmann Siantar, Christine L.; Goldberg, Zelanna

    2008-01-01

    Purpose: The in vivo effects of low-dose low linear energy transfer ionizing radiation on healthy human skin are largely unknown. Using a patient-based tissue acquisition protocol, we have performed a series of genomic analyses on the temporal dynamics over a 24-hour period to determine the radiation response after a single exposure of 10 cGy. Methods and Materials: RNA from each patient tissue sample was hybridized to an Affymetrix Human Genome U133 Plus 2.0 array. Data analysis was performed on selected gene groups and pathways. Results: Nineteen gene groups and seven gene pathways that had been shown to be radiation responsive were analyzed. Of these, nine gene groups showed significant transient transcriptional changes in the human tissue samples, which returned to baseline by 24 hours postexposure. Conclusions: Low doses of ionizing radiation on full-thickness human skin produce a definable temporal response out to 24 hours postexposure. Genes involved in DNA and tissue remodeling, cell cycle transition, and inflammation show statistically significant changes in expression, despite variability between patients. These data serve as a reference for the temporal dynamics of ionizing radiation response following low-dose exposure in healthy full-thickness human skin

  10. Abolition of peroxiredoxin-5 mitochondrial targeting during canid evolution.

    Directory of Open Access Journals (Sweden)

    Valérie Van der Eecken

    Full Text Available In human, the subcellular targeting of peroxiredoxin-5 (PRDX5, a thioredoxin peroxidase, is dependent on the use of multiple alternative transcription start sites and two alternative in-frame translation initiation sites, which determine whether or not the region encoding a mitochondrial targeting sequence (MTS is translated. In the present study, the abolition of PRDX5 mitochondrial targeting in dog is highlighted and the molecular mechanism underlying the loss of mitochondrial PRDX5 during evolution is examined. Here, we show that the absence of mitochondrial PRDX5 is generalized among the extant canids and that the first events leading to PRDX5 MTS abolition in canids involve a mutation in the more 5' translation initiation codon as well as the appearance of a STOP codon. Furthermore, we found that PRDX5 MTS functionality is maintained in giant panda and northern elephant seal, which are phylogenetically closely related to canids. Also, the functional consequences of the restoration of mitochondrial PRDX5 in dog Madin-Darby canine kidney (MDCK cells were investigated. The restoration of PRDX5 mitochondrial targeting in MDCK cells, instead of protecting, provokes deleterious effects following peroxide exposure independently of its peroxidase activity, indicating that mitochondrial PRDX5 gains cytotoxic properties under acute oxidative stress in MDCK cells. Altogether our results show that, although mitochondrial PRDX5 cytoprotective function against oxidative stress has been clearly demonstrated in human and rodents, PRDX5 targeting to mitochondria has been evolutionary lost in canids. Moreover, restoration of mitochondrial PRDX5 in dog MDCK cells, instead of conferring protection against peroxide exposure, makes them more vulnerable.

  11. Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

    Science.gov (United States)

    Cai, Shanbao; Xu, Yi; Cooper, Ryan J; Ferkowicz, Michael J; Hartwell, Jennifer R; Pollok, Karen E; Kelley, Mark R

    2005-04-15

    DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

  12. Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

    DEFF Research Database (Denmark)

    Ferreira, Elisa N; Pires, Lilian C; Parmigiani, Raphael B

    2004-01-01

    The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification ...

  13. SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium.

    Directory of Open Access Journals (Sweden)

    Ya'an Kang

    Full Text Available Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT. The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs. Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGFβ-SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at -3790 bp to -3795 bp within the promoter region of CDH2 was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.

  14. Responses of human cells to ZnO nanoparticles: a gene transcription study†

    Science.gov (United States)

    Moos, Philip J.; Olszewski, Kyle; Honeggar, Matthew; Cassidy, Pamela; Leachman, Sancy; Woessner, David; Cutler, N. Shane; Veranth, John M.

    2013-01-01

    The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-α cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells. PMID:21769377

  15. The Role of S P2, SP3 AND SP4 in The Transcriptional Regulation of The Promoter of Nuclear Encoded Mitochondrial Genes

    International Nuclear Information System (INIS)

    Zaid, A.; Salem, Gh.

    2012-01-01

    The GC-box is an important transcriptional regulatory element present in the promoters of many mammalian genes, and is found in most, if not all, oxidative phosphorylation (OXPHOS) promoters. In the present study we examine the effects of three Spl family members (Sp2, Sp3, and Sp4) on the adenine nucleotide translocase 2, cytochrome cl, Fl-ATPase β-subunit, and the mitochondria transcription factor (mtTFA) promoters in Drosophila SL2 cell line. Sp3, like Spl, strongly activates transcription all four promoters. SP4 stimulates, moderately, but Sp2 had no effect. In addition, Sp3 can, like Spl, inhibit transcription from the proximal promoter of the ANT2 gene through binding to the Cbox GC element. By contrast, Sp4 and Sp2 do not repress promoter activity. Furthermore, since Sp4 and Sp2 bind to the Cbox repression element on the ANT2 promoter, but do not repress transcription, inhibition of transcription cannot be explained by steric hindrance of pre-initiation complex assembly. These data suggest that different Spl family members differentially affect transcription from the OXPHOS promoters.

  16. Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA

    DEFF Research Database (Denmark)

    Rebolledo-Jaramillo, Boris; Su, Marcia Shu-Wei; Stoler, Nicholas

    2014-01-01

    The manifestation of mitochondrial DNA (mtDNA) diseases depends on the frequency of heteroplasmy (the presence of several alleles in an individual), yet its transmission across generations cannot be readily predicted owing to a lack of data on the size of the mtDNA bottleneck during oogenesis......, an order of magnitude higher than for nuclear DNA. Notably, we found a positive association between the number of heteroplasmies in a child and maternal age at fertilization, likely attributable to oocyte aging. This study also took advantage of droplet digital PCR (ddPCR) to validate heteroplasmies...... and confirm a de novo mutation. Our results can be used to predict the transmission of disease-causing mtDNA variants and illuminate evolutionary dynamics of the mitochondrial genome....

  17. HOXB5 cooperates with NKX2-1 in the transcription of human RET.

    Directory of Open Access Journals (Sweden)

    Jiang Zhu

    Full Text Available The enteric nervous system (ENS regulates peristaltic movement of the gut, and abnormal ENS causes Hirschsprung's disease (HSCR in newborns. HSCR is a congenital complex genetic disorder characterised by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET is the major HSCR gene and its expression is crucial for ENS development. We have previously reported that (i HOXB5 transcription factor mediates RET expression, and (ii mouse with defective HOXB5 activity develop HSCR phenotype. In this study, we (i elucidate the underlying mechanisms that HOXB5 mediate RET expression, and (ii examine the interactions between HOXB5 and other transcription factors implicated in RET expression. We show that human HOXB5 binds to the promoter region 5' upstream of the binding site of NKX2-1 and regulates RET expression. HOXB5 and NKX2-1 form a protein complex and mediate RET expression in a synergistic manner. HSCR associated SNPs at the NKX2-1 binding site (-5G>A rs10900296; -1A>C rs10900297, which reduce NKX2-1 binding, abolish the synergistic trans-activation of RET by HOXB5 and NKX2-1. In contrast to the synergistic activation of RET with NKX2-1, HOXB5 cooperates in an additive manner with SOX10, PAX3 and PHOX2B in trans-activation of RET promoter. Taken together, our data suggests that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis- or trans-regulation of RET by HOXB5 could lead to reduction of RET expression and contribute to the manifestation of the HSCR phenotype.

  18. Mitochondrial dysfunction in fatty acid oxidation disorders: insights from human and animal studies

    OpenAIRE

    Wajner, Moacir; Amaral, Alexandre?Umpierrez

    2016-01-01

    Mitochondrial fatty acid oxidation (FAO) plays a pivotal role in maintaining body energy homoeostasis mainly during catabolic states. Oxidation of fatty acids requires approximately 25 proteins. Inherited defects of FAO have been identified in the majority of these proteins and constitute an important group of inborn errors of metabolism. Affected patients usually present with severe hepatopathy, cardiomyopathy and skeletal myopathy, whereas some patients may suffer acute and/or progressive e...

  19. Mitochondrial dysfunction in fatty acid oxidation disorders: insights from human and animal studies.

    Science.gov (United States)

    Wajner, Moacir; Amaral, Alexandre Umpierrez

    2015-11-20

    Mitochondrial fatty acid oxidation (FAO) plays a pivotal role in maintaining body energy homoeostasis mainly during catabolic states. Oxidation of fatty acids requires approximately 25 proteins. Inherited defects of FAO have been identified in the majority of these proteins and constitute an important group of inborn errors of metabolism. Affected patients usually present with severe hepatopathy, cardiomyopathy and skeletal myopathy, whereas some patients may suffer acute and/or progressive encephalopathy whose pathogenesis is poorly known. In recent years growing evidence has emerged indicating that energy deficiency/disruption of mitochondrial homoeostasis is involved in the pathophysiology of some fatty acid oxidation defects (FAOD), although the exact underlying mechanisms are not yet established. Characteristic fatty acids and carnitine derivatives are found at high concentrations in these patients and more markedly during episodes of metabolic decompensation that are associated with worsening of clinical symptoms. Therefore, it is conceivable that these compounds may be toxic. We will briefly summarize the current knowledge obtained from patients and genetic mouse models with these disorders indicating that disruption of mitochondrial energy, redox and calcium homoeostasis is involved in the pathophysiology of the tissue damage in the more common FAOD, including medium-chain acyl-CoA dehydrogenase (MCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies. We will also provide evidence that the fatty acids and derivatives that accumulate in these diseases disrupt mitochondrial homoeostasis. The elucidation of the toxic mechanisms of these compounds may offer new perspectives for potential novel adjuvant therapeutic strategies in selected disorders of this group. © 2016 Authors.

  20. [Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

    Science.gov (United States)

    Zhao, H; Teng, X M; Li, Y F

    2017-11-25

    Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all Pembryo group were significantly higher than those in classⅡ frozen embryo group (both Pembryos of the better quality embryo are higher.

  1. Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

    Science.gov (United States)

    Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

    2012-02-01

    Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

  2. GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene

    Czech Academy of Sciences Publication Activity Database

    Dubot, A.; Godinot, C.; Dumur, V.; Sablonniere, B.; Stojkovic, T.; Cuisset, J. M.; Vojtíšková, Alena; Pecina, Petr; Ješina, Pavel; Houštěk, Josef

    2004-01-01

    Roč. 313, č. 3 (2004), s. 687-693 ISSN 0006-291X R&D Projects: GA MŠk LN00A079; GA MZd NE6533 Grant - others:Fondation Jerome LeJeune(XE) Grant project; GA-(FR) CNRS; GA-(FR) Rhone Alpes Region Institutional research plan: CEZ:AV0Z5011922 Keywords : GUG initiation codon * ATP6 gene * mitochondrial diseases Subject RIV: CE - Biochemistry Impact factor: 2.904, year: 2004

  3. Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

    Directory of Open Access Journals (Sweden)

    Olla Carlo

    2007-08-01

    Full Text Available Abstract Background The Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21 genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy. Results Approximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21 were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects. Conclusion We conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.

  4. Mitochondrial DAMPs induce endotoxin tolerance in human monocytes: an observation in patients with myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Irene Fernández-Ruiz

    Full Text Available Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs, including mitochondrial DNA (mtDNA, induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET, is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage.

  5. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

    Energy Technology Data Exchange (ETDEWEB)

    Wurmb-Schwark, N. von [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)], E-mail: nvonwurmb@rechtsmedizin.uni-kiel.de; Ringleb, A.; Schwark, T. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany); Broese, T.; Weirich, S.; Schlaefke, D. [Clinic of Psychiatry and Psychotherapy, University of Rostock, Gehlsheimer Str. 20, Rostock (Germany); Wegener, R. [Institute of Legal Medicine, St-Georg-Str. 108, University of Rostock, 18055 Rostock (Germany); Oehmichen, M. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)

    2008-01-01

    The 4977 bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or - specifically in skin - external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977 bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

  6. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

    International Nuclear Information System (INIS)

    Wurmb-Schwark, N. von; Ringleb, A.; Schwark, T.; Broese, T.; Weirich, S.; Schlaefke, D.; Wegener, R.; Oehmichen, M.

    2008-01-01

    The 4977 bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or - specifically in skin - external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977 bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process

  7. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  8. HOCOMOCO: a comprehensive collection of human transcription factor binding sites models

    Science.gov (United States)

    Kulakovskiy, Ivan V.; Medvedeva, Yulia A.; Schaefer, Ulf; Kasianov, Artem S.; Vorontsov, Ilya E.; Bajic, Vladimir B.; Makeev, Vsevolod J.

    2013-01-01

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. PMID:23175603

  9. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.; Medvedeva, Yulia A.; Schaefer, Ulf; Kasianov, Artem S.; Vorontsov, Ilya E.; Bajic, Vladimir B.; Makeev, Vsevolod J.

    2012-01-01

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  10. Regulation of the human ADAMTS-4 promoter by transcription factors and cytokines

    International Nuclear Information System (INIS)

    Thirunavukkarasu, Kannan; Pei, Yong; Moore, Terry L.; Wang, He; Yu, Xiao-peng; Geiser, Andrew G.; Chandrasekhar, Srinivasan

    2006-01-01

    ADAMTS-4 (aggrecanase-1) is a metalloprotease that plays a role in aggrecan degradation in the cartilage extracellular matrix. In order to understand the regulation of ADAMTS-4 gene expression we have cloned and characterized a functional 4.5 kb human ADAMTS-4 promoter. Sequence analysis of the promoter revealed the presence of putative binding sites for nuclear factor of activated T cells (NFAT) and Runx family of transcription factors that are known to regulate chondrocyte maturation and differentiation. Using promoter-reporter assays and mRNA analysis we have analyzed the role of chondrocyte-expressed transcription factors NFATp and Runx2 and have shown that ADAMTS-4 is a potential downstream target of these two factors. Our results suggest that inhibition of the expression/function of NFATp and/or Runx2 may enable us to modulate aggrecan degradation in normal physiology and/or in degenerative joint diseases. The ADAMTS-4 promoter would serve as a valuable mechanistic tool to better understand the regulation of ADAMTS-4 expression by signaling pathways that modulate cartilage matrix breakdown

  11. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

    Science.gov (United States)

    Lombraña, Rodrigo; Álvarez, Alba; Fernández-Justel, José Miguel; Almeida, Ricardo; Poza-Carrión, César; Gomes, Fábia; Calzada, Arturo; Requena, José María; Gómez, María

    2016-08-09

    Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6

    International Nuclear Information System (INIS)

    Wu, Tzyy-Choou.

    1989-01-01

    The structure and function of the transcriptional regulatory region of human papillomavirus type 6 (HPV-6) has been investigated. To investigate tissue specific gene expression, a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in plastic-embedded tissue sections of genital and respiratory tract papillomata by using in situ hybridization and immunoperoxidase assays has been developed. This method, using ultrathin sections and strand-specific 3 H labeled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution, and the modified immunocytochemistry provides better sensitivity. The results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. To study the tissue tropism of HPV-6 at the level of regulation of viral gene expression, the polymerase chain reaction was used to isolate the noncoding region (NCR) of HPV-6 in independent isolates. Nucleotide sequence analysis of molecularly cloned DNA identified base substitutions, deletions/insertions and tandem duplications. Transcriptional regulatory elements in the NCR were assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyl transferase

  13. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2012-11-21

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  14. Circulating Human Eosinophils Share a Similar Transcriptional Profile in Asthma and Other Hypereosinophilic Disorders.

    Science.gov (United States)

    Barnig, Cindy; Alsaleh, Ghada; Jung, Nicolas; Dembélé, Doulaye; Paul, Nicodème; Poirot, Anh; Uring-Lambert, Béatrice; Georgel, Philippe; de Blay, Fréderic; Bahram, Seiamak

    2015-01-01

    Eosinophils are leukocytes that are released into the peripheral blood in a phenotypically mature state and are capable of being recruited into tissues in response to appropriate stimuli. Eosinophils, traditionally considered cytotoxic effector cells, are leukocytes recruited into the airways of asthma patients where they are believed to contribute to the development of many features of the disease. This perception, however, has been challenged by recent findings suggesting that eosinophils have also immunomodulatory functions and may be involved in tissue homeostasis and wound healing. Here we describe a transcriptome-based approach-in a limited number of patients and controls-to investigate the activation state of circulating human eosinophils isolated by flow cytometry. We provide an overview of the global expression pattern in eosinophils in various relevant conditions, e.g., eosinophilic asthma, hypereosinophilic dermatological diseases, parasitosis and pulmonary aspergillosis. Compared to healthy subjects, circulating eosinophils isolated from asthma patients differed in their gene expression profile which is marked by downregulation of transcripts involved in antigen presentation, pathogen recognition and mucosal innate immunity, whereas up-regulated genes were involved in response to non-specific stimulation, wounding and maintenance of homeostasis. Eosinophils from other hypereosinophilic disorders displayed a very similar transcriptional profile. Taken together, these observations seem to indicate that eosinophils exhibit non-specific immunomodulatory functions important for tissue repair and homeostasis and suggest new roles for these cells in asthma immunobiology.

  15. Human-Specific Histone Methylation Signatures at Transcription Start Sites in Prefrontal Neurons

    Science.gov (United States)

    Cheung, Iris; Bharadwaj, Rahul; Chou, Hsin-Jung; Houston, Isaac B.; Peter, Cyril J.; Mitchell, Amanda C.; Yao, Wei-Dong; Myers, Richard H.; Chen, Jiang-fan; Preuss, Todd M.; Rogaev, Evgeny I.; Jensen, Jeffrey D.; Weng, Zhiping; Akbarian, Schahram

    2012-01-01

    Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5–1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans. PMID:23185133

  16. E+ subgroup PPR protein defective kernel 36 is required for multiple mitochondrial transcripts editing and seed development in maize and Arabidopsis.

    Science.gov (United States)

    Wang, Gang; Zhong, Mingyu; Shuai, Bilian; Song, Jiandong; Zhang, Jie; Han, Liang; Ling, Huiling; Tang, Yuanping; Wang, Guifeng; Song, Rentao

    2017-06-01

    Mitochondria are semi-autonomous organelles that are the powerhouse of the cells. Plant mitochondrial RNA editing guided by pentatricopeptide repeat (PPR) proteins is essential for energy production. We identify a maize defective kernel mutant dek36, which produces small and collapsed kernels, leading to embryos and/or seedlings lethality. Seed filling in dek36 is drastically impaired, in line with the defects observed in the organization of endosperm transfer tissue. Positional cloning reveals that DEK36, encoding a mitochondria-targeted E+ subgroup PPR protein, is required for mitochondrial RNA editing at atp4-59, nad7-383 and ccmF N -302, thus resulting in decreased activities of mitochondrial complex I, complex III and complex IV in dek36. Loss-of-function of its Arabidopsis ortholog At DEK36 causes arrested embryo and endosperm development, leading to embryo lethality. At_dek36 also has RNA editing defects in atp4, nad7, ccmF N 1 and ccmF N 2 , but at the nonconserved sites. Importantly, efficiency of all editing sites in ccmF N 1 , ccmF N 2 and rps12 is severely decreased in At_dek36, probably caused by the impairment of their RNA stabilization. These results suggest that the DEK36 orthologue pair are essential for embryo and endosperm development in both maize and Arabidopsis, but through divergent function in regulating RNA metabolism of their mitochondrial targets. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  17. Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes

    DEFF Research Database (Denmark)

    Väremo, Leif; Henriksen, Tora Ida; Scheele, Camilla

    2017-01-01

    BACKGROUND: Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize...... in sphingolipid metabolism was transcriptionally regulated. CONCLUSIONS: Our findings identify inherent characteristics in myocytes, as a memory of the in vivo phenotype, without the influence from a diabetic or obese extracellular environment, highlighting their importance in the development of T2D....... intrinsic properties of myocytes, associated independently with T2D or obesity. METHODS: We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor...

  18. (Some) Cellular Mechanisms Influencing the Transcription of Human Endogenous Retrovirus, HERV-Fc1

    DEFF Research Database (Denmark)

    Laska, Magdalena Janina; Nissen, Kari Konstantin; Nexø, Bjørn Andersen

    2013-01-01

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs) is not well...... investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine...... and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza...

  19. Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11.

    Science.gov (United States)

    Herrera, María Georgina; Pignataro, María Florencia; Noguera, Martín Ezequiel; Cruz, Karen Magalí; Santos, Javier

    2018-05-16

    Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.

  20. Methylation and Transcripts Expression at the Imprinted GNAS Locus in Human Embryonic and Induced Pluripotent Stem Cells and Their Derivatives

    Directory of Open Access Journals (Sweden)

    Virginie Grybek

    2014-09-01

    Full Text Available Data from the literature indicate that genomic imprint marks are disturbed in human pluripotent stem cells (PSCs. GNAS is an imprinted locus that produces one biallelic (Gsα and four monoallelic (NESP55, GNAS-AS1, XLsα, and A/B transcripts due to differential methylation of their promoters (DMR. To document imprinting at the GNAS locus in PSCs, we studied GNAS locus DMR methylation and transcript (NESP55, XLsα, and A/B expression in human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs derived from two human fibroblasts and their progenies. Results showed that (1 methylation at the GNAS locus DMRs is DMR and cell line specific, (2 changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation, and (3 interestingly, methylation at A/B DMR is correlated with A/B transcript expression. These results indicate that these models are valuable to study the mechanisms controlling GNAS methylation, factors involved in transcript expression, and possibly mechanisms involved in the pathophysiology of pseudohypoparathyroidism type 1B.

  1. The transcriptional profiling of human in vivo-generated plasma cells identifies selective imbalances in monoclonal gammopathies.

    Directory of Open Access Journals (Sweden)

    Luis M Valor

    Full Text Available Plasma cells (PC represent the heterogeneous final stage of the B cells (BC differentiation process. To characterize the transition of BC into PC, transcriptomes from human naïve BC were compared to those of three functionally-different subsets of human in vivo-generated PC: i tonsil PC, mainly consisting of early PC; ii PC released to the blood after a potent booster-immunization (mostly cycling plasmablasts; and, iii bone marrow CD138+ PC that represent highly mature PC and include the long-lived PC compartment. This transcriptional transition involves subsets of genes related to key processes for PC maturation: the already known protein processing, apoptosis and homeostasis, and of new discovery including histones, macromolecule assembly, zinc-finger transcription factors and neuromodulation. This human PC signature is partially reproduced in vitro and is conserved in mouse. Moreover, the present study identifies genes that define PC subtypes (e.g., proliferation-associated genes for circulating PC and transcriptional-related genes for tonsil and bone marrow PC and proposes some putative transcriptional regulators of the human PC signatures (e.g., OCT/POU, XBP1/CREB, E2F, among others. Finally, we also identified a restricted imbalance of the present PC transcriptional program in monoclonal gammopathies that correlated with PC malignancy.

  2. ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1

    International Nuclear Information System (INIS)

    Ou Ying; Wang Shenqiu; Cai Zhenyu; Wang Yuequn; Wang Canding; Li Yongqing; Li Fang; Yuan Wuzhou; Liu Bisheng; Wu Xiushan; Liu Mingyao

    2005-01-01

    The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C 2 H 2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions

  3. Effects of butyltin exposures on MAP kinase dependent transcription regulators in human natural killer cells

    Science.gov (United States)

    Person, Rachel J.; Whalen, Margaret M.

    2010-01-01

    Natural Killer (NK) cells are a major immune defense mechanism against cancer development and viral infection. The butyltins (BTs), tributyltin (TBT) and dibutyltin (DBT) have been widely used in industrial and other applications and significantly contaminate the environment. Both TBT and DBT have been detected in human blood. These compounds inhibit the lytic and binding function of human NK cells and thus could increase the incidence of cancer and viral infections. Butyltin (BT)-induced loss of NK function is accompanied by activation of mitogen activated protein kinases (MAPKs) and decreases in expression of cell-surface and cytolytic proteins. MAPKs activate components of the transcription regulator AP-1 and activate the transcription regulator Elk-1. Based on the fact that BTs activate MAPKs and alter protein expression, the current study examined the effect of BT exposures on the levels and phosphorylation states of the components of AP-1 and the phosphorylation state of Elk-1. Exposure to 300 nM TBT for 10 min increased the phosphorylation of c-Jun in NK cells. 1 h exposures to 300 nM and 200 nM TBT increased the phosphorylation and overall level of c-Jun. During a 300 nM treatment with TBT for 1 h the binding activity of AP-1 was significantly decreased. There were no significant alterations of AP-1 components or of Elk-1 with DBT exposures. Thus, it appears that TBT-induced alterations on phosphorylation, total levels and binding activity of c-Jun might contribute to, but are not fully responsible for, TBT-induced alterations of NK protein expression. PMID:20370538

  4. [Effect of 3-bromopyruvate on mitochondrial membrane potential and apoptosis of human breast carcinoma SK-BR-3 cells].

    Science.gov (United States)

    Zhang, Yuanyuan; Liu, Zhe; Zhang, Qianwen; Chao, Zhenhua; Zhang, Pei; Xia, Fei; Jiang, Chenchen; Liu, Hao; Jiang, Zhiwen

    2013-09-01

    To study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism. MTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay. MTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells. 3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.

  5. Azoxystrobin Induces Apoptosis of Human Esophageal Squamous Cell Carcinoma KYSE-150 Cells through Triggering of the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Xiao-ke Shi

    2017-05-01

    Full Text Available Recent studies indicate that mitochondrial pathways of apoptosis are potential chemotherapeutic target for the treatment of esophageal cancer. Azoxystrobin (AZOX, a methoxyacrylate derived from the naturally occurring strobilurins, is a known fungicide acting as a ubiquinol oxidation (Qo inhibitor of mitochondrial respiratory complex III. In this study, the effects of AZOX on human esophageal squamous cell carcinoma KYSE-150 cells were examined and the underlying mechanisms were investigated. AZOX exhibited inhibitory effects on the proliferation of KYSE-150 cells with inhibitory concentration 50% (IC50 of 2.42 μg/ml by 48 h treatment. Flow cytometry assessment revealed that the inhibitory effect of AZOX on KYSE-150 cell proliferation occurred with cell cycle arrest at S phase and increased cell apoptosis in time-dependent and dose-dependent manners. Cleaved poly ADP ribose polymerase (PARP, caspase-3 and caspase-9 were increased significantly by AZOX. It is worth noted that the Bcl-2/Bax ratios were decreased because of the down-regulated Bcl-2 and up-regulated Bax expression level. Meanwhile, the cytochrome c release was increased by AZOX in KYSE-150 cells. AZOX-induced cytochrome c expression and caspase-3 activation was significantly blocked by Bax Channel Blocker. Intragastric administration of AZOX effectively decreased the tumor size generated by subcutaneous inoculation of KYSE-150 cells in nude mice. Consistently, decreased Bcl-2 expression, increased cytochrome c and PARP level, and activated caspase-3 and caspase-9 were observed in the tumor samples. These results indicate that AZOX can effectively induce esophageal cancer cell apoptosis through the mitochondrial pathways of apoptosis, suggesting AZOX or its derivatives may be developed as potential chemotherapeutic agents for the treatment of esophageal cancer.

  6. Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells.

    Science.gov (United States)

    Feng, Yi; Wang, Yonghua; Jiang, Chengrui; Fang, Zishui; Zhang, Zhiqiang; Lin, Xiaoying; Sun, Liwei; Jiang, Weiying

    2017-07-15

    Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

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    Ubaid Ullah

    2018-02-01

    Full Text Available Regulatory T (Treg cells are critical in regulating the immune response. In vitro induced Treg (iTreg cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1 as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function.

  8. A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

    OpenAIRE

    Saxena, Kapil; Simon, Lukas M.; Zeng, Xi-Lei; Blutt, Sarah E.; Crawford, Sue E.; Sastri, Narayan P.; Karandikar, Umesh C.; Ajami, Nadim J.; Zachos, Nicholas C.; Kovbasnjuk, Olga; Donowitz, Mark; Conner, Margaret E.; Shaw, Chad A.; Estes, Mary K.

    2017-01-01

    Understanding host?enteric virus interactions has been limited by the inability to culture nontransformed small intestinal epithelial cells and to infect animal models with human viruses. We report epithelial responses in human small intestinal enteroid cultures from different individuals following infection with human rotavirus (HRV), a model enteric pathogen. RNA-sequencing and functional assays revealed type III IFN as the dominant transcriptional response that activates interferon-stimula...

  9. Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Nishio, Sachiyo; Ohira, Takahito; Sunamura, Naohiro; Oshimura, Mitsuo; Ryoke, Kazuo; Kugoh, Hiroyuki

    2015-01-01

    Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.

  10. Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nishio, Sachiyo [Division of Oral and Maxillofacial Biopathological Surgery, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503 (Japan); Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503 (Japan); Ohira, Takahito; Sunamura, Naohiro [Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503 (Japan); Oshimura, Mitsuo [Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, 683-8503 (Japan); Ryoke, Kazuo [Division of Oral and Maxillofacial Biopathological Surgery, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503 (Japan); Kugoh, Hiroyuki, E-mail: kugoh@med.tottori-u.ac.jp [Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503 (Japan); Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, 683-8503 (Japan)

    2015-10-30

    Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.

  11. Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types

    DEFF Research Database (Denmark)

    Ecker, Simone; Chen, Lu; Pancaldi, Vera

    2017-01-01

    Background: A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. Results: We apply a novel analytical approach to measure and compare transcriptional and epigenetic v...

  12. Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARgamma.

    Directory of Open Access Journals (Sweden)

    Anil K Singh

    2010-03-01

    Full Text Available Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.We show that the minimal promoter of human resistin lies within approximately 80 bp sequence upstream of the transcriptional start site (-240 whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha, activating transcription factor 2 (ATF-2 and activator protein 1 (AP-1 transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1 binding site (-276 to -295 is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARgamma is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARgamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARgamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARgamma interaction. Chromatin immunoprecipitation (ChIP assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARgamma, chromatin modifier histone deacetylase 1 (HDAC1 and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistin gene transcription.Our findings suggest a complex interplay of Sp1 and PPARgamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

  13. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed

    2012-01-01

    Highlights: ► Butachlor exhibited strong binding affinity with DNA and produced 8-oxodG adducts. ► Butachlor induced DNA strand breaks and micronuclei formation in PBMN cells. ► Butachlor induced ROS and dissipation of mitochondrial membrane potential in cells. ► Butachlor resulted in cell cycle arrest and eventually caused cellular necrosis. -- Abstract: Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka = 1.2 × 10 4 M −1 ) and binding capacity (n = 1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2′,7′-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24 h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow

  14. Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

    International Nuclear Information System (INIS)

    Chantarawong, Wipa; Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki; Pradermwong, Kantimanee; Shibahara, Shigeki

    2014-01-01

    Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure

  15. Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chantarawong, Wipa [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Inter Departmental Multidisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok (Thailand); Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Pradermwong, Kantimanee [Department of Zoology, Faculty of Science, Kasetsart University, Bangkok (Thailand); Shibahara, Shigeki, E-mail: shibahar@med.tohoku.ac.jp [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan)

    2014-11-28

    Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.

  16. Overexpression of octamer transcription factors 1 or 2 alone has no effect on HIV-1 transcription in primary human CD4 T cells

    International Nuclear Information System (INIS)

    Zhang Mingce; Genin, Anna; Cron, Randy Q.

    2004-01-01

    We explored the binding of octamer (Oct) transcription factors to the HIV-1 long terminal repeat (LTR) by gel shift assays and showed none of the previously identified four potential Oct binding sites bound Oct-1 or Oct-2. Overexpression of Oct-1 or Oct-2 had no effect on HIV-1 LTR activity in transiently transfected primary human CD4 T cells. Next, primary human CD4 T cells were co-transfected with a green fluorescent protein (GFP)-expression vector and an Oct-1 or Oct-2 expression plasmid. The transfected cells were stimulated for 2 days and then infected with the NL4-3 strain of HIV-1. After 3 days of infection, there were no differences in HIV-1 p24 supernatant levels. Apoptosis of infected or bystander cells overexpressing Oct-1 or Oct-2 compared to control was also unaffected. Our studies demonstrate that Oct-1 and Oct-2 fail to bind to the HIV-1 LTR and have no effect on HIV-1 transcription in primary human CD4 T cells

  17. In vitro activation of transcription by the human T-cell leukemia virus type I Tax protein.

    Science.gov (United States)

    Matthews, M A; Markowitz, R B; Dynan, W S

    1992-05-01

    The human T-cell leukemia virus type I (HTLV-I) regulatory protein Tax activates transcription of the proviral long terminal repeats and a number of cellular promoters. We have developed an in vitro system to characterize the mechanism by which Tax interacts with the host cell transcription machinery. Tax was purified from cells infected with a baculovirus expression vector. Addition of these Tax preparations to nuclear extracts from uninfected human T lymphocytes activated transcription of the HTLV-I long terminal repeat approximately 10-fold. Transcription-stimulatory activity copurified with the immunoreactive 40-kDa Tax polypeptide on gel filtration chromatography, and, as expected, the effect of recombinant Tax was diminished in HTLV-I-infected T-lymphocyte extracts containing endogenous Tax. Tax-mediated transactivation in vivo has been previously shown to require 21-bp-repeat Tax-responsive elements (TxREs) in the promoter DNA. Stimulation of transcription in vitro was also strongly dependent on these sequences. To investigate the mechanism of Tax transactivation, cellular proteins that bind the 21-bp-repeat TxREs were prepared by DNA affinity chromatography. Recombinant Tax markedly increased the formation of a specific host protein-DNA complex detected in an electrophoretic mobility shift assay. These data suggest that Tax activates transcription through a direct interaction with cellular proteins that bind to the 21-bp-repeat TxREs.

  18. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  19. Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

    Science.gov (United States)

    Galeano, Eva; Nieto, Elena; García-Pérez, Ana Isabel; Delgado, M Dolores; Pinilla, Montserrat; Sancho, Pilar

    2005-10-01

    Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

  20. Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA

    International Nuclear Information System (INIS)

    Mariottini, P.; Chomyn, A.; Riley, M.; Cottrell, B.; Doolittle, R.F.; Attardi, G.

    1986-01-01

    In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH 2 -terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH 2 -terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells

  1. Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

    Science.gov (United States)

    Dores-Silva, Paulo R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2015-01-01

    The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

  2. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    Science.gov (United States)

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  3. Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response

    Directory of Open Access Journals (Sweden)

    Hemantkumar Chavan

    2017-01-01

    Full Text Available Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

  4. Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells.

    Science.gov (United States)

    Wang, Kai; Hu, Dan-Ning; Lin, Hui-Wen; Yang, Wei-En; Hsieh, Yi-Hsien; Chien, Hsiang-Wen; Yang, Shun-Fa

    2018-05-01

    Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma. © 2018 Wiley Periodicals, Inc.

  5. Papillomavirus genomes in human cervical carcinoma: Analysis of their integration and transcriptional activity

    International Nuclear Information System (INIS)

    Matulic, M.; Soric, J.

    1994-01-01

    Eighty-four biopsies derived from cervical tissues were analyzed for the presence of human papillomavirus (HPV) DNA types 6, 16 and 18 using Southern blot hybridization. HPV 6 was found in none of the cervical biopsies, and HPV types 16 and 18 were found in 44% of them. The rate of HPV 16/18 positive samples increased proportionally to the severity of the lesion. In normal tissue there were no positive samples, in mild and moderate dysplasia HPV 16/18 was present in 20% and in severe dysplasia and invasive carcinomas in 37 and 50%, respectively. In biopsies from 13 cases with squamous cell carcinoma of the uterine cervix and CIN III lesions HPV 16 was integrated within the host genome. It was concluded that the virus could be integrated at variable, presumably randomly selected chromosomal loci and with different number of copies. Transcription of HPV 16 and 18 was detected in one cervical cancer in HeLa cells, respectively. These results imply that HPV types 16 and 18 play an etiological role in the carcinogenesis of human cervical epithelial cells. (author)

  6. Architecture of the human and yeast general transcription and DNA repair factor TFIIH

    Science.gov (United States)

    Luo, Jie; Cimermancic, Peter; Viswanath, Shruthi; Ebmeier, Christopher C.; Kim, Bong; Dehecq, Marine; Raman, Vishnu; Greenberg, Charles H.; Pellarin, Riccardo; Sali, Andrej; Taatjes, Dylan J.; Hahn, Steven; Ranish, Jeff

    2015-01-01

    Summary TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved “topological regions” that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with Xeroderma pigmentosum and Trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit. PMID:26340423

  7. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

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    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrow