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Theodore Friedmann - One of the best experts on this subject based on the ideXlab platform.

  • the Housekeeping Gene hypoxanthine guanine phosphoribosyltransferase hprt regulates multiple developmental and metabolic pathways of murine embryonic stem cell neuronal differentiation
    PLOS ONE, 2013
    Co-Authors: Tae Hyuk Kang, Yongjin Park, Joel S Bader, Theodore Friedmann
    Abstract:

    The mechanisms by which mutations of the purinergic Housekeeping Gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of Gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial Gene expression and dysregulates expression of Sox2 and its regulator, Genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodeGenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathoGenesis and for devising new potential therapeutic targets in developmental and Genetic disease.

  • Purinergic signaling in human pluripotent stem cells is regulated by the Housekeeping Gene encoding hypoxanthine guanine phosphoribosyltransferase.
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Lina Mastrangelo, Jieun Kim, Atsushi Miyanohara, Tae Hyuk Kang, Theodore Friedmann
    Abstract:

    Lesch–Nyhan disease (LND) is an X-linked Genetic disorder caused by mutations of the hypoxanthine guanine phosphoribosyltransferase (HPRT) purine biosynthesis Gene and characterized by aberrant purine metabolism, deficient basal ganglia dopamine levels, dystonia, and severe neurobehavioral manifestations, including compulsive self-injurious behavior. Although available evidence has identified important roles for purinergic signaling in brain development, the mechanisms linking HPRT deficiency, purinergic pathways, and neural dysfunction of LND are poorly understood. In these studies aimed at characterizing purinergic signaling in HPRT deficiency, we used a lentivirus vector stably expressing an shRNA targeted to the HPRT Gene to produce HPRT-deficient human CVB induced pluripotent stem cells and human HUES11 embryonic stem cells. Both CVB and HUES11 cells show >99% HPRT knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA and protein down-regulation by HPRT knockdown is refractory to activation by the P2Y1 receptor agonist ATP and shows aberrant purinergic signaling, as reflected by marked deficiency of the transcription factor pCREB and constitutive activation of the MAP kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells also demonstrate marked reduction of phosphorylated β-catenin. These results indicate that the Housekeeping Gene HPRT regulates purinergic signaling in pluripotent human stem cells, and that this regulation occurs at least partly through aberrant P2Y1-mediated expression and signaling. We propose that such mechanisms may play a role in the neuropathology of HPRT-deficiency LND and may point to potential molecular targets for modulation of this intractable neurological phenotype.

  • deficiency of the Housekeeping Gene hypoxanthine guanine phosphoribosyltransferase hprt dysregulates neuroGenesis
    Molecular Therapy, 2010
    Co-Authors: Ghiabehenri Guibinga, Stephen K Hsu, Theodore Friedmann
    Abstract:

    Neuronal transcription factors play vital roles in the specification and development of neurons, including dopaminergic (DA) neurons. Mutations in the Gene encoding the purine biosynthetic enzyme hypoxanthine–guanine phosphoribosyltransferase (HPRT) cause the resulting intractable and largely untreatable neurological impairment of Lesch–Nyhan disease (LND). The disorder is associated with a defect in basal ganglia DA pathways. The mechanisms connecting the purine metabolic defect and the central nervous system (CNS) phenotype are poorly understood but have been presumed to reflect a developmental defect of DA neurons. We have examined the effect of HPRT deficiency on the differentiation of neurons in the well-established human (NT2) embryonic carcinoma neuroGenesis model. We have used a retrovirus expressing a small hairpin RNA (shRNA) to knock down HPRT Gene expression and have examined the expression of a number of transcription factors essential for neuronal differentiation and marker Genes involved in DA biosynthetic pathway. HPRT-deficient NT2 cells demonstrate aberrant expression of several transcription factors and DA markers. Although differentiated HPRT-deficient neurons also demonstrate a striking deficit in neurite outgrowth during differentiation, resulting neurons demonstrate wild-type electrophysiological properties. These results represent direct experimental evidence for aberrant neuroGenesis in HPRT deficiency and suggest developmental roles for other Housekeeping Genes in neurodevelopmental disease.

Claudio Orlando - One of the best experts on this subject based on the ideXlab platform.

  • quantitative real time reverse transcription polymerase chain reaction normalization to rrna or single Housekeeping Genes is inappropriate for human tissue biopsies
    Analytical Biochemistry, 2002
    Co-Authors: Carmela Tricarico, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, V Distante, Mario Pazzagli, Stephen A Bustin, Claudio Orlando
    Abstract:

    Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a Housekeeping Gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used Housekeeping Genes and 18SrRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated Gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular Housekeeping Gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single Housekeeping Genes or rRNA is inappropriate for studies involving tissue biopsies.

  • quantitative real time reverse transcription polymerase chain reaction normalization to rrna or single Housekeeping Genes is inappropriate for human tissue biopsies
    Analytical Biochemistry, 2002
    Co-Authors: Carmela Tricarico, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, V Distante, Mario Pazzagli, Stephen A Bustin, Claudio Orlando
    Abstract:

    Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a Housekeeping Gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used Housekeeping Genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated Gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular Housekeeping Gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single Housekeeping Genes or rRNA is inappropriate for studies involving tissue biopsies.

Stephen B. Goodwin - One of the best experts on this subject based on the ideXlab platform.

  • Variable genome evolution in fungi after transposon-mediated amplification of a Housekeeping Gene
    Mobile DNA, 2019
    Co-Authors: Braham Dhillon, Gert H. J. Kema, Richard C. Hamelin, Burt H. Bluhm, Stephen B. Goodwin
    Abstract:

    Background Transposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact Gene and genome function are largely unknown. Previous analyses revealed that TE-mediated Gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase Gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola ) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis , a histone H3 Gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided Gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded genome of the banana pathogen, Pseudocercospora fijiensis , was analyzed in greater detail. Results These analyses identified a Housekeeping Gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 Gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal Genera Cercospora , Pseudocercospora and Sphaerulina . Conclusions These results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified Genes, with variable effects on Gene and genome evolution.

  • variable genome evolution in fungi after transposon mediated amplification of a Housekeeping Gene
    Mobile Dna, 2019
    Co-Authors: Braham Dhillon, Gert H. J. Kema, Richard C. Hamelin, Burt H. Bluhm, Stephen B. Goodwin
    Abstract:

    Transposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact Gene and genome function are largely unknown. Previous analyses revealed that TE-mediated Gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase Gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis, a histone H3 Gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided Gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded genome of the banana pathogen, Pseudocercospora fijiensis, was analyzed in greater detail. These analyses identified a Housekeeping Gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 Gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal Genera Cercospora, Pseudocercospora and Sphaerulina. These results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified Genes, with variable effects on Gene and genome evolution.

  • variable genome evolution in fungi after transposon mediated amplification of a Housekeeping Gene
    bioRxiv, 2019
    Co-Authors: Braham Dhillon, Gert H. J. Kema, Richard C. Hamelin, Burt H. Bluhm, Stephen B. Goodwin
    Abstract:

    Transposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact Gene and genome function are largely unknown. Previous analyses revealed that TE-mediated Gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase Gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis, a histone H3 Gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided Gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded Pseudocercospora fijiensis genome was analyzed in greater detail. These analyses identified a Housekeeping Gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 Gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal Genera Cercospora, Pseudocercospora and Sphaerulina. These results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified Genes, with variable effects on Gene and genome evolution.

Carmela Tricarico - One of the best experts on this subject based on the ideXlab platform.

  • quantitative real time reverse transcription polymerase chain reaction normalization to rrna or single Housekeeping Genes is inappropriate for human tissue biopsies
    Analytical Biochemistry, 2002
    Co-Authors: Carmela Tricarico, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, V Distante, Mario Pazzagli, Stephen A Bustin, Claudio Orlando
    Abstract:

    Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a Housekeeping Gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used Housekeeping Genes and 18SrRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated Gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular Housekeeping Gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single Housekeeping Genes or rRNA is inappropriate for studies involving tissue biopsies.

  • quantitative real time reverse transcription polymerase chain reaction normalization to rrna or single Housekeeping Genes is inappropriate for human tissue biopsies
    Analytical Biochemistry, 2002
    Co-Authors: Carmela Tricarico, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, V Distante, Mario Pazzagli, Stephen A Bustin, Claudio Orlando
    Abstract:

    Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a Housekeeping Gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used Housekeeping Genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated Gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular Housekeeping Gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single Housekeeping Genes or rRNA is inappropriate for studies involving tissue biopsies.

Katsutaka Oishi - One of the best experts on this subject based on the ideXlab platform.

  • impact of denervation induced muscle atrophy on Housekeeping Gene expression in mice
    Muscle & Nerve, 2015
    Co-Authors: Reiko Nakao, Saori Yamamoto, Yuki Yasumoto, Koji Kadota, Katsutaka Oishi
    Abstract:

    Introduction: Immobilization induced by experimental denervation leads to rapid and progressive alterations in structural and biochemical properties of skeletal muscle. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a popular method of elucidating the molecular mechanisms involved in muscle atrophy. Identification of suitable reference Genes that are not affected by experimental conditions is a critical step in accurate normalization of real-time RT-PCR. Methods: We investigated the impact of denervation-induced muscle atrophy for 2 weeks on the expression of common Housekeeping Genes. Results: Denervation differentially affected the expression levels of these Genes. RefFinder software identified TATA box binding protein (Tbp) as the most stable Gene and showed that the stability of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and hypoxanthine guanine phosphoribosyl transferase (Hprt) Genes was low, even though they are widely used for normalization. Conclusions: The appropriate reference Gene for normalization of Genes of interest in denervated muscle is Tbp. Muscle Nerve 51: 276–281, 2015