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Robert M. Tanguay - One of the best experts on this subject based on the ideXlab platform.
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Identification of human Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase.
The Journal of biological chemistry, 2011Co-Authors: Haymo Pircher, Robert M. Tanguay, Grit Daniela Straganz, Daniela Ehehalt, Geneviève Morrow, Pidder Jansen-dürrAbstract:The human Fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg2+ was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.
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involvement of endoplasmic reticulum stress in hereditary tyrosinemia type i
Journal of Biological Chemistry, 2006Co-Authors: Anne Bergeron, Rossana Jorquera, Diana Orejuela, Robert M. TanguayAbstract:Abstract Hereditary tyrosinemia type I (HTI) is the most severe disease of the tyrosine degradation pathway. HTI is caused by a deficiency of Fumarylacetoacetate hydrolase (FAH), the enzyme responsible for the hydrolysis of Fumarylacetoacetate (FAA). As a result, there is an accumulation of metabolites such as maleylacetoacetate, succinylacetone, and FAA. The latter was shown to display mutagenic, cytostatic, and apoptogenic activities and to cause chromosomal instability. Herein, we demonstrate that FAA also causes a cellular insult leading to the endoplasmic reticulum (ER) stress signaling. Treatment of V79 Chinese hamster lung cells with an apoptogenic dose of FAA (100 μm) causes an early induction of the ER resident chaperone GRP78/BiP and a simultaneous phosphorylation of the eIF2α. FAA treatment also causes a subsequent induction of the proapoptotic CHOP (CEBP homologous protein) transcription factor as well as a late activation of caspase-12. Data obtained from fah–/– mice taken off the therapeutic 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione drug are similar. However, in this mouse model, there is also an increase in proteasome activity indicative of ER-associated degradation. This difference observed between the two models may be due to the fact that the murine model measures the effects of all metabolites accumulating in hereditary tyrosinemia type I as opposed to the cellular model that only measures the effects of exogenous FAA.
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A minor alternative transcript of the Fumarylacetoacetate hydrolase gene produces a protein despite being likely subjected to nonsense-mediated mRNA decay
BMC molecular biology, 2005Co-Authors: Natacha Dreumont, Antonella Maresca, Jean François Boisclair-lachance, Anne Bergeron, Robert M. TanguayAbstract:Coupling of alternative splicing with nonsense-mediated mRNA decay (NMD) may regulate gene expression. We report here the identification of a nonsense alternative transcript of the Fumarylacetoacetate hydrolase (fah) gene, which produces a protein despite the fact that it is subject to NMD.
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Cytoplasmic nonsense-mediated mRNA decay for a nonsense (W262X) transcript of the gene responsible for hereditary tyrosinemia, Fumarylacetoacetate hydrolase.
Biochemical and biophysical research communications, 2004Co-Authors: Natacha Dreumont, Antonella Maresca, Edward W. Khandjian, Faouzi Baklouti, Robert M. TanguayAbstract:Messenger RNAs containing premature stop codons are generally targeted for degradation through the nonsense-mediated mRNA decay (NMD) pathway. The subcellular localization of the NMD process in higher eukaryotes remains controversial. While many mRNAs are subjected to NMD prior to their release from the nucleus, a few display cytoplasmic NMD. To understand the possible impact of NMD on the pathogenesis of hereditary tyrosinemia type I, a severe metabolic disease caused by Fumarylacetoacetate hydrolase (FAH) deficiency, we examined the metabolism of FAH mRNA harboring a nonsense mutation, W262X, in lymphoblastoid cell lines derived from patients and their parents. W262X-FAH transcripts show a approximately 20-fold reduction in abundance in mutant cells, which is translation-dependent. Cellular fractionation shows that this down-regulation of the W262X transcript occurs in the cytoplasm. Thus, the W262X FAH is another example of nonsense mRNAs subjected to the NMD pathway in the cytoplasm.
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Fumarylacetoacetate the metabolite accumulating in hereditary tyrosinemia activates the erk pathway and induces mitotic abnormalities and genomic instability
Human Molecular Genetics, 2001Co-Authors: Rossana Jorquera, Robert M. TanguayAbstract:Patients suffering from the metabolic disease hereditary tyrosinemia type I (HT1), caused by Fumarylacetoacetate hydrolase deficiency, have a high risk of developing liver cancer. We report that a sub-apoptogenic dose of Fumarylacetoacetate (FAA), the mutagenic metabolite accumulating in HT1, induces spindle disturbances and segregational defects in both rodent and human cells. Mitotic abnormalities, such as distorted spindles, lagging chromosomes, anaphase/telophase chromatin bridges, aberrant karyokinesis/cytokinesis and multinucleation were observed. Some mitotic asters displayed a large pericentriolar material cloud and/or altered distribution of the spindle pole-associated protein NuMA. FAA-treated cells developed micronuclei which were predominantly CREST-positive, suggesting chromosomal instability. The Golgi complex was rapidly disrupted by FAA, without evident microtubules/tubulin alterations, and a sustained activation of the extracellular signal-regulated protein kinase (ERK) was also observed. Primary skin fibroblasts derived from HT1 patients, not exogenously treated with FAA, showed similar mitotic-derived alterations and ERK activation. Biochemical data suggest that FAA causes ERK activation through a thiol-regulated and tyrosine kinase-dependent, but growth factor receptor- and protein kinase C-independent pathway. Pre-treatment with the MEK inhibitor PD98059 and the Ras farnesylation inhibitor B581 decreased the formation of CREST-positive micronuclei by approximately 75%, confirming the partial contribution of the Ras/ERK effector pathway to the induction of chromosomal instability by FAA. Replenishment of intracellular glutathione (GSH) with GSH monoethylester abolished ERK activation and reduced the chromosomal instability induced by FAA by 80%. Together these results confirm and extend the previously reported genetic instability occurring in cells from HT1 patients and allow us to speculate that this tumorigenic-related phenomenon may rely on the biochemical/cellular effects of FAA as a thiol-reacting and organelle/mitotic spindle-disturbing agent.
Markus Grompe - One of the best experts on this subject based on the ideXlab platform.
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Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51
2010Co-Authors: Nicole K. Paulk, Milton J Finegold, Karsten Wursthorn, Zhongya Wang, Mark A. Kay, Markus GrompeAbstract:Adeno-associated virus (AAV) vectors are ideal for performing gene repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to gene repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation–based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable gene repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for repairing the mutated Fah (Fumarylacetoacetate hydrolase) gene. Hepatic gene repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful gene repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10 �3 were achieved and highly reproducible within typical dose ranges. In this model, repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. Conclusion: AAV-mediated gene repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates. (HEPATOLOGY 2010;51: 1200-1208.) Gene therapy is a promising means to cure many monogenic diseases. However, traditional gene therapies are best suited to treat diseases of deficient or absent gene products rather than those diseases Abbreviations: AAV, adeno-associated virus; AST, aspartate aminotransferase; dGE, diploid genome equivalent; FAH, Fumarylacetoacetate hydrolase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hAAT, human alpha-1 antitrypsin; HTI, hereditary tyrosinemia type I; LD-PCR, long-distance polymerase chain reaction
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robust expansion of human hepatocytes in fah rag2 il2rg mice
Nature Biotechnology, 2007Co-Authors: Hisaya Azuma, Milton J Finegold, Nicole K. Paulk, Mark A. Kay, Muhsen Aldhalimy, Stephen C. Strom, Aarati Ranade, Craig Dorrell, Ewa Ellis, Markus GrompeAbstract:Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, Fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.
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Cell fusion is the principal source of bone-marrow-derived hepatocytes
Nature, 2003Co-Authors: Xin Wang, Yassmine Akkari, Yumi Torimaru, Holger Willenbring, Milton J Finegold, Mark Foster, Eric Lagasse, Susan B. Olson, Muhsen Al-dhalimy, Markus GrompeAbstract:Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with Fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.
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Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
The American journal of pathology, 1997Co-Authors: Ken Overturf, Milton J Finegold, Muhsen Al-dhalimy, Markus GrompeAbstract:Previous work has shown that adult mouse hepatocytes can divide at least 18 times in vivo. To test whether this represents the upper limit of their regenerative capacity, we performed serial transplantation of hepatocytes in the Fumarylacetoacetate hydrolase deficiency murine model of liver repopulation. Hepatocytes from adult donors were serially transplanted in limiting numbers six times and resulted in complete repopulation during each cycle. This corresponds to a minimal number of 69 cell doublings or a 7.3 x 10(20)-fold expansion. No evidence for abnormal liver function or altered hepatic architecture was found in repopulated animals. We conclude that a fraction of adult mouse hepatocytes have growth potential similar to that of hematopoietic stem cells.
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Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I
Nature genetics, 1996Co-Authors: Ken Overturf, Milton J Finegold, Robert M. Tanguay, Mark L. Brantly, Muhsen Al-dhalimy, Markus GrompeAbstract:Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of Fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.
Pidder Jansen-dürr - One of the best experts on this subject based on the ideXlab platform.
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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins.
Journal of visualized experiments : JoVE, 2019Co-Authors: Alexander K. H. Weiss, Max Holzknecht, Elia Cappuccio, Ilaria Dorigatti, Karin Kreidl, Andreas Naschberger, Bernhard Rupp, Hubert Gstach, Pidder Jansen-dürrAbstract:Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (>98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.
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The Fumarylacetoacetate hydrolase (FAH) superfamily of enzymes: multifunctional enzymes from microbes to mitochondria.
Biochemical Society transactions, 2018Co-Authors: Alexander K. H. Weiss, Klaus R Liedl, Hubert Gstach, Johannes R. Loeffler, Pidder Jansen-dürrAbstract:Prokaryotic and eukaryotic Fumarylacetoacetate hydrolase (FAH) superfamily members, sharing conserved regions that form the so-called FAH-domain, catalyze a remarkable variety of reactions. These enzymes are essential in the metabolic pathways to degrade aromatic compounds in prokaryotes and eukaryotes. It appears that prokaryotic FAH superfamily members evolved mainly to allow microbes to generate energy and useful metabolites from complex carbon sources. We review recent findings, indicating that both prokaryotic and eukaryotic members of the FAH superfamily also display oxaloacetate decarboxylase (ODx) activity. The identification of human FAH domain-containing protein 1 as mitochondrial ODx regulating mitochondrial function supports the new concept that, during evolution, eukaryotic FAH superfamily members have acquired important regulatory functions beyond catabolism of complex carbon sources. Molecular studies on the evolution and function of FAH superfamily members are expected to provide new mechanistic insights in their physiological roles.
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Identification of human Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase.
The Journal of biological chemistry, 2011Co-Authors: Haymo Pircher, Robert M. Tanguay, Grit Daniela Straganz, Daniela Ehehalt, Geneviève Morrow, Pidder Jansen-dürrAbstract:The human Fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg2+ was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.
Alexander K. H. Weiss - One of the best experts on this subject based on the ideXlab platform.
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inhibitors of Fumarylacetoacetate hydrolase domain containing protein 1 fahd1
Molecules, 2021Co-Authors: Alexander K. H. Weiss, Klaus R Liedl, Johannes R. Loeffler, Richard Wurzer, Patrycia Klapec, Manuel Philip Eder, Susanne Von Grafenstein, Stefania Monteleone, Pidder Jansendurr, Hubert GstachAbstract:FAH domain containing protein 1 (FAHD1) acts as oxaloacetate decarboxylase in mitochondria, contributing to the regulation of the tricarboxylic acid cycle. Guided by a high-resolution X-ray structure of FAHD1 liganded by oxalate, the enzymatic mechanism of substrate processing is analyzed in detail. Taking the chemical features of the FAHD1 substrate oxaloacetate into account, the potential inhibitor structures are deduced. The synthesis of drug-like scaffolds afforded first-generation FAHD1-inhibitors with activities in the low micromolar IC50 range. The investigations disclosed structures competing with the substrate for binding to the metal cofactor, as well as scaffolds, which may have a novel binding mode to FAHD1.
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structural and functional comparison of Fumarylacetoacetate domain containing protein 1 in human and mouse
Bioscience Reports, 2020Co-Authors: Alexander K. H. Weiss, Max Holzknecht, Elia Cappuccio, Andreas Naschberger, Bernhard Rupp, Christina Metzger, Lorenza Mottes, Jill Von Velsen, Matthew W Bowler, Pidder JansendurrAbstract:FAH domain containing protein 1 (FAHD1) is a mammalian mitochondrial protein, displaying bifunctionality as acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. We report the crystal structure of mouse FAHD1 and structural mapping of the active site of mouse FAHD1. Despite high structural similarity with human FAHD1, a rabbit monoclonal antibody (RabMab) could be produced that is able to recognize mouse FAHD1, but not the human form, whereas a polyclonal antibody recognized both proteins. Epitope mapping in combination with our deposited crystal structures revealed that the epitope overlaps with a reported SIRT3 deacetylation site in mouse FAHD1.
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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins.
Journal of visualized experiments : JoVE, 2019Co-Authors: Alexander K. H. Weiss, Max Holzknecht, Elia Cappuccio, Ilaria Dorigatti, Karin Kreidl, Andreas Naschberger, Bernhard Rupp, Hubert Gstach, Pidder Jansen-dürrAbstract:Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (>98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.
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The Fumarylacetoacetate hydrolase (FAH) superfamily of enzymes: multifunctional enzymes from microbes to mitochondria.
Biochemical Society transactions, 2018Co-Authors: Alexander K. H. Weiss, Klaus R Liedl, Hubert Gstach, Johannes R. Loeffler, Pidder Jansen-dürrAbstract:Prokaryotic and eukaryotic Fumarylacetoacetate hydrolase (FAH) superfamily members, sharing conserved regions that form the so-called FAH-domain, catalyze a remarkable variety of reactions. These enzymes are essential in the metabolic pathways to degrade aromatic compounds in prokaryotes and eukaryotes. It appears that prokaryotic FAH superfamily members evolved mainly to allow microbes to generate energy and useful metabolites from complex carbon sources. We review recent findings, indicating that both prokaryotic and eukaryotic members of the FAH superfamily also display oxaloacetate decarboxylase (ODx) activity. The identification of human FAH domain-containing protein 1 as mitochondrial ODx regulating mitochondrial function supports the new concept that, during evolution, eukaryotic FAH superfamily members have acquired important regulatory functions beyond catabolism of complex carbon sources. Molecular studies on the evolution and function of FAH superfamily members are expected to provide new mechanistic insights in their physiological roles.
Muhsen Al-dhalimy - One of the best experts on this subject based on the ideXlab platform.
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Cell fusion is the principal source of bone-marrow-derived hepatocytes
Nature, 2003Co-Authors: Xin Wang, Yassmine Akkari, Yumi Torimaru, Holger Willenbring, Milton J Finegold, Mark Foster, Eric Lagasse, Susan B. Olson, Muhsen Al-dhalimy, Markus GrompeAbstract:Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with Fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.
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Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
The American journal of pathology, 1997Co-Authors: Ken Overturf, Milton J Finegold, Muhsen Al-dhalimy, Markus GrompeAbstract:Previous work has shown that adult mouse hepatocytes can divide at least 18 times in vivo. To test whether this represents the upper limit of their regenerative capacity, we performed serial transplantation of hepatocytes in the Fumarylacetoacetate hydrolase deficiency murine model of liver repopulation. Hepatocytes from adult donors were serially transplanted in limiting numbers six times and resulted in complete repopulation during each cycle. This corresponds to a minimal number of 69 cell doublings or a 7.3 x 10(20)-fold expansion. No evidence for abnormal liver function or altered hepatic architecture was found in repopulated animals. We conclude that a fraction of adult mouse hepatocytes have growth potential similar to that of hematopoietic stem cells.
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Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I
Nature genetics, 1996Co-Authors: Ken Overturf, Milton J Finegold, Robert M. Tanguay, Mark L. Brantly, Muhsen Al-dhalimy, Markus GrompeAbstract:Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of Fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.
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Mutations of the Fumarylacetoacetate hydrolase gene in four patients with tyrosinemia, type I
Human mutation, 1993Co-Authors: Markus Grompe, Muhsen Al-dhalimyAbstract:Tyrosinemia type I is an autosomal recessive inborn error of metabolism caused by deficiency of the enzyme fumaryl acetoacetate hydrolase (FAH, EC 3.7.1.2.). We have used reverse transcription and the polymerize chain reaction to amplify the peptide coding region of the FAH cDNA from four patients with tyrosinemia type I. Chemical mismatch cleavage analysis and DNA sequencing were utilized to determine mutant alleles in all cases. A French Canadian patient was homozygous for a splice error mutation in the 3′ portion of the gene. A second patient, from a consanguineous pedigree in Iran, had the identical splice alteration. The third patient has a missense mutation, changing valine to glycine in codon 166. And finally two nonsense mutations in codons 357 and 364 were found in the fourth patient. © 1993 Wiley-Liss, Inc.
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Nucleotide sequence of a cDNA encoding murine Fumarylacetoacetate hydrolase
Biochemical medicine and metabolic biology, 1992Co-Authors: Markus Grompe, Muhsen Al-dhalimyAbstract:Hereditary tyrosinemia type I is caused by deficiency of the enzyme Fumarylacetoacetate hydrolase (FAH) (EC 3.7.1.2), the final step in tyrosine degradation. We report here the cloning and sequencing of a full length cDNA coding for murine FAH. This cDNA is highly homologous to the previously cloned human and rat genes.
