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1.
Dev Biol ; 409(1): 152-165, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26546974

RESUMO

Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5' monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.


Assuntos
Sistema Nervoso Entérico/irrigação sanguínea , Sistema Nervoso Entérico/embriologia , Face/embriologia , IMP Desidrogenase/metabolismo , Crista Neural/embriologia , Crista Neural/enzimologia , Crânio/embriologia , Alelos , Animais , Bromodesoxiuridina/metabolismo , Sistema Nervoso Entérico/enzimologia , Sistema Nervoso Entérico/patologia , Feminino , Feto/anormalidades , Feto/embriologia , Deleção de Genes , Genes Reporter , Doença de Hirschsprung/patologia , IMP Desidrogenase/deficiência , Marcação In Situ das Extremidades Cortadas , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Especificidade de Órgãos , RNA não Traduzido/metabolismo , Recombinação Genética/genética , Crânio/metabolismo , Proteína Wnt1/metabolismo
2.
Mol Biochem Parasitol ; 180(2): 123-6, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21907738

RESUMO

Leishmania cannot synthesize purines de novo and therefore must scavenge purines from its host for survival and growth. Biochemical and genomic analyses have indicated that Leishmania species express three potential routes for the synthesis of guanylate nucleotides: (1) a two-step pathway that converts IMP to GMP; (2) a three-step pathway that starts with the deamination of guanine to xanthine, followed by phosphoribosylation to XMP and then conversion to GMP; or (3) direct guanine phosphoribosylation by HGPRT. To determine the role of the first of these pathways to guanylate nucleotide synthesis, an L. donovani line deficient in IMP dehydrogenase (IMPDH), the first step in the IMP to GMP pathway, was constructed by targeted gene replacement. The Δimpdh lesion triggered a highly restrictive growth phenotype in promastigotes in culture but did not impact parasitemias in mice. The dispensability of IMPDH in vivo is the first definitive demonstration that intracellular L. donovani amastigotes have access to a sufficient pool of guanine, xanthine, or guanylate precursors from the host.


Assuntos
IMP Desidrogenase/deficiência , Leishmania donovani/enzimologia , Leishmania donovani/crescimento & desenvolvimento , Leishmania infantum/parasitologia , Proteínas de Protozoários/metabolismo , Animais , Guanosina Monofosfato/metabolismo , Humanos , IMP Desidrogenase/genética , Leishmania donovani/genética , Leishmania donovani/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Proteínas de Protozoários/genética , Ribonucleotídeos/metabolismo , Xantina
3.
PLoS One ; 5(8): e12179, 2010 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-20808934

RESUMO

BACKGROUND: Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5'-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance. METHODOLOGY/PRINCIPAL FINDINGS: Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2. CONCLUSIONS: IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Osteossarcoma/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Técnicas de Silenciamento de Genes , Humanos , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/deficiência , Concentração Inibidora 50 , Metotrexato/farmacologia , Ácido Micofenólico/farmacologia , Osteossarcoma/genética
4.
Proc Natl Acad Sci U S A ; 106(42): 17817-22, 2009 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-19822744

RESUMO

Degenerative retinopathies, including age-related macular degeneration, diabetic retinopathy, and hereditary retinal disorders--major causes of world blindness--are potentially treatable by using low-molecular weight neuroprotective, antiapoptotic, or antineovascular drugs. These agents are, however, not in current systemic use owing to, among other factors, their inability to passively diffuse across the microvasculature of the retina because of the presence of the inner blood-retina barrier (iBRB). Moreover, preclinical assessment of the efficacies of new formulations in the treatment of such conditions is similarly compromised. We describe here an experimental process for RNAi-mediated, size-selective, transient, and reversible modulation of the iBRB in mice to molecules up to 800 Da by suppression of transcripts encoding claudin-5, a protein component of the tight junctions of the inner retinal vasculature. MRI produced no evidence indicative of brain or retinal edema, and the process resulted in minimal disturbance of global transcriptional patterns analyzed in neuronal tissue. We show that visual function can be improved in IMPDH1(-/-) mice, a model of autosomal recessive retinitis pigmentosa, and that the rate of photoreceptor cell death can be reduced in a model of light-induced retinal degeneration by systemic drug delivery after reversible barrier opening. These findings provide a platform for high-throughput drug screening in models of retinal degeneration, and they ultimately could result in the development of a novel "humanized" approach to therapy for conditions with little or no current forms of treatment.


Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Sistemas de Liberação de Medicamentos , Oligopeptídeos/administração & dosagem , Animais , Calpaína/antagonistas & inibidores , Claudina-5 , Inibidores de Cisteína Proteinase/administração & dosagem , Modelos Animais de Doenças , Eletrorretinografia , Guanosina Trifosfato/administração & dosagem , Guanosina Trifosfato/metabolismo , Humanos , IMP Desidrogenase/deficiência , IMP Desidrogenase/genética , Imageamento por Ressonância Magnética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/genética , Retina/efeitos dos fármacos , Retina/metabolismo , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo
5.
Pharmacogenet Genomics ; 17(4): 283-90, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17496727

RESUMO

BACKGROUND AND OBJECTIVE: Inosine 5'-monophosphate dehydrogenase 2 is required for purine synthesis in activated lymphocytes. Variants in the IMPDH2 gene may account for the large inter-individual variability in baseline enzyme activity, immunosuppressive efficacy and side effects in transplant recipients receiving mycophenolic acid. Therefore, the objective of this study was to identify and functionally characterize IMPDH2 variants. METHODS: DNA samples from 152 solid organ transplant patients were screened at exons and exon/intron junctions of the IMPDH2 genes by PCR amplification followed by bidirectional direct DNA sequencing. Genetic variant was constructed by site-directed mutagenesis and transformed to an inosine 5'-monophosphate dehydrogenase-deficient strain of Escherichia coli h712. Proteins were purified to homogeneity and the enzymatic activity was measured by reduced nicotinamide adenine dinucleotide production. RESULTS: Nine genetic variants were identified in the IMPDH2 gene, with frequencies of the rarer alleles ranging from 0.5 to 10.2%. A novel nonsynonymous variant L263F was identified, and the kinetic assay demonstrated that the inosine 5'-monophosphate dehydrogenase activity of L263F variant was decreased to 10% of the wild-type. The Ki for mycophenolic acid inhibition of the L263F variant was comparable with the wild-type, and the variant Km for inosine 5'-monophosphate and nicotinamide adenine dinucleotide did not change significantly. CONCLUSIONS: IMPDH2 has low genetic diversity, but the nonsynonymous variant L263F has a significant impact on inosine 5'-monophosphate dehydrogenase activity. This novel functional variant may be one of the factors contributing to the inter-individual difference of baseline inosine 5'-monophosphate dehydrogenase activity as well as drug efficacy and adverse events in transplant patients.


Assuntos
IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Éxons , Frequência do Gene , Variação Genética , Humanos , IMP Desidrogenase/deficiência , Imunossupressores/farmacologia , Técnicas In Vitro , Íntrons , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Micofenólico/farmacologia , Farmacogenética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Imunologia de Transplantes/efeitos dos fármacos , Imunologia de Transplantes/genética
6.
Mol Cell Biol ; 23(18): 6702-12, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12944494

RESUMO

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.


Assuntos
IMP Desidrogenase/genética , Linfócitos/fisiologia , Animais , Divisão Celular/genética , Feminino , Marcação de Genes , Engenharia Genética/métodos , Guanina/biossíntese , Guanosina Trifosfato/metabolismo , Heterozigoto , Homozigoto , Hipoxantina Fosforribosiltransferase/genética , IMP Desidrogenase/deficiência , IMP Desidrogenase/metabolismo , Interleucina-4 , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mutação , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
7.
J Clin Invest ; 106(4): 599-606, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10953035

RESUMO

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, which are also synthesized from guanine by a salvage reaction catalyzed by the X chromosome-linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Since inhibitors of IMPDH are in clinical use as immunosuppressive agents, we have examined the consequences of knocking out the IMPDH type II enzyme by gene targeting in a mouse model. Loss of both alleles of the gene encoding this enzyme results in very early embryonic lethality despite the presence of IMPDH type I and HPRT activities. Lymphocytes from IMPDH II(+/-) heterozygous mice are normal with respect to subpopulation distribution and respond normally to a variety of mitogenic stimuli. However, mice with an IMPDH II(+/-), HPRT(-/o) genotype demonstrate significantly decreased lymphocyte responsiveness to stimulation with anti-CD3 and anti-CD28 antibodies and show a 30% mean reduction in GTP levels in lymphocytes activated by these antibodies. Furthermore, the cytolytic activity of their T cells against allogeneic target cells is significantly impaired. These results demonstrate that a moderate decrease in the ability of murine lymphocytes to synthesize guanine nucleotides during stimulation results in significant impairment in T-cell activation and function.


Assuntos
IMP Desidrogenase/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Sequência de Bases , Primers do DNA/genética , Resistência a Medicamentos/genética , Feminino , Heterozigoto , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/fisiologia , IMP Desidrogenase/deficiência , IMP Desidrogenase/genética , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mitógenos/farmacologia , Nucleotídeos de Purina/metabolismo , Linfócitos T/efeitos dos fármacos
8.
Adv Exp Med Biol ; 165 Pt A: 373-9, 1984.
Artigo em Inglês | MEDLINE | ID: mdl-6144250

RESUMO

These studies with wild-type and mutant cells defective in IMP dehydrogenase and the previous data with the adenylosuccinate synthetase-deficient cell line suggest that among the clinical population with dominantly inherited hyperuricemia, patients with partial deficiencies in these enzymes exist. It is hoped that these pharmacogenetic cell culture models for overproduction hyperuricemia will lead to the initiation of a search for hyperuricemia patients with either of these deficiencies. If such patients are found it may be possible to design chemotherapeutic regimens by which effectors (inhibitors) of purine synthesis might ameliorate the overproduction of purines by the de novo pathway.


Assuntos
Gota/metabolismo , IMP Desidrogenase/deficiência , Cetona Oxirredutases/deficiência , Linfoma/metabolismo , Mutação , Ribonucleotídeos/metabolismo , Ácido Úrico/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Ácido Micofenólico/farmacologia
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