RESUMO
The importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.
Assuntos
Anormalidades Congênitas/genética , Embrião de Mamíferos/metabolismo , Epigênese Genética , Ferredoxina-NADP Redutase/genética , Retardo do Crescimento Fetal/genética , Ácido Fólico/metabolismo , Animais , Cruzamentos Genéticos , Metilação de DNA , Feminino , Ferredoxina-NADP Redutase/metabolismo , Masculino , Camundongos , MutaçãoRESUMO
Carnitine is an essential cofactor for mitochondrial import and oxidation of fatty acids. High-dose chemotherapy and radiation, often required for hematopoietic stem cell transplant (HSCT), leads to tissue damage, mitochondrial dysfunction, and alterations in carnitine metabolism. The aim of this pilot cohort study was to describe plasma and urinary carnitine profiles during pediatric HSCT and their relationships with clinical outcomes. Plasma and urinary carnitine samples were collected from 22 pediatric patients before and through day 180 post-HSCT. Associations were observed between graft-versus-host disease and an elevated plasma total carnitine (P=0.019), and also increased plasma acyl:free carnitine ratio with veno-occlusive disease (P=0.016). Mortality was observed in those with their highest urinary total carnitine losses on day 0 (P=0.005), and in those with an abnormal day 28 plasma ratio either above or below the reference range (P=0.007). Changes in carnitine profiles were more reflective of metabolic stress and negative outcomes than of inadequate dietary intake. Associations observed direct larger studies to assess the validity of carnitine profiles as a prognostic indicator and also to assess whether prophylactic carnitine supplementation pre-HSCT could reduce mitochondrial injury and urinary losses and help mitigate inflammatory and metabolic comorbidities of HSCT.
Assuntos
Biomarcadores/análise , Carnitina/sangue , Carnitina/urina , Doença Enxerto-Hospedeiro/diagnóstico , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Doenças Vasculares/diagnóstico , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Lactente , Masculino , Projetos Piloto , Prognóstico , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismoRESUMO
Vitamin B12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [14C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Caenorhabditis elegans/metabolismo , Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans/genética , Citosol/enzimologia , Citosol/metabolismo , Humanos , Lisossomos/metabolismo , Espectrometria de Massas , Ácido Metilmalônico/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Mitocôndrias/enzimologia , Proteína 2 Associada à Farmacorresistência Múltipla , Mutação , Propionatos/metabolismoRESUMO
Parasitic protozoa employ a salvage pathway to synthesize purines and generate essential active nucleotides, whereas mammals are capable of their de novo biosynthesis. This difference provides opportunity for the design of potential new antiprotozoan compounds. A series of 47 adenosine analogues was prepared with modifications at the 2-, 6- and 5'-positions, based on the hypothesis that such compounds would serve as substrates for protozoan nucleoside salvage enzymes, while remaining refractory in mammalian cells. The nucleosides were designed to produce toxic metabolites upon cleavage to the corresponding purine base by the parasite. Three 7-deazaguanosine derivatives were prepared with similar objectives. All of these compounds were tested in vitro against T. brucei (African sleeping sickness), T. cruzi (Chagas' disease), L. donovani (leishmaniasis) and P. falciparum (malaria). In order to determine the therapeutic selectivity indices (SI) of the antiprotozoan nucleosides, their cytotoxicities toward a rat myoblast cell line were also determined. One adenosine derivative proved highly effective against P. falciparum (IC50=110nM and SI=1010, while a modified guanosine displayed potent activities against L. donovani (IC50=60nM, SI=2720) and T. brucei (IC50=130nM, SI=1250), as well as moderate activity against T. cruzi (IC50=3.4µM, SI=48). These results provide proof of concept for the nucleoside-based antiprotozoan strategy, as well as potential lead compounds for further optimization and validation.
Assuntos
Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Animais , Antiprotozoários/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Concentração Inibidora 50 , Leishmania donovani/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria Infravermelho , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The malaria-causing parasite Plasmodium falciparum employs a salvage pathway for the biosynthesis of nucleotides, in contrast to de novo biosynthesis that is utilized by the human host. A series of twenty-two 2-, 6- and 5'-modified adenosine ribonucleosides was synthesized, with the expectation that these compounds would generate toxic metabolites instead of active nucleotides by the pathogen, while remaining inert in host cells. Bioassays with P. falciparum (K1 strain) indicated IC50 values as low as 110nM and a selectivity index with respect to cytotoxicity toward an L6 rat myoblast cell line of >1000 for the most potent analogue.
Assuntos
Antimaláricos/farmacologia , Malária/tratamento farmacológico , Mioblastos/efeitos dos fármacos , Nucleosídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/síntese química , Antimaláricos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Mioblastos/parasitologia , Nucleosídeos/síntese química , Nucleosídeos/química , Testes de Sensibilidade Parasitária , Ratos , Relação Estrutura-AtividadeRESUMO
We report serial neurodegenerative changes on neuroimaging in a rare peroxisomal disease called D-bifunctional protein deficiency. The pattern of posterior to anterior demyelination with white matter disease resembles X-linked adrenoleukodystrophy. We feel this case is important to (1) highlight that D-bifunctional protein deficiency should be considered in cases where the neuroimaging resembles X-linked adrenoleukodystrophy, (2) to show different stages of progression to help identify this disease using neuroimaging in children, and (3) to show that neuroimaging suggesting a leukodystrophy can warrant peroxisomal beta-oxidation studies in skin fibroblasts even when plasma very long chain fatty acids are normal.
Assuntos
17-Hidroxiesteroide Desidrogenases/deficiência , Encéfalo/patologia , Hidroliases/deficiência , Imageamento por Ressonância Magnética/métodos , Doenças Neurodegenerativas/congênito , Doenças Neurodegenerativas/patologia , Transtornos Peroxissômicos/diagnóstico , Criança , Pré-Escolar , Humanos , Lactente , Estudos Longitudinais , Masculino , Proteína Multifuncional do Peroxissomo-2RESUMO
Purine nucleoside phosphorylase had previously been engineered to accept 6-amino substituted purine nucleosides by two active site substitutions, Asn243Asp; Lys244Gln. In the present study, recombinant adenosine phosphorylase (AP) has been conjugated to branched polyethylene glycol (PEG) polymers of approximately 42.5 kDa. Matrix-assisted laser desorption/ionization analysis and SDS acrylamide electrophoresis analysis indicated a subunit composition of greater than 205 kDa consistent with the conjugation of as many as four PEG molecules per AP subunit. The PEG-conjugated enzyme retained greater than 90% of the native catalytic activity. Administration of the enzyme to mice demonstrated the PEG-AP to have a 67-fold increased plasma half-life compared to the native enzyme, 65.1+/-2.9 h versus 57.8+/-1.1 min, respectively. PEG-AP was principally confined to the plasma with minimal activity detected in tissues and of these spleen had the greatest activity and essentially no activity was found in urine. PEG-AP has retained activity with inosine and its injection into PNP-deficient mice resulted in a 2.7-fold increase in urine urate. AP was also shown to protect human CEM cells in culture from the toxic effects of 2'-deoxyadenosine. These studies provide evidence for consideration of PEG-AP as an alternative enzyme therapy for the inherited deficiency of adenosine deaminase.
Assuntos
Adenosina Desaminase/deficiência , Polietilenoglicóis/química , Purina-Núcleosídeo Fosforilase/química , Animais , Células Cultivadas , Desenho de Fármacos , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Meia-Vida , Camundongos , Purina-Núcleosídeo Fosforilase/farmacocinética , Purina-Núcleosídeo Fosforilase/uso terapêutico , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Distribuição TecidualRESUMO
The genetic and environmental factors influencing catabolism of homocysteine in end-stage renal disease (ESRD) patients remain poorly understood. This study investigated how genetic and nutritional influences affect the response to high-dose vitamin B(12) and folate treatment in ESRD patients with hyperhomocysteinemia. We studied 81 hemodialysis patients with hyperhomocysteinemia (> 16 micromol/L) on varied doses of a multivitamin containing 1 mg of folic acid per day. After screening blood work, all patients were switched to daily multivitamin therapy including 1 mg of folic acid for 4 weeks. Vitamin B(12), 1 mg/d, was added for an additional 4 weeks. Patients were then randomized to receive folic acid or placebo. The influence of the 3 methylenetetrahydrofolate reductase (MTHFR) 677 C-->T genotypes on the efficacy of vitamin therapy was assessed. In addition, we investigated how the metabolic complications of ESRD, including the relationship between methylmalonic acid (MMA) and circulating glycine, may contribute to hyperhomocysteinemia. There was no significant difference in total homocysteine (tHcy) levels between the MTHFR 677 C-->T genotypes during the screening phase of the trial. Treatment with a daily multivitamin containing 1 mg folate significantly lowered tHcy levels in all patients by 19.2%. Further supplementation with 1 mg vitamin B(12) resulted in greater tHcy reduction among subjects with the MTHFR 677 T/T genotype (P<.01, T/T v C/C or C/T) while lowering MMA equally in all MTHFR genotypes. There was a significant positive correlation between plasma glycine levels and MMA (P <.05). High-dose vitamin therapy significantly lowers, but does not normalize, MMA and tHcy levels. The MTHFR genotype, while influencing homocysteine levels, was not responsible for the majority of the elevation in plasma tHcy.
Assuntos
Homocisteína/sangue , Falência Renal Crônica/sangue , Falência Renal Crônica/tratamento farmacológico , Ácido Metilmalônico/sangue , Vitamina B 12/uso terapêutico , Cisteína , Genótipo , Glicina/sangue , Humanos , Hiper-Homocisteinemia/etiologia , Falência Renal Crônica/complicações , Metilenotetra-Hidrofolato Redutase (NADPH2) , Mutação/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , TreoninaRESUMO
A media has been developed which enables the assessment of mitochondrial function in fibroblasts by measuring proliferation as an end point.
Assuntos
Biologia Celular , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Biópsia , Proliferação de Células , Células Cultivadas , Meios de Cultura/farmacologia , Glutamina/metabolismo , Humanos , Inosina/metabolismo , Oxigênio/metabolismo , Pentoses/metabolismoRESUMO
A metabolomic analysis of plasma amino acids and acylcarnitines was applied to four disorders of nucleotide metabolism. Multivariate analysis gave score plots that show segregation of hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase deficient plasma from controls with equivocal results for adenosine deaminase and dihydropyrimidine dehydrogenase deficiencies. Loadings plots revealed the principal metabolites responsible for the discrimination between these classes. There were increases for HPRT in C4-, C6-, and C3-DC (malonyl)-carnitines, and decreased serine. For APRT there were increases in C4- to C10- and C3-DC to C6-DC-carnitines, urea, 1-methylhistidine, 3-methylhistidine, and decreased tryptophan. For ADA deficiency there were increases in C4- and C6-carnitines, taurine, and isoleucine.
Assuntos
Doenças Metabólicas/metabolismo , Nucleotídeos/metabolismo , Adenina Fosforribosiltransferase/deficiência , Adenina Fosforribosiltransferase/metabolismo , Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Aminoácidos/sangue , Carnitina/análogos & derivados , Carnitina/sangue , Deficiência da Di-Hidropirimidina Desidrogenase , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Doenças Genéticas Inatas/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/deficiência , Hipoxantina Fosforribosiltransferase/metabolismo , Ninidrina/metabolismoRESUMO
Guanine deaminase catalyses the conversion of guanine to xanthine and ammonia, thereby irreversibly removing the guanine base from the pool of guanine-containing metabolites. We have identified five alleles at the mouse guanine deaminase locus by cDNA sequencing. These alleles were defined by single-nucleotide polymorphisms at a total of 19 positions. For each allele the representative strains are as follows: Gda(a), C57BL/6J and DBA/2J; Gda(b), A/J; Gda(c), MOLF/Ei; Gda(d), CAST/Ei; and Gda(e), SPRET-1. The only codon change resulting in an amino acid substitution was found at nucleotide 523, where GAT was replaced by AAT in Mus spretus resulting in the deduced substitution of Asp-174 by Asn. The single-nucleotide difference between the a and b alleles was also typed by allele-specific oligonucleotide amplification for 17 common strains of Mus musculus susbp. musculus. By typing the AxB and BxA recombinant inbred (RI) strain sets, Gda was mapped to mouse chromosome 19, a region syntenic with human chromosome 9q11-q22.