RESUMEN
Cultured diploid fibroblasts from a patient with a previously undescribed inborn error of cobalamin metabolism accumulate unmetabolized, nonprotein-bound vitamin B12 in lysosomes. These cells are able to endocytose the transcobalamin II-B12 complex and to release B12 from transcobalamin II. The freed vitamin B12 is not released from lysosomes into the cytoplasm of the cell. This suggests that there is a specific lysosomal transport mechanism for vitamin B12 in the human.
Asunto(s)
Lisosomas/metabolismo , Errores Innatos del Metabolismo/metabolismo , Vitamina B 12/metabolismo , Transporte Biológico , Compartimento Celular , Células Cultivadas , Citoplasma/metabolismo , Endocitosis , Femenino , Humanos , LactanteRESUMEN
The mut0 mutation resulting in methylmalonyl CoA mutase (MCM) apoenzyme deficiency and methylmalonic aciduria is characterized by undetectable enzyme activity in cell extracts and low incorporation of propionate into cultured cells which is not stimulated by hydroxycobalamin. A mut0 fibroblast cell line (WG1681) from an African-American male infant complemented another mut0 cell line (WG 1130). Cloning and sequencing of cDNA from WG 1681 demonstrated compound heterozygosity for two novel changes at highly conserved sites: G623R and G703R. In addition, two previously described homozygous polymorphisms, H532R and V671I, were found. Hybridization of allele-specific oligonucleotides to PCR amplified MCM exons from the proband and family members identified a clinically normal mother, half-sister, and half-brother as carriers of the G703R change in cis with both polymorphisms. Transfection of each change into a mut0 cell line with very low MCM mRNA (GM1673) demonstrated a lack of stimulation of propionate uptake in the absence and presence of hydroxycobalamin. Cotransfection of each mutation with the previously identified R93H mutation of WG 1130 stimulated propionate uptake, indicating that G623R and G703R are independently capable of complementing the R93H mutation.
Asunto(s)
Prueba de Complementación Genética , Ácido Metilmalónico/orina , Metilmalonil-CoA Mutasa/genética , Mutación , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Propionatos/metabolismoRESUMEN
By converting methotrexate (MTX) into poly-gamma-glutamyl derivatives, cultured human fibroblasts accumulated high intracellular levels of drug. Once polyglutamates had been formed, DNA synthesis and cell growth remained suppressed even after MTX had been removed from the culture medium. Co-cultivation of cells with MTX and folinic acid reversed the effect of MTX on polyglutamate formation, DNA synthesis, and cell growth. However, if folinic acid was added to the culture medium following a preincubation in methotrexate, DNA synthesis initially remained inhibited and cell growth was only gradually restored. Co-cultivation of cells with 0.67 mM glycine, 37.5 micrometers adenosine, and 41.3 micrometers thymidine (GAT) and MTX did not prevent polyglutamate formation but allowed cells to grow. If GAT was removed from the culture medium along with MTX, cell growth and DNA synthesis were inhibited. If GAT was added to the culture medium following growth in MTX, cell growth recovered. These studies differentiate the effects of GAT and folinic acid treatment. Folinic acid prevented MTX polyglutamate accumulation and reversed the effects of MTX on cell growth when present along with MTX in the cultures. Folinic acid was only partially effective in circumventing the MTX-induced block in folate metabolism when added after pretreatment with MTX. In contrast, GAT allowed growth of cells both in the presence of MTX and after a preincubation in MTX. In contrast, GAT allowed growth of cells both in the presence of MTX and after a preincubation in MTX. However, co-incubation in MTX plus GAT resulted in the accumulation of polyglutamates and a sustained inhibition of cell growth and DNA synthesis upon removal of both MTX and GAT from the culture medium.
Asunto(s)
Adenosina/farmacología , Glicina/farmacología , Leucovorina/farmacología , Metotrexato/metabolismo , Timidina/farmacología , ADN/biosíntesis , Desoxiuridina/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Metotrexato/toxicidad , Ácido Poliglutámico/metabolismo , Factores de TiempoRESUMEN
Methylmalonic aciduria (MMA) is an autosomal recessive inborn error of metabolism that results from functional defects in methylmalonyl CoA mutase (MCM), a nuclear-encoded, mitochondrial enzyme that uses the vitamin B12 derivative, adenosylcobalamin (AdoCbl) as a cofactor. To date, 23 mutations have been identified at the MUT locus on the short arm of chromosome 6, causing the mut forms of MMA (mut complementation group; mut MMA, McKusick #251000). We now report seven novel mutations. Three were found inmut0 patients: R228Q (c759G-->A) was found as a heterozygous change; G312V (c1011G-->T) and 346delL (c1112delCTT) were both found as homozygous changes. Four mutations were found in mut patients: A191E (c648C-->A) and V633G (c1974T-->G) were found in the same patient; 684insL (c2128insCTC) and L685R (c2130T-->G) were both found as homozygous changes. The recent modelling of the human methylmalonyl CoA mutase allowed for an interpretation of the identified mutations.