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1.
J Clin Invest ; 75(1): 40-8, 1985 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3880775

RESUMO

Phenylketonuria provides a human model for the study of the effect of phenylalanine on brain function. Although irreversible mental retardation is preventable through newborn diagnosis and dietary phenylalanine restriction, controversy exists regarding the effects of increased concentrations of phenylalanine in older patients. We have studied ten older, treated, phenylketonuric patients using a triple-blind, multiple trials, crossover design. Each patient was tested at the end of each of three 1-wk periods of high or low phenylalanine intakes. Tests included a repeatable battery of neuropsychological tests, analysis of plasma amino acids, and measurement of urine amino acids, phenyl organic acids, dopamine, and serotonin. In all 10 patients plasma phenylalanine rose (900-4,000 microM). In 9 of 10 patients there was an inverse relationship between plasma phenylalanine and urine dopamine excretion. When blood phenylalanine was elevated, these patients had prolonged performance times on neuropsychological tests of higher but not lower integrative function. Urinary serotonin fell during phenylalanine loading in six patients. The concentration of phenylacids in the urine was not proportional to the plasma phenylalanine at concentrations below 1.5 mM. In one patient, neither performance time nor dopamine excretion varied as blood phenylalanine rose or fell. We interpret these data as follows: blood phenylalanine above 1.3 mM impairs performance on neuropsychological tests of higher integrative function, this effect is reversible, and one mechanism may involve impaired biogenic amine synthesis.


Assuntos
Encéfalo/fisiologia , Fenilalanina/sangue , Fenilcetonúrias/sangue , Adolescente , Adulto , Transporte Biológico , Criança , Ensaios Clínicos como Assunto , Dopamina/urina , Feminino , Humanos , Túbulos Renais/metabolismo , Masculino , Testes Neuropsicológicos , Serotonina/urina , Triptofano/metabolismo , Tirosina/metabolismo
2.
Biochim Biophys Acta ; 840(2): 143-52, 1985 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-3922429

RESUMO

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.


Assuntos
Colágeno/síntese química , Oxigenases de Função Mista/metabolismo , Fragmentos de Peptídeos/síntese química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Células Cultivadas , Cromatografia/métodos , Cromatografia em Camada Fina , Colágeno/metabolismo , Síndrome de Ehlers-Danlos/enzimologia , Fibroblastos/enzimologia , Humanos , Marcação por Isótopo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pele/enzimologia , Trítio
3.
Am J Med Genet ; 55(1): 21-6, 1995 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-7702090

RESUMO

We report on two unrelated patients with different presentations of mannosidosis. One patient was affected in early childhood with a severe phenotype characteristic of type I mannosidosis. The other was diagnosed with type II mannosidosis only after the onset of progressive neurologic deterioration in late adulthood. Both were detected by non-invasive urinary screening of oligosaccharides. Lymphoblasts transformed from both patients' blood cells had markedly reduced lysosomal alpha-mannosidase activity. Kinetic analyses showed that alpha-mannosidase from the type I patient had a 400-fold reduction in affinity while that from the type II patient was reduced 40-fold. Lymphoblasts from all 4 parents had reduced alpha-mannosidase activity, but there were overlapping activities among these type I and type II obligate heterozygotes. We conclude that screening urinary oligosaccharides will detect mannosidosis over a wide range of phenotypes, that lymphoblasts transformed from affected heterozygotes have decreased enzymatic activity, and that the severity of clinical expression is related to the degree of enzyme impairment.


Assuntos
Manosidases/deficiência , alfa-Manosidose/diagnóstico , Adulto , Células Cultivadas , Criança , Pré-Escolar , Ensaios Enzimáticos Clínicos , Feminino , Genes Recessivos , Humanos , Lactente , Cinética , Linfócitos/enzimologia , Masculino , Oligossacarídeos/urina , Especificidade por Substrato , alfa-Manosidase , alfa-Manosidose/genética , beta-N-Acetil-Hexosaminidases/sangue
4.
Metabolism ; 36(7): 687-91, 1987 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3110540

RESUMO

We studied two unrelated individuals with Ehlers-Danlos syndrome type VI, which is characterized by congenital hypotonia, lax joints, severe kyphoscoliosis, friable skin, and hemorrhagic hypotrophic scars. The diagnosis was confirmed by decreased hydroxylysine residues in dermal collagen and decreased collagen lysyl hydroxylase activities in their cultured skin fibroblasts. Despite the diminished hydroxylysine residues in dermal collagen from the probands, we found no differences in hydroxylysyl residues of collagen synthesized by fibroblasts in culture. When patient 1 was given oral sodium ascorbate (5 g/d) for 3 weeks, ascorbate concentrations increased two-fold in plasma and 300-fold in urine. Urinary excretion of hydroxylysine and hydroxyproline increased during ascorbate administration. After a 1-year interval, bleeding time, wound healing, and muscle strength improved. Ascorbate supplementation (50 micrograms/mL) to confluent fibroblasts cultured from the two patients and controls increased hydroxyprolyl and hydroxylysyl residues of fibroblasts four to seven and three to four-fold respectively. Total protein associated with the cell layer increased 14% to 32% without concomitant change in cellular DNA. Total soluble collagenous material recovered from culture media increased 61% to 103% with ascorbate supplementation. These studies demonstrate that ascorbate improves the clinical status of patients with impaired collagen lysyl hydroxylase activity by enhancing lysyl and prolyl hydroxylation and total collagen production.


Assuntos
Ácido Ascórbico/farmacologia , Colágeno/biossíntese , Síndrome de Ehlers-Danlos/metabolismo , Aminoácidos/metabolismo , Criança , Pré-Escolar , DNA/metabolismo , Síndrome de Ehlers-Danlos/classificação , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/deficiência , Pele/metabolismo
5.
Metabolism ; 47(2): 207-11, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9472972

RESUMO

Elevated total plasma homocysteine (tHcy) is recognized as an independent risk factor for occlusive vascular disease. However, it is not known how much of the observed hyperhomocysteinemia in patients with vascular disease is due to heterozygosity for cystathionine-beta-synthase (CbetaS) deficiency, because a clinically useful screening method is unavailable. To determine this, parents of children who are homozygous for CbetaS deficiency (affected with homocystinuria) and a control population were compared for tHcy, total plasma cysteine (tCys), plasma folate, and plasma vitamin B12. The group of obligate heterozygotes had increased tHcy (P < or = .01), decreased tCys (P < or = .01), and decreased plasma folate (P < or = .01). The calculated ratios of tHcy/tCys (P = .01) and tHcy/plasma folate (P = .003) were the best metabolic discriminants for genotype. These ratios are likely to prove useful in heterozygote screening for CPS deficiency and in the development of rational treatment strategies for patients with increased tHcy.


Assuntos
Cistationina beta-Sintase/deficiência , Cisteína/sangue , Ácido Fólico/sangue , Triagem de Portadores Genéticos , Homocisteína/sangue , Homocistinúria/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vitamina B 12/sangue
6.
Clin Biochem ; 23(1): 91-6, 1990 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2110043

RESUMO

Two methods for analysis of urinary glycosaminoglycans (GAGs) have been modified and improved for efficient screening of mucopolysaccharidoses. Urinary excretion of GAGs was estimated by spectrophometric measurement of alcian blue complex formation and was used in conjunction with qualitative analysis by thin layer chromatography. After normal variation in GAG excretion was established using 120 urine samples, these screening methods were applied to a total of 2057 urine samples over 4 years. Six patients with abnormal urinary GAG excretion had a mean of 50.5 +/- 20.6 mg GAG/mmol creatinine compared to 3.4 +/- 2.9 for age-matched controls. Qualitative analysis by thin layer chromatography using alternating solvent systems identified the GAGs excreted in excess and facilitated selection of specific enzyme assays for final confirmation. Six cases were diagnosed prospectively and demonstrate these quantitative and qualitative methods to be economical, efficient, and suitable for clinical use.


Assuntos
Mucopolissacaridoses/urina , Cromatografia em Camada Fina , Glicosaminoglicanos/urina , Humanos , Programas de Rastreamento/métodos , Mucopolissacaridoses/prevenção & controle , Solventes
8.
Blood ; 57(6): 1125-31, 1981 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-6784792

RESUMO

Hemoglobin beta-chain synthesis by rabbit erythroid cells was tested for dependence on availability of complementary alpha-chains. Reticulocytes and bone marrow cells were obtained from variant rabbits that have hemoglobin with isoleucine in alpha-chains but not in beta-chains. This characteristic permits the use of L-O-methylthreonine, a specific isoleucine antagonist, to inhibit selectively the synthesis of hemoglobin alpha-chains without directly affecting that of beta-chains. Study of hemoglobin synthesis by bone marrow cells presents two problems that require careful management: (A) the fragility of the globin-synthesizing apparatus and (B) the isolation of globin from the various proteins made by the mixture of nucleated cells. Disruption of synthetic activity was minimized by collecting the bone marrow in autologous plasma then removing fat and connective tissue while the cells were suspended in this medium. Purification involved gel filtration of hemoglobin and globin then CM-cellulose chromatography of globin chains. Absence of radioactive isoleucine in beta-chains demonstrated the efficacy of this scheme in removing isoleucine-containing proteins that otherwise elute with beta-chains on CM-cellulose columns. In reticulocytes, when synthesis of alpha-chains is inhibited by 30%--80%, that of beta-chains is stimulated by 20%--60%, but in marrow cells, incorporation into beta-chains stays at control level when alpha incorporation is inhibited. The data indicate that beta-chain synthesis is independent of the availability of complementary alpha-chains.


Assuntos
Eritrócitos/análise , Hemoglobinas/biossíntese , Animais , Proteínas Sanguíneas , Células da Medula Óssea , Separação Celular , Globinas/isolamento & purificação , Hemoglobinas/metabolismo , Coelhos , Reticulócitos/análise , Treonina/análogos & derivados , Treonina/farmacologia
9.
Eur J Biochem ; 58(2): 339-50, 1975 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1183442

RESUMO

We have examined the relationship between alpha and beta globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The alpha globin chain contains three isoleucyl residues while the beta chain of certain rabbits contains no isoleucine. O-Methyl-L-threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into alpha chains in rabbit reticulocytes. When alpha chain synthesis is inhibited by 50-85%, beta synthesis is stimulated by 15-50%. The excess labeled beta chains are not distinguishable from authentic beta chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate-urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine-containing tryptic peptides. The stimulation of beta synthesis continues after the pool of excess alpha chains has been exhausted by preincubation with O-methyl-L-threonine. The stimulation does not occur, however, when 1 mM 2-mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of beta chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate-limiting step in increased beta chain production. The stimulation of beta synthesis when alpha synthesis is inhibited is interpreted as resulting from relief of competition between alpha and beta mRNAs for limiting components of the protein synthetic apparatus.


Assuntos
Globinas/biossíntese , Aminoácidos/metabolismo , Animais , Heterozigoto , Homozigoto , Isoleucina/metabolismo , Lisina/metabolismo , Mercaptoetanol/farmacologia , Metionina/metabolismo , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , Polirribossomos/metabolismo , Coelhos , Reticulócitos/metabolismo , Treonina/análogos & derivados , Treonina/farmacologia
10.
Pediatr Res ; 24(1): 9-13, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3137519

RESUMO

Although dietary leucine restriction and supplemental glycine are used to treat patients with isovaleric acidemia [deficient isovaleryl-CoA-dehydrogenase (E.C.1.3.99.10)], little quantitative information is available regarding their optimum relationship. Herein we compare different glycine supplements and quantitate isovalerylglycine produced in two patients with clinically different forms of isovaleric acidemia during restricted leucine intake and during oral leucine loading. We found that under stable conditions of leucine restriction, 150 mg glycine/kg/day is an optimum glycine supplement and that glycine supplements of more than 250 mg/kg/day may result in reduced isovalerylglycine production; that when isovaleric acid accumulation is increased, glycine supplements to 600 mg/kg/day will increase isovalerylglycine production; and that the phenotype of isovaleric acidemia is related not only to the extent of impaired isovaleryl-CoA dehydrogenase, but also the ability to detoxify accumulated isovaleryl CoA to isovalerylglycine.


Assuntos
Glicina/uso terapêutico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/deficiência , Ácidos Pentanoicos/sangue , Valeratos/sangue , Dióxido de Carbono/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Glicina/análogos & derivados , Glicina/sangue , Glicina/urina , Hemiterpenos , Humanos , Isovaleril-CoA Desidrogenase , Leucina/administração & dosagem , Leucina/sangue , Masculino
11.
Pediatr Res ; 20(11): 1112-6, 1986 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3797105

RESUMO

Phenylketonuria is a human model for the study of the effects of phenylalanine on brain function. We found previously a correlation between high blood phenylalanine, prolonged performance times on neuropsychological tests of higher integrative function, and decreased urinary dopamine in 10 patients. In this protocol we examine changes in triplicate of plasma dihydroxyphenylalanine (L-DOPA) and the mean power frequency of the electroencephalogram in eight additional older patients with phenylketonuria using longer intervals in a blinded, cross-over design. Mean power frequency was obtained by Fourier transform of the power spectrum from traditional eight channel electroencephalograms. Plasma L-DOPA was quantitated by radioenzymatic methods. In all patients statistically significant decreases were found in the mean power frequency of the electroencephalogram and in plasma L-DOPA when plasma L-phenylalanine increased. These findings were reversible and correlated in the reverse direction when plasma L-phenylalanine was reduced. Thus changes in the mean power frequency of electroencephalograms and circulating L-DOPA offer sensitive parameters of human brain function in vivo. These findings indicate reversible effects of elevated plasma phenylalanine on electrical function of the brain which may be mediated in part through inhibition of catecholamine synthesis.


Assuntos
Eletroencefalografia , Levodopa/sangue , Fenilalanina/sangue , Fenilcetonúrias/fisiopatologia , Adolescente , Análise de Variância , Criança , Método Duplo-Cego , Humanos , Fenilalanina/fisiologia , Fenilcetonúrias/sangue , Fenilcetonúrias/tratamento farmacológico , Análise de Regressão
12.
Am J Hum Genet ; 60(2): 366-72, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9012409

RESUMO

Human orythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the "Los Angeles (LA) phenotype," which has increased GALT enzyme activity. We evaluated GALT enzyme activity and screened the GALT genes of 145 patients with one or more N314D-containing alleles. We found seven with the LA biochemical phenotype, and all had a 1721C-->T transition in exon 7 in cis with the N314D missense mutation. The 1721C-->T transition is a neutral polymorphism for leucine at amino acid 218 (L218L). In pedigree analyses, this 1721C-->T transition segregated with the LA phenotype of increased GALT activity in three different biochemical phenotypes (LA/N, LA/G, and LA/D). To determine the mechanism for increased activity of the LA variant, we compared GALT mRNA, protein abundance, and enzyme thermal stability in lymphoblast cell lines of D and LA phenotypes with comparable genotypes. GALT protein abundance was increased in LA compared to D alleles, but mRNA was similar among all genotypes. When LA/D and D/D GALT biochemical phenotypes were compared to N/N GALT phenotypes, both had 50%, as compared to 21%, reduction in GALT activity in the wild type (N/N) after exposure at identical initial enzyme activity to 50 degrees C for 15 min. We conclude that the codon change N314D in cis with the base-pair transition 1721C-->T produces the LA variant of galactosemia and that this nucleotide change increases GALT activity by increasing GALT protein abundance without increasing transcription or decreasing thermal lability. A favorable codon bias for the mutated codon with consequently increased translation rates is postulated as the mechanism.


Assuntos
Galactosemias/genética , Variação Genética , Mutação , UTP-Hexose-1-Fosfato Uridililtransferase/genética , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , Alelos , Linhagem Celular , Códon , Estabilidade Enzimática , Feminino , Galactosemias/enzimologia , Genótipo , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo Conformacional de Fita Simples , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Temperatura , UTP-Hexose-1-Fosfato Uridililtransferase/química
13.
Eur J Pediatr ; 154(7 Suppl 2): S21-7, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7671959

RESUMO

Classical galactosemia (G/G) is caused by the lack of galactose-1-phosphate uridyltransferase (GALT) activity. A more common clinical variant, Duarte/Classical (D/G) produces partial enzymatic impairment. Although neonatal death due to G/G galactosemia has been largely eliminated by population-based screening and intervention, long-term outcome in some is associated with impaired growth, ovarian failure, dyspraxic speech and neurologic deficits. At least 32 variants in the nucleotide sequence of the GALT gene have been identified and 9 have transferred impaired GALT activity to transformed cells in transfection experiments. We here define the prevalence and biochemical phenotype of two mutations. An A to G transition in exon 6 of the GALT gene converts a predicted glutamine at codon 188 to an arginine (Q188R), and introduces a new HpaII cut site into the gene which enables population screening by polymerase chain reaction. An A to G transition in exon 10 in the GALT gene produces a codon change converting an asparagine to aspartic acid at codon 314 (N314D) and adds an AVA II cut site. We screened a large population for the Q188R and N314D sequence changes to investigate the prevalence of Q188R in G/G galactosemia, the effect of homozygosity for Q188R on outcome, and the prevalence and biochemical phenotype of the N314D sequence change. We found that the Q188R mutation has a prevalence of 62% in a predominately Caucasian population of 107 patients with G/G galactosemia. Homozygosity for Q188R was associated with a poor clinical outcome in a subgroup of these patients. The N314D mutation is associated with the Duarte biochemical phenotype with extraordinary concordance.


Assuntos
Galactosemias/genética , Sequência de Bases , Códon , Éxons , Humanos , Dados de Sequência Molecular , Mutação , Transfecção , UTP-Hexose-1-Fosfato Uridililtransferase/genética
14.
J Neurochem ; 43(5): 1257-64, 1984 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6208326

RESUMO

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.


Assuntos
Cálcio/farmacologia , Proteína Básica da Mielina/metabolismo , Fosfolipídeos/farmacologia , Proteínas Quinases/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , AMP Cíclico/farmacologia , Fosforilação , Especificidade por Substrato , Suínos
15.
Am J Hum Genet ; 36(4): 783-90, 1984 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6089551

RESUMO

Collagen lysyl and prolyl hydroxylase activities were measured in cultured fibroblasts from a child with clinical features of Ehlers-Danlos syndrome. Lysyl-to-prolyl hydroxylase activity ratios in cells from the proband, mother, father, and control were .24, .86, .52, and 1.00, respectively, providing a biochemical diagnosis of Ehlers-Danlos syndrome type VI and indicating an autosomal recessive mode of inheritance in this family. Prenatal assessment of lysyl hydroxylase deficiency was requested and accomplished for the first time during a subsequent pregnancy in the family. A series of control cultures established lysyl hydroxylase activity to be similar in cultured amniotic fluid cells (AF and F cells) and in cultured dermal fibroblasts. Cultured F and AF cells from the monitored pregnancy had enzyme activity similar to controls, indicating that the fetus should not be affected by lysyl hydroxylase deficiency. This finding was confirmed by demonstration of normal lysyl hydroxylase activity in fibroblasts cultured from the newborn baby. These studies show that cells cultured from second trimester amniotic fluid have collagen lysyl hydroxylase activity similar to that in dermal fibroblasts, making prenatal diagnosis of lysyl hydroxylase deficiency possible.


Assuntos
Colágeno/metabolismo , Síndrome de Ehlers-Danlos/diagnóstico , Oxigenases de Função Mista/deficiência , Diagnóstico Pré-Natal , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/deficiência , Pele/enzimologia , Adulto , Líquido Amniótico/enzimologia , Células Cultivadas , Criança , Ensaios Enzimáticos Clínicos , Síndrome de Ehlers-Danlos/genética , Feminino , Fibroblastos/enzimologia , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Gravidez , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/genética , Pele/citologia
16.
Hum Mutat ; 11(1): 28-38, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9450900

RESUMO

The Duarte allele (D) is a missense mutation (N314D) that produces a characteristic isoform and partial impairment of galactose-1-phosphate uridyltransferase (GALT) in human erythrocytes, fibroblasts, and transformed lymphoblasts. The position of this amino acid is distant, however, from presumptive catalytic site(s) as deduced from a three-dimensional model of crystallized Escherichia coli galT protein. To evaluate the mechanism(s) involved in the partial impairment of enzymatic activity, we compared the activity, abundance, biological stability, and mRNA of GALT in human lymphoblastoid cell lines cultured from individuals homozygous for wild-type (WT/WT) and Duarte alleles (N314D/N314D). No other nucleotide differences were present in their GALT genes. The apparent Vmax was reduced in N314D/N314D cells to 31 +/- 3.6 compared to WT/WT of 54 +/- 6.5 nmole UDP-galactose formed/g cell protein/hour. Both genotypes had similar apparent KMs for UDP-glucose of 0.142 +/- 0.057 mM and 0.133 +/- 0.056 mM. This reduced Vmax was associated with a reduced abundance of the 86kD GALT dimer as determined by Western blots and densitometry. Using RNase protection assays, this reduced GALT protein in the N314D/N314D cell lines was not associated with reduced abundance of GALT mRNA. Using cycloheximide (3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide) inhibition of de novo protein synthesis, GALT enzyme activity, and its dimeric protein had a biological T1/2 of approximately 24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50 degrees C for 15 minutes, N314D/ N314D lymphoblasts retained 45% of GALT activity, whereas controls retained 77% activity. Reduced activity and thermal sensitivity caused by the N314D mutation reverted to control values when a lysine was substituted for a glutamic acid at amino acid 203 in cis (E203K). In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity, dimeric protein abundance, biological, and thermal stability. We conclude that the substitution of aspartate for asparagine at amino acid 314 in the human GALT protein reduces the biostability of the active enzyme in human lymphoblasts.


Assuntos
Alelos , Galactosemias/genética , Linfócitos/enzimologia , Mutação , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Substituição de Aminoácidos , Linhagem Celular Transformada , Dimerização , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Galactosemias/enzimologia , Temperatura Alta , Humanos , Cinética , RNA Mensageiro/sangue , UTP-Hexose-1-Fosfato Uridililtransferase/sangue
17.
Am J Hum Genet ; 54(6): 1030-6, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8198125

RESUMO

The human cDNA and gene for galactose-1-phosphate uridyl transferase (GALT) have been cloned and sequenced. A prevalent mutation (Q188R) is known to cause classic galactosemia (G/G). G/G galactosemia has an incidence of 1/38,886 in 1,396,766 Georgia live-born infants, but a more common variant of galactosemia, Duarte, has an unknown incidence. The proposed Duarte biochemical phenotypes of GALT are as follows: D/N, D/D, and D/G, which have approximately 75%, 50%, and 25% of normal GALT activity respectively. In addition, the D allele has isoforms of its enzyme that have more acidic pI than normal. Here we systematically determine (a) the prevalence of an A-to-G transition at base pair 2744 of exon 10 in the GALT gene, transition that produces a codon change converting asparagine to aspartic acid at position 314 (N314D), and (b) the association of this mutation with the Duarte biochemical phenotype. The 2744G nucleotide change adds an AvaII (SinI) cut site, which was identified in PCR-amplified DNA. In 111 biochemically unphenotyped controls with no history of galactosemia, 13 N314D alleles were identified (prevalence 5.9%). In a prospective study, 40 D alleles were biochemically phenotyped, and 40 N314D alleles were found. By contrast, in 36 individuals known not to have the Duarte biochemical phenotype, no N314D alleles were found. We conclude that the N314D mutation is a common allele that probably causes the Duarte GALT biochemical phenotype and occurs in a predominantly Caucasian, nongalactosemic population, with a prevalence of 5.9%.


Assuntos
Alelos , Galactosemias/genética , Mutação Puntual/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Sequência de Bases , DNA/sangue , Análise Mutacional de DNA , Eritrócitos/enzimologia , Feminino , Galactosemias/enzimologia , Galactosemias/epidemiologia , Humanos , Isoenzimas/sangue , Dados de Sequência Molecular , Fenótipo , Prevalência , UDPglucose-Hexose-1-Fosfato Uridiltransferase/sangue
18.
J Pediatr ; 128(1): 89-95, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8551426

RESUMO

OBJECTIVE: To define the mutation causing galactosemia in patients of black American origin who have no galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes but good clinical outcome. METHODS: We discovered a mutation caused by a C-->T transition at base-pair 1158 of the GALT gene that results in a serine-to-leucine substitution at codon 135 (S135L). We developed a method with which to screen populations for its prevalence. We compared galactose-1-phosphate uridyltransferase among erythrocytes, leukocytes, and transformed lymphoblasts, as well as total body oxidation of D-(13C)-galactose to 13CO2 among three genotypes for GALT (S135L/S135L, Q188R/Q188R, and Normal/Normal). RESULTS: We found a 48% prevalence of the S135L mutation among 17 black American patients with classic galactosemia and a 1% prevalence in a population of 50 black Americans without galactosemia. The S135L mutation was not found in 84 white patients with G/G galactosemia nor in 87 white control subjects without galactosemia. We found normal whole body oxidation of D-(13C)-galactose by the patient homozygous for S135L and various degrees of enzyme impairment among different tissues. CONCLUSIONS: The S135L mutation in the GALT gene is a prevalent cause of galactosemia among black patients. Because GALT activity varies in different tissues of patients homozygous for S135L, they may have a better clinical outcome than patients who are homozygous for Q188R when both are treated from infancy.


Assuntos
População Negra/genética , Galactosemias/genética , Mutação Puntual , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Sequência de Bases , Eritrócitos/enzimologia , Feminino , Galactosemias/dietoterapia , Genótipo , Homozigoto , Humanos , Recém-Nascido , Leucócitos/enzimologia , Dados de Sequência Molecular , Fenótipo , Vigilância da População , Prevalência
19.
Genet Med ; 1(1): 34-9, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-11261427

RESUMO

PURPOSE: Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport and manifests as nonketotic hypoglycemia or skeletal or heart myopathy. METHODS: To define the mechanisms producing partially reduced plasma carnitine levels in the parents of affected patients, we examined carnitine transport in vivo and in the fibroblasts of a new patient and his heterozygous parents. RESULTS: Kinetic analysis of carnitine transport in fibroblasts revealed an absence of saturable carnitine transport in the proband's cells and a partially impaired carnitine transport in fibroblasts from both parents, whose cells retained normal Km values toward carnitine (6-9 microM) but reduced Vmax. At steady state, normal fibroblasts accumulated carnitine to a concentration that was up to 80 times the extracellular value (0.5 microM). By contrast, cells from the proband had minimal carnitine accumulation, and cells from both parents had intermediate values of carnitine accumulation. Plasma carnitine levels were slightly below normal in both heterozygous, yet clinically normal, parents and in the paternal grandfather and the maternal grandmother. To define the mechanism producing partially decreased carnitine levels, we studied urinary carnitine losses in heterozygous parents compared with controls. Urinary losses increased linearly (P < 0.05) with plasma carnitine levels in normal controls. When urinary carnitine losses were normalized to plasma carnitine levels, a significant difference was observed between controls and heterozygous individuals (P < 0.01). CONCLUSIONS: These results indicate that fibroblasts from heterozygotes for primary carnitine deficiency have a decreased capacity to accumulate carnitine and that heterozygotes have increased urinary losses, which may contribute to their reduced plasma carnitine levels.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Carnitina/deficiência , Carnitina/urina , Heterozigoto , Erros Inatos do Metabolismo dos Aminoácidos/urina , Carnitina/sangue , Humanos , Recém-Nascido , Cinética , Masculino
20.
Pediatr Res ; 19(10): 1011-6, 1985 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3903643

RESUMO

We measured the biochemical response for four patients with maple syrup disease to pharmacologic doses of thiamine, and correlated their response to their branched chain alpha-ketoacid dehydrogenase activity. We observed a linear correlation between the concentrations of each plasma branched-chain amino acid and its corresponding ketoacid analogue. In addition, the renal tubular reabsorption of branched-chain amino and ketoacids was nearly complete within these physiologic concentrations. Three children responded to thiamine therapy with a reduction in concentration of plasma and urinary branched-chain amino and ketoacids. Each responder had at least 5% activity for branched chain alpha-ketoacid dehydrogenase in their mononuclear blood cells and in whole cell fibroblasts from cultured skin when compared to the activity in normal control cells. We propose that each child with maple syrup urine disease be assessed for their response to thiamine by quantifying the concentration of branched-chain amino acids in plasma before and after vitamin supplementation.


Assuntos
Doença da Urina de Xarope de Bordo/metabolismo , Tiamina/uso terapêutico , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida) , Aminoácidos de Cadeia Ramificada/sangue , Aminoácidos de Cadeia Ramificada/urina , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Cetoácidos/sangue , Cetoácidos/urina , Cetona Oxirredutases/análise , Túbulos Renais/metabolismo , Leucina/sangue , Masculino , Doença da Urina de Xarope de Bordo/tratamento farmacológico , Monócitos/enzimologia , Complexos Multienzimáticos/análise
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