Lipoic acid as an antioxidant. The role of dihydrolipoamide dehydrogenase.
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Animals↗
Biomedical subjects
M S Patel
Publications and source records attributed to M S Patel.
The golem of osteoporosis genetics.
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Arthritis, Rheumatoid↗
Deficiency of dihydrolipoamide dehydrogenase due to two mutant alleles (E340K and G101del). Analysis of a family and prenatal testing.
A male child with metabolic acidosis was diagnosed as having dihydrolipoamide dehydrogenase (E3) deficiency. E3 activity of the proband's cultured fibroblasts and blood lymphocytes was 3-9% of normal, while in the parent's lymphocytes it was about 60% of normal. The proband's pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex activities from cultured skin fibroblasts were 12% and 6% of normal, respectively. PDC activity in the parents cultured fibroblasts was 25-31% of normal. Western and Northern blot analyses showed similar quantities of E3 protein and mRNA in cultured fibroblasts from the proband and his parents. DNA sequencing of cloned full-length E3 cDNAs, from the proband and the parents, showed two mutations on different alleles of proband were inherited from the parents. One mutation is a three nucleotide (AGG) deletion, from the mother, resulting in deletion of Gly101 in the FAD binding domain. The other mutation is a nucleotide substitution (G to A), from the father, leading to substitution of Lys for Glu340 in the central domain. The same deletion mutation was found in E3 cDNA from a chorionic villus sample and cultured fibroblasts obtained from the mother's subsequent offspring. This finding illustrates the possibility of successful prenatal diagnosis of E3 deficiency utilizing mutations characterized prior to initiation of pregnancy.
Acidosis↗
Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality.
The Dld gene product, known as dihydrolipoamide dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched-chain alpha-ketoacid dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine Dld gene (Dldtm1mjp). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the Dld-/- class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The Dld-/- embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides in vivo evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.
Animals↗
New guidelines for management and prevention of meningococcal disease in Australia. Meningococcal Disease Working Party of the National Health and Medical Research Council.
The incidence of invasive meningococcal disease in Australia has increased over the past decade, and in April 1997 the National Health and Medical Research Council published guidelines for management of patients with meningococcal disease and their contacts. These guidelines emphasise the need for immediate intravenous antibiotic treatment of patients with suspected meningococcal disease, before transfer to hospital or lumbar puncture. When possible, blood for culture should be collected before antibiotic therapy, if this does not delay treatment.
Adolescent↗
Characterization of the mouse dihydrolipoamide dehydrogenase (Dld) gene: genomic structure, promoter sequence, and chromosomal localization.
The mouse dihydrolipoamide dehydrogenase (Dld) gene has been cloned, characterized, and mapped. This nuclear gene encodes a mitochondrial protein that is shared among several alpha-keto acid dehydrogenase complexes and the glycine cleavage system. The Dld gene is contained within an approximately 21-kb region and consists of 14 exons ranging in size from 69 to 521 nucleotides. The open reading frame codes for a preprotein of 509 amino acids with a predicted mature protein of 474 amino acids that is highly conserved among mammalian species (> 90% identical). Primer extension analyses have shown the gene to have transcription initiation sites with tissue-specific differences in relative utilization. The 5' flanking region is G-C rich and lacks a TATA box, but does contain initiator element and multiple transcription factor-binding consensus sequences. Northern blot analysis shows that the Dld mRNA in various tissues is approximately 2.4 kb in size. The Dld gene has been localized to the proximal region of chromosome 12, approximately 21 cM from the centromere.
Amino Acid Sequence↗
Outcome of pyruvate dehydrogenase deficiency treated with ketogenic diets. Studies in patients with identical mutations.
Inborn errors of the pyruvate dehydrogenase complex (PDC) are associated with lactic acidosis, neuroanatomic defects, developmental delay, and early death. PDC deficiency is a clinically heterogeneous disorder, with most mutations located in the coding region of the X-linked alpha subunit of the first catalytic component, pyruvate dehydrogenase (E1). Treatment of E1 deficiency hs included cofactor replacement, activation of PDC with dichloroacetate, and ketogenic diets. In this report, we describe the outcome of ketogenic diet treatment in seven boys with E1 deficiency. These patients were divided into two groups based on their mutations (R349H, three patients; and R234G, four patients, two sibling pairs). All seven patients received ketogenic diets with varying degrees of carbohydrate restriction. Clinical outcome was compared within each group and between siblings as related to the intensity and duration of dietary intervention. Subjects who either had the diet initiated earlier in life or who were placed on greater carbohydrate restriction had increased longevity and improved mental development. Based on the improved outcomes of patients with identical mutations, it appears that a nearly carbohydrate-free diet initiated shortly after birth may be useful in the treatment of E1 deficiency.
Child Development↗
Pharmacy education and practice in France.
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Education, Pharmacy↗
Tissue-specific expression of the human pyruvate dehydrogenase alpha (Pdha-1)/chloramphenicol acetyltransferase fusion gene in transgenic mice.
A chimeric gene (-763/+33Pdha-1/CAT) containing -763/+33 nucleotides o f the human pyruvate dehydrogenase gene (Pdha-1) and the structural gene of chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. CAT activity was detected predominantly in the brain followed in decreasing order by adipose tissue, spleen, heart, kidney and liver. Developmental expression of CAT activity in the testes was similar to that of the endogenous Pdha-1 subunit expression in the testes. Dietary regulation of the transgene was comparable to the expression of endogenous pyruvate dehydrogenase complex in kidney and adipose tissue. Thus, the -763/+33 bp segment of the human Pdha-1 gene is transcriptionally active in vivo and can direct the expression of CAT in several tissues.
Adipose Tissue↗
Three new mutations of the pyruvate dehydrogenase alpha subunit: a point mutation (M181V), 3 bp deletion (-R282), and 16 bp insertion/frameshift (K358SVS-->TVDQS).
Three novel mutations in the coding region of E1 alpha gene were found in three PDC-deficient male patients, including a missense mutation (M181V), a 3 bp deletion (AGA, corresponding to R282), and a 16 bp insertion (CAGTGGATCAAGTTTA), causing a frameshift starting with lysine 358 and resulting in decrease of both E1 subunits.
Cells, Cultured↗
Loglinear models using capture-recapture methods to estimate the size of a measles epidemic.
Cases of measles reported separately by doctors, hospitals, laboratories, and others are treated as separate captures and releases of the population of children aged up to 10 years who have been infected by measles in a recent epidemic. Loglinear models are used to assess the adequacy of fit of various models for capture-recapture and the size of the epidemic in different age groups is estimated.
Australia↗
Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency.
An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.
Alleles↗
Dihydrolipoamide dehydrogenase: structural and mechanistic aspects.
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Amino Acid Sequence↗
Glycogen synthase regulation in hyperinsulinemic/obese progeny of rats fed a high carbohydrate formula in their infancy.
OBJECTIVE: To evaluate the effects of chronic hyperinsulinemia/obesity on the proximal events leading to the activation of glycogen synthase. DESIGN: 100 d old second generation of chronically hyperinsulinemic/obese rats born to mothers which were artificially reared on a high carbohydrate (HC) milk formula in their infancy were used for this study and compared with mother-fed (MF) controls. MEASUREMENTS: Glycogen, glycogen synthase, protein phosphatase-1 (PP-1), mitogen-activated protein kinase (MAPK), insulin-stimulated protein kinase (ISPK) and protein kinase A (PKA) were measured in liver and muscle of both MF and HC rats. RESULTS: Glycogen content, glycogen synthase and PP-1 activities were significantly reduced in liver and muscle of HC rats compared to MF controls while trypsin released PP-1 activity was elevated. The activities of both MAPK and ISPK were also decreased in the HC rats. In contrast PKA activity was increased. CONCLUSIONS: Glycogen synthase activity in the basal state may be impaired in the hyperinsulinemic HC rats in two ways: (i) by a decrease in the activities of the kinases that presumably activate PP-1 and (ii) by increased activity of PKA which inactivates glycogen synthase directly by phosphorylation and indirectly by its effects on PP-1.
Animals↗
Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase.
We have modified bovine pyruvate dehydrogenase (E1), the first catalytic component of the pyruvate dehydrogenase complex, with pyreneglyoxal. Treatment of E1 with pyreneglyoxal resulted in the loss of enzyme activity. Pyruvate plus thiamin pyrophosphate (TPP) afforded approximately 80% protection against this inactivation and protected two arginine residues per mol of E1 tetramer (alpha 2 beta 2) from modification. Circular dichroism spectral analysis indicated absence of any gross structural changes in the enzyme as a result of modification. Comparison of the peptide maps, monitored at 345 nm of unprotected and pyruvate plus TPP protected E1s after V8 digestion revealed that a peptide in the protected enzyme was labeled by pyreneglyoxal to a lesser extent than its counterpart in the unprotected enzyme. Sequence analysis of the peptide demonstrated that it corresponded precisely to amino-acid residues 235 to 246 in the human E1 beta sequence, with arginine residues at positions 239 and 242. Since Arg-239 is conserved in the beta-subunit of all presently known sequences of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex, it is strongly suggested that Arg-239 in the human E1 beta sequence is at or near the active site of bovine E1.
Amino Acid Sequence↗
Pretranslational regulation of pyruvate dehydrogenase complex subunits in white adipose tissue during the suckling-weaning transition in the rat.
Total pyruvate dehydrogenase complex activity is low in white adipose tissue during the suckling period and increases markedly at weaning on to a high-carbohydrate diet. This is concomitant with an increase in the E1 alpha, E1 beta and E2 subunit protein concentration and their respective mRNAs, suggesting a pretranslational control of this phenomenon. The most marked change is seen for the E1 alpha subunit (17-fold increase in protein concentration). The changes in pyruvate dehydrogenase complex activity and subunit abundance induced by weaning on to a high-carbohydrate diet are precluded if the animals are weaned on to a high-fat diet, suggesting that the nutritional and/or related hormonal changes rather than a developmental stage are responsible for the observed adipose-tissue pyruvate dehydrogenase complex pattern.
Adipose Tissue↗
Spectroscopic studies of the characterization of recombinant human dihydrolipoamide dehydrogenase and its site-directed mutants.
In this paper, we report the overexpression and single-step purification of recombinant wild-type and site-directed mutants of human dihydrolipoamide dehydrogenase in Escherichia coli and detailed spectroscopic studies aimed at understanding the catalytic mechanism of this enzyme. One mutation (K37E) has been identified in a patient lacking dihydrolipoamide dehydrogenase activity and has been reported previously (Liu, T.-C., Kim, H., Arizmendi, C., Kitano, A., and Patel, M. S. (1993) Proc. Natl. Acad. Sci. USA. 90, 5186-5190), while the other two mutations were previously generated specifically to address the role of the active-site base (His-452) and its ion pair (Glu-457). Circular dichroic and fluorescence spectroscopic data illustrate the role of these amino acids in maintaining the structure and function of human dihydrolipoamide dehydrogenase. While mutant H452Q is severely crippled in catalysis of the physiological reaction, the reverse reaction is affected in the E457Q mutant. The K37E mutant shows very little deviation from the wild-type enzyme.
Base Sequence↗
Mutagenesis studies of the phosphorylation sites of recombinant human pyruvate dehydrogenase. Site-specific regulation.
Mammalian pyruvate dehydrogenase (alpha 2 beta 2) (E1) is regulated by phosphorylation-dephosphorylation, catalyzed by the E1-kinase and the phospho-E1-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E1 alpha, several human E1 mutants were made with single, double, and triple mutations by changing Ser to Ala. Mutation at site 1 but not at sites 2 and/or 3 decreased E1 specific activity and also increased Km values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E1 mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E1 kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E1. However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E1-phosphatase resulting in complete reactivation of the E1. The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.
Alanine↗