Genetic defects in E3 component of alpha-keto acid dehydrogenase complexes.
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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Biomedical subjects
M S Patel
Publications and source records attributed to M S Patel.
Targeting E3 component of alpha-keto acid dehydrogenase complexes.
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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Three novel SALL1 mutations extend the mutational spectrum in Townes-Brocks syndrome.
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Abnormalities, Multiple↗
Adaptive changes in insulin secretion by islets from neonatal rats raised on a high-carbohydrate formula.
Artificial rearing of neonatal rats on a high-carbohydrate (HC) milk formula resulted in the immediate onset of hyperinsulinemia. This study examines, in islets of 12-day-old HC rats, adaptive changes that support the hyperinsulinemic state. Increases in plasma glucagon-like peptide-1 (GLP-1) levels and islet GLP-1 receptor mRNA supported increased insulin secretion by HC islets. Isolated HC islets, but not mother-fed (MF) islets, secreted moderate amounts of insulin in a glucose- and Ca(2+)-independent manner. Under stringent Ca(2+)-free conditions and in the presence of glucose, GLP-1 plus acetylcholine augmented insulin release to a larger extent in HC islets. Levels of adenylyl cyclase type VI mRNA and activities of protein kinase A, protein kinase C, and calcium calmodulin kinase II were increased in HC islets. A tenfold increase in norepinephrine concentration was required to inhibit insulin secretion in HC islets compared with MF islets, indicating reduced sensitivity to adrenergic signals. This study shows that significant alterations at proximal and distal sites of the insulin secretory pathway in HC islets may support the hyperinsulinemic state of these rats.
Adaptation, Physiological↗
Alleles of the estrogen receptor alpha-gene and an estrogen receptor cotranscriptional activator gene, amplified in breast cancer-1 (AIB1), are associated with quantitative calcaneal ultrasound.
Quantitative bone ultrasound (QUS) has a significant heritable component. Because estrogen is required for attainment of peak bone mass, we studied alleles of two genes, estrogen receptor alpha (ER1) and amplified in breast cancer-1 (AIB1), for their association with QUS. In a volunteer sample of 663 white women aged 18-35 years, bone ultrasound attenuation (BUA), speed of sound (SOS), and heel stiffness index (SI), the latter consisting of the component measures of BUA and SOS, were measured at the right calcaneus by QUS. Subjects were genotyped for the ER1 polymorphisms Xba I and Pvu II and for the AIB1 polyglutamine tract polymorphism. In a multiple regression analysis, ER1 genotype was an independent predictor of QUS-SI (p = 0.03). Because AIB1 and ER1 enhance gene expression in a coordinate manner, we also searched for interactions. A gene-by-gene interaction effect was seen for QUS-SI (p = 0.009), QUS-BUA (p = 0.03), and QUS-SOS (p = 0.004). These remained significant after the inclusion of clinically relevant variables into the final regression model. Overall, these clinical and genetic factors accounted for up to 16% of the variance in peak QUS; the genetic markers alone accounted for 4-7%. This is the first demonstration of specific genetic effects on calcaneal QUS encoded by alleles of genes directly involved in mediating estrogen effects on bone.
Adolescent↗
Metabolic programming by nutrition during early development.
Incidence of obesity and diabetes is increasing at an alarming rate not only among the populations of the affluent nations but also amongst the populations of the developing nations. Understanding the mechanisms that cause the onset of these pathological conditions is a requisite to effectively tackling this problem. In this context the role of early nutritional experiences as a causative factor is being extensively investigated. This article briefly reviews the field of metabolic programming vis-a-vis an altered nutritional milieu during perinatal period and consequent adaptive metabolic patterning and metabolic imprinting in adult and/or consequent offspring.
Adaptation, Physiological↗
Characterization of the regulatory region of the human testis-specific form of the pyruvate dehydrogenase alpha-subunit (PDHA-2) gene.
The alpha-subunit of human pyruvate dehydrogenase (E(1)) is encoded by two separate genes. The gene located on chromosome X (PDHA-1) is expressed in somatic tissues, whereas the second gene (PDHA-2), located on chromosome 4, is expressed only in post-meiotic spermatogenic cells. A genomic fragment harboring the human gene encoding PDHA-2 has been isolated and approximately 800 nucleotides of the promoter region have been characterized. Functional studies of the promoter indicate the presence of both enhancer and repressor elements that are common to other genes that are only expressed in mature sperm.
Base Sequence↗
Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvate dehydrogenase catalysis.
Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.
Catalytic Domain↗
Early-infantile galactosialidosis: prenatal presentation and postnatal follow-up.
Galactosialidosis (GS) is an autosomal recessive condition caused by combined deficiency of the lysosomal enzymes beta-galactosidase and alpha-neuraminidase. The combined deficiency has been found to result from a defect in protective protein/cathepsin A (PPCA), an intralysosomal protein which protects these enzymes from premature proteolytic processing. The most severe form of GS, the early-infantile form, results in early onset of edema, ascites, visceromegaly, and skeletal dysplasia. We report a case of early-infantile GS in a male infant who presented with nonimmune fetal hydrops (NIH), "coarse" facial appearance, massive fluid-filled inguinal hernias, multiple telangiectasia, and diffuse hypopigmentation; he subsequently developed visceromegaly. The diagnosis of GS was confirmed biochemically and the defect in PPCA characterized at the protein level. Examination of fetal peripheral blood smears sampled at 30 weeks gestation demonstrated vacuolation of lymphocytes, suggesting blood film examination may be a useful screening tool for cases of NIH where a metabolic disorder is suspected. Skeletal radiography at birth demonstrated punctate epiphyses of the femora, calcanei, and sacrum. We present a discussion of and differential diagnosis for this radiographic finding. To the best of our knowledge, this is the first case of early-infantile GS presenting with stippled epiphyses.
Blotting, Western↗
Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.
Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.
2,6-Dichloroindophenol↗
Cloning and characterization of a 5.9 kb promoter region of the human pyruvate dehydrogenase alpha subunit gene.
A human genomic clone containing a 5.9 kb promoter region of the human pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) was isolated from a human X-chromosome library. The nucleotide sequence showed two Alu repeats at the -2880 and -2200 bp regions. Comparison between the -1400 bp E1alpha promoter and the -1241 bp E1beta promoter revealed a 57% homology, with a high degree of homology at the putative protein binding regions in these two promoters. Computer-aided transcription factor binding consensus sequence analysis revealed the presence of PPAR, HOXD, MyoD and other tissue-specific transcription factor binding sites. Promoter function analysis using the chloramphenicol acetyltransferase reporter gene indicated that the -2.2 kb/-1.7 kb and -5.9 kb/-5.2 kb regions of the E1alpha promoter may possess negative regulatory elements which are likely to function in a tissue-specific manner.
Amino Acid Sequence↗
Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene.
The 5'-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) gene was characterized for its promoter activity. DNase I footprinting analysis of the promoter region (-545 bp to +41 bp) revealed six major protein-binding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast nuclear extracts. Transient transfection assays, using a series of nested deletions of the 2.5 kb 5'-flanking region ligated to the chloramphenicol acetyltransferase reporter gene, identified that the -42-bp to +41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional activity. When the 5'-flanking region was extended from -42 bp to -82 bp, there was an approx. 5-fold increase in promoter activity. To identify further the cis elements involved in transcription of the Dld gene (-82 bp to +41 bp), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobility shift assays. Mutation or deletion of the CACGAC direct repeat, located from -61 bp to -46 bp, resulted in minimal promoter activity. Mutation of the Ets motif, located from -37 bp to -32 bp, reduced the minimal promoter activity by approx. 50%, whereas the deletion of this motif almost abolished the promoter activity. These results indicate that: (i) the Ets motif is required for the minimal promoter activity and (ii) the CACGAC direct repeat enhances promoter activity. Database searches failed to identify the direct repeat with the CACGAC motif and hence the CACGAC sequence may represent a novel motif. The requirement of both the Ets motif and the direct repeat element for optimal promoter activity represents a unique combination for gene transcription.
3T3 Cells↗
Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate.
The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.
Base Sequence↗
The inhibitory effects of lipoic compounds on mammalian pyruvate dehydrogenase complex and its catalytic components.
To examine the stereospecific effects of lipoic compounds on pyruvate metabolism, the effects of R-lipoic acid (R-LA), S-lipoic acid (S-LA) and 1,2-diselenolane-3-pentanoic acid (Se-LA) on the activities of the mammalian pyruvate dehydrogenase complex (PDC) and its catalytic components were investigated. Both S-LA and R-LA markedly inhibited PDC activity; whereas Se-LA displayed inhibition only at higher concentrations. Examination of the effects on the individual catalytic components indicated that Se-LA inhibited the pyruvate dehydrogenase component; whereas R-LA and S-LA inhibited the dihydrolipoamide acetyltransferase component. The three lipoic compounds lowered dihydrolipoamide dehydrogrenase (E3) activity in the forward reaction by about 30 to 45%. The kinetic data of E3 showed that both R-LA and Se-LA are used as substrates by E3 for the reverse reaction. Decarboxylation of [1-14C]pyruvate via PDC by cultured HepG2 cells was not affected by R-LA, but moderately decreased with S-LA and Se-LA. These findings indicate that (i) purified PDC and its catalytic components are affected by lipoic compounds based on their stereoselectivity; and (ii) the oxidation of pyruvate by intact HepG2 cells is not inhibited by R-LA. The later finding with the intact cells is in support of therapeutic role of R-LA as an antioxidant.
Acetyltransferases↗
Evaluating the use of penicillin to control outbreaks of acute poststreptococcal glomerulonephritis.
INTRODUCTION: Outbreaks of acute poststreptococcal glomerulonephritis (APSGN), occur every few years in remote Australian Aboriginal communities. Intervention with benzathine penicillin G (BPG) to all children is effective in reducing streptococcal carriage in a community, but its effectiveness in arresting outbreaks of APSGN has not been established. OBJECTIVE: To study nine recent community outbreaks of APSGN in Australia's Northern Territory and compare them with outbreaks reported in the literature to assess the impact of intervention with BPG. METHODS: Because randomized controlled trials have not been conducted for this purpose, we assessed data from published observational studies and relevant experiences in the Northern Territory (NT). Eight of the nine outbreaks in the NT were studied prospectively. An outbreak was defined as two or more clinical cases of APSGN occurring within 1 week in a single community. Three intervention methods were used: intramuscular BPG to all children ages 3 to 15 years; BPG only to children with skin lesions; and BPG only to child contacts of clinical cases. The attack rates, number of clinical cases before and after the interventions were documented and the coverage of children with penicillin were estimated. RESULTS: A review of the literature found very little evidence either for or against the effectiveness of intervention with BPG. In our study four communities used the first method of intervention. The community with the lowest uptake of penicillin continued to have cases in untreated children for 9 further weeks, two communities had no new cases from 3 weeks after the intervention and the fourth had a single further case after 4 weeks. The one community that used the second method had a high initial attack rate but no further cases from 1 week after the intervention. Three communities used the third method and in one community no intervention was attempted. CONCLUSION: Our observational study supports the use of BPG in the community to prevent new cases of APSGN. It suggests that targeted treatment of children with skin sores and household contacts of cases, rather than attempted treatment of all children in the community, could be an effective method of intervention.
Acute Disease↗
Basal cell carcinoma with lung metastasis diagnosed by fine-needle aspiration biopsy.
Basal cell carcinoma of the skin is one of the most common types of cancer. Its natural history is one of local recurrence rather than metastatic spread. Certain histologic features and primary tumor size seem to be risk factors for metastases. The diagnosis of metastatic disease imparts a poor prognosis with a short median survival. Treatment is usually in the form of systemic chemotherapy with cisplatin-based combination described as most active agent.
Aged↗
A dietary intervention (high carbohydrate) during the neonatal period causes islet dysfunction in rats.
Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats and compared with age-matched mother-fed (MF) pups. The plasma insulin and glucagon contents were increased sixfold and twofold, respectively, in HC rats compared with MF rats. There was a distinct leftward shift in the glucose-stimulated insulin secretory pattern for HC islets. HC islets secreted insulin in the absence of any added glucose and in the presence of Ca(2+) channel inhibitors. The activities of glucokinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase complex were significantly increased in HC islets compared with MF islets. The protein contents of GLUT-2 and hexokinase were significantly increased in HC islets. These findings indicate that a nutritional intervention in the form of a HC formula only during the suckling period has a profound influence on pancreatic function, causing the onset of hyperinsulinemia.
Age Factors↗
Regulation of mammalian pyruvate dehydrogenase alpha subunit gene expression by glucose in HepG2 cells.
We report the effect of glucose on the expression of the gene encoding the pyruvate dehydrogenase (E1) alpha subunit (E1alpha) in human hepatoma (HepG2) cells. Total pyruvate dehydrogenase complex activity as well as the levels of protein and mRNA of the E1alpha subunit were significantly increased in HepG2 cells cultured in medium containing 16.7 mM glucose compared with 1.0 mM glucose for a period of 4 weeks. The level of E1alpha mRNA was elevated approx. 2-fold in HepG2 cells cultured for 24 h in medium containing 16.7 mM glucose compared with 1 mM glucose. This effect was specific to glucose and independent of insulin. Nuclear run-on assays and promoter analysis indicate that the glucose-induced increases in the levels of E1alpha mRNA in HepG2 cells are due to increased transcription of the human E1alpha (PDHA1) gene. Mutational analysis of the E1alpha promoter region has identified two regions, from -78 to -73 bp (CCCCTG) and from -8 to -3 bp (GCGGTG), that are responsible for the effect of glucose on promoter activity; the former exhibits a larger effect. These two sequences represent new variations of the carbohydrate-response element that has been identified in other genes. The stimulation of E1alpha promoter activity by glucose was abolished by okadaic acid at 100 nM but not at 5 nM, suggesting that glucose-mediated regulation of pyruvate dehydrogenase complex E1alpha gene transcription involves a phosphorylation/dephosphorylation mechanism, possibly involving protein phosphatase-1.
Chloramphenicol O-Acetyltransferase↗