PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “LIPOAMIDE DEHYDROGENASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources
PubMed2002

The E1beta and E2 subunits of the Bacillus subtilis pyruvate dehydrogenase complex are involved in regulation of sporulation.

The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1alpha and E1beta), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of the pdhC gene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1alpha and E1beta. Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1alpha is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1beta and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.

Bacillus subtilis↗
PubMed1997

Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

3T3 Cells↗
PubMed2005

The SR protein SC35 is responsible for aberrant splicing of the E1alpha pyruvate dehydrogenase mRNA in a case of mental retardation with lactic acidosis.

Pyruvate dehydrogenase (PDH) complex deficiency is a major cause of lactic acidosis and Leigh's encephalomyelopathies in infancy and childhood, resulting in early death in the majority of patients. Most of the molecular defects have been localized in the coding regions of the E1alpha PDH gene. Recently, we identified a novel mutation of the E1alpha PDH gene in a patient with an encephalopathy and lactic acidosis. This mutation, located downstream of exon 7, activates a cryptic splice donor and leads to the retention of intronic sequences. Here, we demonstrate that the mutation results in an increased binding of the SR protein SC35. Consistently, ectopic overexpression of this splicing factor enhanced the use of the cryptic splice site, whereas small interfering RNA-mediated reduction of the SC35 protein levels in primary fibroblasts from the patient resulted in the almost complete disappearance of the aberrantly spliced E1alpha PDH mRNA. Our findings open the exciting prospect for a novel therapy of an inherited disease by altering the level of a specific splicing factor.

Acidosis, Lactic↗
PubMed1993

X chromosome inactivation and the diagnosis of X linked disease in females.

In studies of female patients with suspected deficiency of the E1 alpha subunit of the pyruvate dehydrogenase complex, we have found that X inactivation ratios of 80:20 or greater occur at sufficient frequency in cultured fibroblasts to make exclusion of the diagnosis impossible in about 25% of cases. Pyruvate dehydrogenase E1 alpha subunit deficiency is an X linked inborn error of metabolism which is well defined biochemically and is unusual in that most heterozygous females manifest the condition. The diagnosis is usually established by measurement of enzyme activity and the level of immunoreactive protein and these analyses are most commonly performed on cultured fibroblasts from the patients. Skewed patterns of X chromosome inactivation make it impossible to exclude the diagnosis if the normal X chromosome is expressed in the majority of cells. While most of the observed variation appears to be the expected consequence of random X inactivation, it may be further exaggerated by sampling and subsequent expansion of the cells for analysis.

Cells, Cultured↗
PubMed2002

Detection of pyruvate dehydrogenase E1 alpha-subunit deficiencies in females by immunohistochemical demonstration of mosaicism in cultured fibroblasts.

Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1 alpha-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1 alpha deficiency. We also show that anti-E1 alpha and anti-E2 MAbs, when used in immunocytochemical analysis, can detect mosaicism in cell cultures from female patients in which as few as 2-5% of cells express the deficiency. This immunocytochemical approach, which is fast, reliable, and quantitative, will be particularly useful in identifying females with PDH E1 alpha-subunit deficiency as a precursor to mutation analysis.

Antibodies, Monoclonal↗
PubMed1991

Demonstration of an unstable variant of pyruvate dehydrogenase protein (E1) in cultured fibroblasts from a patient with congenital lactic acidemia.

The deficiency of pyruvate dehydrogenase enzyme complex causes congenital lactic acidemia and devastating neurologic abnormalities in newborns and children. In the majority of cases, the basic defect appears to be in the pyruvate dehydrogenase (E1) component, which consists of two subunits, alpha and beta. Whereas some patients are deficient of a single subunit, in other patients both subunits of E1 are missing. To find out why two proteins were deficient, we investigated the cultured fibroblasts of a female patient who had missing E1-alpha and E1-beta protein bands on Western blot. Radiolabeling-immunoprecipitation studies with 35S-methionine revealed that patient fibroblasts synthesized normal sized precursor E1-alpha and E1-beta proteins, which were presumably transported into mitochondria and processed into normal sized mature proteins. However, pulse-chase analysis showed that alpha- and beta-proteins were degraded rapidly compared to normal. Our findings proved that alpha- and beta-subunits were synthesized and processed normally but failed to form a stable structure for incorporation into the pyruvate dehydrogenase complex.

Cells, Cultured↗
PubMed1994

Aberrant splicing of exon 6 in the pyruvate dehydrogenase-E1 alpha mRNA linked to a silent mutation in a large family with Leigh's encephalomyelopathy.

Pyruvate dehydrogenase (PDH)-E1 alpha deficiency has recently been studied at the molecular-genetic level. The gene is situated on the X chromosome. We report on an unusual mutation in a familial E1 alpha deficiency. In fibroblasts, PDH deficiency was diagnosed in a young infant presenting with Leigh's encephalomyelopathy and in a maternal nephew with episodes of "malaises." In the two affected children as well as their mothers we found a silent mutation in exon 6 of the PDH-E1 alpha and an aberrant splicing of exon 6 in some of the cDNA clones. This case emphasizes the need for both genomic and cDNA analysis in cases where a PDH-E1 alpha deficiency is strongly suspected.

Base Sequence↗
PubMed2000

Analysis of exonic mutations leading to exon skipping in patients with pyruvate dehydrogenase E1 alpha deficiency.

The pyruvate dehydrogenase (PDH) complex is situated at a key position in energy metabolism and is responsible for the conversion of pyruvate to acetyl CoA. In the literature, two unrelated patients with a PDH complex deficiency and splicing out of exon 6 of the PDH E1 alpha gene have been described, although intronic/exonic boundaries on either side of exon 6 were completely normal. Analysis of exon 6 in genomic DNA of these patients revealed two exonic mutations, a silent and a missense mutation. Although not experimentally demonstrated, the authors in both publications suggested that the exonic mutations were responsible for the exon skipping. In this work, we were able to demonstrate, by performing splicing experiments, that the two exonic mutations described in the PDH E1 alpha gene lead to aberrant splicing. We observed a disruption of the predicted wild-type pre-mRNA secondary structure of exon 6 by the mutated sequences described. However, when we constructed mutations that either reverted or disrupted the wild-type predicted pre-mRNA secondary structure of exon 6, we were unable to establish a correlation between the aberrant splicing and disruption of the predicted structure. The mutagenic experiments described here and the silent mutation found in one of the patients suggest the presence of an exonic splicing enhancer in the middle region of exon 6 of the PDH E1alpha gene.

Amino Acid Substitution↗
PubMed2003

Differential effect of DCA treatment on the pyruvate dehydrogenase complex in patients with severe PDHC deficiency.

Dichloroacetate (DCA) is a structural analog of pyruvate that has been recommended for the treatment of primary lactic acidemia, particularly in patients with pyruvate dehydrogenase (PDHC) deficiency. Recent reports have demonstrated that the response to DCA may depend on the type of molecular abnormality. In this study, we investigated the response to DCA in various PDHC-deficient cell lines and tried to determine the mechanism involved. The effect of chronic 3-d DCA treatment on PDHC activity was assessed in two PDHC-deficient cell lines, each with a different point mutation in the E1alpha subunit gene (R378C and R88C), and one cell line in which an 8-bp tandem repeat was deleted (W383 del). Only two (R378C and R88C) of the three PDHC-deficient cell lines with very low levels of PDHC activity and unstable polypeptides were sensitive to chronic DCA treatment. In these cell lines, DCA treatment resulted in an increase in PDHC activity by 125 and 70%, respectively, with concomitant increases of 121 and 130% in steady-state levels of immunoreactive E1alpha. DCA treatment reduced the turnover of the E1alpha subunit in R378C and R88C mutant cells with no significant effect on the E1beta subunit. Chronic DCA treatment significantly improved the metabolic function of PDHC in digitonin-permeabilized R378C and R88C fibroblasts. The occurrence of DCA-sensitive mutations suggests that DCA treatment is potentially useful as an adjuvant to ketogenic and vitamin treatment in PDHC-deficient patients.

Cells, Cultured↗
PubMed2006

A Korean female patient with thiamine-responsive pyruvate dehydrogenase complex deficiency due to a novel point mutation (Y161C)in the PDHA1 gene.

Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations in the X-linked E1alpha subunit gene (PDHA1). Some of the patients with PDHC deficiency showed clinical improvements with thiamine treatment. We report the results of biochemical and molecular analysis in a female patient with lactic acidemia. The PDHC activity was assayed at different concentrations of thiamine pyrophosphate (TPP). The PDHC activity showed null activity at low TPP concentration (1 x 10(-3) mM), but significantly increased at a high TPP concentration (1 mM). Sequencing analysis of PDHA1 gene of the patient revealed a substitution of cysteine for tyrosine at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient's serum lactate concentration and dramatic clinical improvement. Biochemical, molecular, and clinical data suggest that this patient has a thiamine-responsive PDHC deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine responsiveness it is necessary to measure activities of PDHC not only at high but also at low concentration of TPP.

Cells, Cultured↗