ABSTRACT
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.
Subject(s)
Ketone Bodies/biosynthesis , Pyruvate Dehydrogenase Complex Deficiency Disease/diet therapy , Pyruvate Dehydrogenase Complex Deficiency Disease/metabolism , Child Development , Dietary Carbohydrates/administration & dosage , Humans , Infant, Newborn , Longevity , Male , Mutation , Pyruvate Dehydrogenase Complex Deficiency Disease/genetics , Treatment OutcomeSubject(s)
Point Mutation , Pyruvate Dehydrogenase Complex Deficiency Disease/genetics , Pyruvate Dehydrogenase Complex/genetics , Thiamine Pyrophosphate/metabolism , Base Sequence , Binding Sites , Biopsy , Cells, Cultured , DNA, Complementary/chemistry , Fibroblasts/chemistry , Fibroblasts/enzymology , Humans , Immunoblotting , Infant , Male , Molecular Sequence Data , Muscle, Skeletal/chemistry , Muscle, Skeletal/enzymology , Myocardium/chemistry , Myocardium/enzymology , Polymerase Chain Reaction , Pyruvate Dehydrogenase Complex/chemistry , Pyruvate Dehydrogenase Complex/metabolism , Pyruvate Dehydrogenase Complex Deficiency Disease/etiology , RNA, Messenger/analysis , Sequence Analysis, DNA , Skin/cytologyABSTRACT
Defects in pyruvate dehydrogenase, the first catalytic component of the pyruvate dehydrogenase complex, are the most common cause of pyruvate dehydrogenase complex deficiency. A family with variable pyruvate dehydrogenase complex deficiency had been described in which cultured skin fibroblasts of affected family members had normal pyruvate dehydrogenase complex activity, but different tissues and blood lymphocytes had significantly diminished activities. Enzymatic activity and immunoblot studies indicated that pyruvate dehydrogenase was affected. Further evidence is presented here showing that the defect affecting pyruvate dehydrogenase complex activity is posttranscriptional. Sequencing of the coding region of the alpha-subunit of pyruvate dehydrogenase revealed a point mutation in the codon for amino acid 234 resulting in a substitution of glycine for arginine. Study of other members of the family suggested that this mutation is inherited in a sex-linked mode. The point mutation is located in a highly conserved region of the pyruvate dehydrogenase alpha-subunit gene that contains both hydrophobic and positively charged amino acid residues. Variable expression of pyruvate dehydrogenase complex deficiency in this case may be due to instability of the pyruvate dehydrogenase heterotetramer in specific tissues because of a disruption in subunit-subunit interaction.
Subject(s)
Pyruvate Dehydrogenase Complex Deficiency Disease/genetics , Pyruvate Dehydrogenase Complex/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA/genetics , DNA Mutational Analysis , Gene Expression , Humans , Male , Molecular Sequence Data , Protein Conformation , Pyruvate Dehydrogenase Complex/chemistry , Pyruvate Dehydrogenase Complex Deficiency Disease/enzymology , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
Gram-negative spiral organisms, currently referred to as Helicobacter pylori, are associated with primary gastritis and duodenal ulceration. The organisms colonise gastric mucus and adhere to epithelial cells of inflamed antra. To further examine the binding of H. pylori to human gastric epithelial cells, we developed and characterised an in-vitro bacterial adherence assay. Scanning electronmicroscopy suggested that spiral-shaped bacteria were adherent to the surface of KATO-III cells which were derived from a human gastric adenocarcinoma. Transmission electronmicroscopy confirmed the attachment of H. pylori to these epithelial cells in tissue culture. Some bacteria were adherent to intact microvilli, others were closely adherent to the plasma membrane in regions where microvilli were effaced. In studies with radiolabelled H. pylori, adherence to epithelial cells in tissue culture contrasted with minimal binding of bacteria to polystyrene wells alone. Incubation of bacteria with gastric cells at 4 degrees C significantly reduced adherence of H. pylori. We conclude that adherence of H. pylori to gastric epithelial cells in tissue culture involved "attachment and effacement mechanisms". This assay could serve as a suitable in-vitro model for the study of the bacterial adhesins and host receptors which mediate attachment of H. pylori to gastric epithelial cell surfaces.
Subject(s)
Bacterial Adhesion , Gastric Mucosa/microbiology , Helicobacter pylori/physiology , Amino Acids/metabolism , Child , Epithelial Cells , Epithelium/microbiology , Epithelium/ultrastructure , Gastric Mucosa/cytology , Helicobacter pylori/growth & development , Helicobacter pylori/ultrastructure , Humans , Kinetics , Microscopy, Electron , Microscopy, Electron, Scanning , Tumor Cells, CulturedABSTRACT
Amino acid sequence comparison of 8 alpha and 6 beta subunits of the alpha-keto acid dehydrogenase (E1) component of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex form multiple species was performed by computer analysis. In addition to 2 previously recognized regions of homology in the alpha subunit, a 3rd region of extensive homology was identified in E1 alpha, and may be one of the sites involved in subunit interaction. E1 beta contains 4 regions of extensive homology. Region 1 contains 10 amino acids that are homologous to a 10-amino acid stretch in Escherichia coli E1. Regions 2 and 3 have sequence homologies with other dehydrogenases suggesting that these regions may be involved in catalysis.
