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B H Robinson

Publications and source records attributed to B H Robinson.

At least 163 records · Page 9Linked to original sources
PubMed1987

Clinical presentation of mitochondrial respiratory chain defects in NADH-coenzyme Q reductase and cytochrome oxidase: clues to pathogenesis of Leigh disease.

Measurement of pyruvate and lactate produced from glucose by confluent skin fibroblast cultures from 95 patients with lactic acidemia revealed 10 in whom the lactate/pyruvate ratio (L/P) was increased (L/P = 57 to 232) compared with that observed in control cell lines (L/P = 18 to 35). Mitochondria prepared from these cells revealed two types of respiratory chain defect. In four patients the deficient activity was present in NADH-coenzyme Q reductase (14% to 21% of controls), and in six the deficiency was in cytochrome c oxidase (21% to 28% of controls). The four patients with NADH-coQ reductase deficiency presented early with lactic acidosis, respiratory failure, anorexia, and hypotonia; all four died within 7 months. The group with cytochrome oxidase deficiency had a somewhat later (18 months to 2 years of age) presentation with milder lactic acidemia, but also with hypotonia and anorexia. They had delayed development, beginning to walk and talk at 18 to 24 months, and then slowly regressed. Although an investigation of central nervous system disorders in this latter group has not been possible, the clinical progression fits into the broad category of Leigh disease. We conclude that in these two groups respiratory chain defects can be detected and localized by the use of skin fibroblast cultures.

Brain Diseases, Metabolic↗
PubMed1987

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient.

Acidosis, Lactic↗
PubMed1987

The French and North American phenotypes of pyruvate carboxylase deficiency, correlation with biotin containing protein by 3H-biotin incorporation, 35S-streptavidin labeling, and Northern blotting with a cloned cDNA probe.

Cultured skin fibroblasts from 16 patients with either French or American pyruvate carboxylase (PC) deficiency were examined for their ability to incorporate 3H-biotin into proteins. Cell extracts were also examined for the presence of biotin-containing proteins with 35S-streptavidin, immunoreactive protein with anti-PC antibody, and PC mRNA by Northern blotting with a PC cDNA probe. All the North American presentation patients showed a 3H-biotin protein, a streptavidin protein, and an anti-PC precipitable protein at 125 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins. They also showed a detectable mRNA species for PC on Northern blotting. Of the French presentation patients, five showed very low or absent 3H-biotin protein, streptavidin protein, and anti-PC precipitable protein at 125 kilodaltons. Three French presentation patients showed PC protein to be present on the basis of these techniques. Similarly, five showed either very low or absent mRNA for PC on Northern blotting whereas three gave evidence of the presence of PC-specific mRNA. Thus, whereas the North American presentation of PC deficiency is associated with the presence of a mature biotin containing protein of the correct molecular weight, the French presentation may, in some (but not in all) cases, have both absent PC protein and absent PC mRNA.

Bacterial Proteins↗
PubMed1987

Deficient fumarase activity in an infant with fumaricacidemia and its distribution between the different forms of the enzyme seen on isoelectric focusing.

A male infant, whose parents were first cousins, presented at 6 mo of age with hypotonia, microcephaly, and delayed development. He was found to have large amounts of fumaric and succinic acids present in the urine. In lysed cultured skin-fibroblast preparations, the activity of fumarase was found to be 22.7% of that in controls. Cell fractionation by homogenization and by digitonin treatment indicated that the residual activity in the cells of the patient was primarily located in the mitochondrial fraction rather than in the cytosolic fraction. Isoelectric focusing of fibroblast extracts showed that six bands of fumarase activity were discernible in control cell lines, two of them cytosolic with pI's of 5.53 and 5.60 and four of them mitochondrial with a pI of 5.65-6.8. In contrast, isoelectric focusing of fibroblast extracts from the fumarase-deficient patient showed only a single band of activity with a pI corresponding to the mitochondrial type seen in the controls. Immunoprecipitation of proteins with rabbit antifumarase antibody in (35S)-methionine-labeled fibroblasts indicated that a protein of correct size (Mr = 44,000 daltons) corresponding to fumarase was synthesized in similar amounts in both the patients and controls. It is proposed that in the patient's cells a single active species of fumarase that is mitochondrial in location is synthesized. Since it is known that mitochondrial and cytosolic fumarases are encoded by the same gene but differ slightly in amino acid sequence, it is possible that a point mutation might explain these findings.

Abnormalities, Multiple↗
PubMed1986

Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lacticacidemia.

The cultured skin fibroblasts from three patients with lacticacidemia were found to have low rates of 1-[14C]pyruvate oxidation in the face of normal pyruvate dehydrogenase activity. After incubation with 1 mM glucose, these three cell strains also exhibited lactate/pyruvate ratios which were three times greater than those of controls. In two of the patients, both ATP and oxygen consumption in fibroblast mitochondrial preparations was deficient with NAD-linked substrates but normal with succinate and ascorbate/N'N'N'N' tetramethyl phenylene diamine. In the third patient, ATP synthesis in mitochondrial preparations was deficient with all substrates tested. Measurement of Rotenone-sensitive NADH-cytochrome c reductase in mitochondrial preparations from skin fibroblasts showed that two of the patients had 14 and 18%, respectively, of control activity. In the third patient, cytochrome oxidase activity was 15% of that in controls. We conclude that respiratory chain defects can be demonstrated in cultured skin fibroblasts with consistency using a number of different techniques.

Adenosine Triphosphate↗
PubMed1985

Immunoextraction of lipoamide dehydrogenase from cultured skin fibroblasts in patients with combined alpha-ketoacid dehydrogenase deficiency.

Combined deficiency of the pyruvate, alpha-ketoglutarate and branched-chain keto acid dehydrogenase complexes is a rare condition in which activity of lipoamide dehydrogenase is either reduced or grossly deficient. Activities in three cell strains from patients with excretion of branched chain ketoacids and alpha-ketoglutarate and lactic-acidemia showed decreased levels of the three alpha-ketoacid dehydrogenases. Lipoamide dehydrogenase activity was 5% of normal in one cell stain and 50-60% in the other two. Antiserum raised against lipoamide dehydrogenase was used to immunoprecipitate labelled lipoamide dehydrogenase from fibroblasts grown on [35S]methionine. After separation of cell proteins from control fibroblasts by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and fluorography, a prominent 55 kilodalton band was evident in cell extracts treated with the antiserum which corresponded to lipoamide dehydrogenase. In the cell lines from patients with combined alpha-ketoacid dehydrogenase deficiency immunoprecipitation of lipoamide dehydrogenase showed that this protein was present in similar amounts to that seen in control cell lines and was also of the correct molecular weight.

Antibodies, Monoclonal↗
PubMed1985

Simultaneous preparation of the three biotin-containing mitochondrial carboxylases from rat liver.

Affinity chromatography on avidin-Sepharose column was used to bind the biotin-containing carboxylases from rat liver. With a biotin gradient (0-0.3 mM), peaks of activity of pyruvate, propionly CoA and beta-methylcrotonyl CoA carboxylases co-eluted. Subsequent separation of the three carboxylases was attained using DEAE-Sepharose chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed each of the enzymes to be pure, with pyruvate carboxylase giving a single subunit band (Mr 130 000), propionyl-CoA carboxylase giving two bands (Mr 73 000 and 56 500) and beta-methylcrotonyl-CoA carboxylase giving two bands (Mr 75 000 and 60 000). The specific activity of propionyl-CoA carboxylase (15.8 munits/mg) and beta-methylcrotonyl-CoA carboxylase (24.2 munits/mg) were comparable with reported activities for these purified enzymes, while that of pyruvate carboxylase (1.25 munits/mg) was low. This is a suitable method for the simultaneous preparation of purified carboxylases for the specific purpose of raising antisera to these enzymes.

Animals↗
PubMed1985

Pyruvate carboxylase deficiencies: complementation studies between "French" and "American" phenotypes in cultured fibroblasts.

Pyruvate carboxylase (PC) deficiencies (McKusick 26615) are heterogeneous clinically and biochemically. We performed a complementation study with fibroblast strains from seven patients, (four patients with "French" phenotype, two patients with "American" phenotype, one patient with biotin responsive multiple carboxylase deficiency, MCD). The six isolated pyruvate carboxylase mutants (two cross-reacting material CRM -ve and four CRM +ve) failed to complement each other, but did complement a form of biotin responsive MCD.

Amino Acid Metabolism, Inborn Errors↗
PubMed1985

Brain amino acid abnormalities in pyruvate carboxylase deficiency.

Amino acids were measured in several regions of autopsied brain from an infant who died with congenital lactic acidosis due to pyruvate carboxylase deficiency (McKusick 26615), as well as in cerebrospinal fluid (CSF) and plasma of four living infants with this disorder. Glutamine content was greatly reduced in all brain regions, while glutamic acid and proline contents were elevated. The gamma-aminobutyric acid (GABA) content was normal in brain. Glutamine concentrations in CSF and plasma were also decreased in the living patients. Glutamine may serve as a pool to provide glutamate and GABA for use as neurotransmitters, and to provide alpha-ketoglutarate for the tricarboxylic acid cycle when oxaloacetate can no longer be formed directly from pyruvate.

Acidosis, Lactic↗
PubMed1985

Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia.

A small-for-gestational-age female infant born at term developed severe lactic acidosis and died on day 13 of life. Two previous sibs had also died of overwhelming lactic acidosis in the neonatal period. The lactate-to-pyruvate and 3-hydroxybutyrate-to-acetoacetate ratios were elevated at 136 and 42 to one, respectively. The activities of the pyruvate dehydrogenase complex and pyruvate carboxylase in cultured skin fibroblasts were normal but a defect in respiration was indicated by the low rates of conversion of 1-[14C]pyruvate, glutamate, and lactate to 14CO2 in these cells. Skin fibroblast cultures also displayed an elevated lactate-to-pyruvate ratio (72:1) when incubated with glucose as substrate compared to control cell cultures (20:1). When mitochondrial preparations of skin fibroblasts (prepared by digitonin extraction) were tested for their ability to synthesize ATP from a variety of substrates, it was found that those of the patient made adequate amounts of ATP with either succinate or ascorbate/tetramethyl-phenylenediamine as substrate but not with the NAD-linked substrates pyruvate, isocitrate, and palmitoyl carnitine. We propose that this is indicative of a defect in the respiratory chain between NADH and coenzyme Q, for the first time demonstrable in cultured skin fibroblasts.

Acidosis↗
PubMed1984

The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases.

Two spin-labeled analogues of NAD+ were synthesized with an 15N and perdeuterated nitroxide radical, 4-amino-2,2,6,6-[2H17, 15N]tetramethylpiperidone-1-oxyl, which was attached to either the C-6 or C-8 position of the purine ring. The EPR spectra of these derivatives exhibit an approx. 6-fold increase in sensitivity compared with the corresponding 14N, protonated analogues due to a decrease in both the number of nuclear manifolds (from three to two) and the linewidth. The enhanced spectral resolution obtained with (2H17, 15N)spin-labeled-NAD+ analogues has facilitated simulation of the EPR lineshape of the nucleotide bound to lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). The spin-label moiety exhibits highly constrained motion indicative of a single environment. The motion of the spin label does not reflect the overall motion of the enzyme; rather, it is characteristic of some limited mobility relative to the lactate dehydrogenase. By contrast, the spin label on the membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase (D-beta-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30), is completely immobilized and exhibits two distinct spectral components for spin-labeled NAD+, which appear to differ in the polarity of the environment of the nitroxide.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Interactions and spatial arrangement of spin-labeled NAD+ bound to glyceraldehyde-3-phosphate dehydrogenase. Comparison of EPR and X-ray modeling data.

The spatial arrangement of coenzyme NAD+ in remote and adjacent binding sites in various stoichiometric complexes with tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was examined via EPR spectroscopy. An adenosine N6-15N,2H17 spin-labeled derivative of coenzyme NAD+ (SL-NAD+) was chemically synthesized for this work. The spectral simplifications and narrow line widths afforded by 15N and 2H substitution enabled experimental EPR spectra to be deconvoluted into their three component spectra: (a) unbound coenzyme, (b) bound coenzyme without adjacent site occupied, and (c) bound coenzyme with adjacent site occupied. Binding of SL-NAD+ in adjacent active centers of R axis-related subunits resulted in resolved dipolar interactions which characterized intersubunit distances. Binding to distant subunits related by the P and Q axes gave no dipolar interaction. Once the first NAD+ site was occupied, EPR spectra at various stoichiometries provided evidence for nonpreferential spatial binding of SL-NAD+ to the three unoccupied sites. EPR spectral simulations indicated a separation of 12.8 A for the unpaired electrons of spin label moieties of R axis-related coenzymes. Molecular modeling based on x-ray crystallographic data predicted 11-13 A. The angles and distance relating to interacting spin-labels were calculated from atomic coordinates based on molecular modeling of both anti-anti and anti-syn (adenine-ribose) conformations of SL-NAD+. Computer-generated line shapes indicated best agreement with experimental EPR results when the anti-anti geometry was employed. Comparison of EPR spectra from soluble and ammonium sulfate-precipitated enzymes indicated that the NAD+-binding domains are positioned equivalently in the two physical states. Since the observed dipolar line shapes are critically dependent on the distance and geometry relating to the interacting SL-NAD+, these data provide direct evidence for a high degree of conservation of quaternary structure of the enzyme in the hydrated crystalline state. Studies on the enzyme isolated from human erythrocytes also indicated a close correlation with the rabbit muscle enzyme in both the arrangement of NAD+-binding domains and negative cooperativity of coenzyme binding.

Animals↗
PubMed1984

Lactic acidaemia.

Congenital childhood lactic acidaemia is a poorly understood group of genetic diseases. The most common underlying inherited defect encountered in this group is deficiency of the pyruvate dehydrogenase complex. Of 23 cases we have diagnosed, 18 have a deficiency in the first component of the complex, the E1 decarboxylase, while the other five have multiple alpha-keto acid dehydrogenase deficiency due to a defect in lipoamide dehydrogenase. In addition to the lactic acidosis associated with pyruvate decarboxylase deficiency, ten of the cases showed evidence of facial dysmorphism consisting of a narrow head, wide nasal bridge and flared nostrils or gross microcephaly. Two further patients had agenesis of the corpus callosum. Isolated pyruvate carboxylase deficiency was found to present in two different forms, one with lactic acidaemia and mental retardation, the other with lactic acidaemia, hyperammonaemia citrullinaemia and hyperlysinaemia. The former presentation we have shown to be associated with the presence of a biotinylated pyruvate carboxylase protein of the correct subunit molecular weight (125 kd) which has no catalytic activity (CRM + ve). The latter we have shown to be associated with the absence of any recognizable pyruvate carboxylase protein (CRM - ve).

Abnormalities, Multiple↗
PubMed1984

3-Hydroxy-3-methylglutaric aciduria.

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglycemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.

Cells, Cultured↗
PubMed1984

The molecular basis for the two different clinical presentations of classical pyruvate carboxylase deficiency.

Eight cases of isolated human pyruvate carboxylase deficiency were examined from seven families. Although all patients presented with a chronic lacticacidemia, two particular patients presented with the added features of hyperammonemia, citrullinemia, and hyperlysinemia. When cultured skin fibroblasts from these patients were examined for their ability to synthesize [3H]biotin-containing proteins, it was found that the two patients who presented with hyperammonemia, citrullinemia, and hyperlysinemia did not synthesise a protein of the correct subunit molecular weight (Mr = 125 K daltons) corresponding to pyruvate carboxylase. In addition, when skin fibroblast proteins were labeled with [35S]methionine, cross-reacting material (CRM) corresponding to pyruvate carboxylase was immunoprecipitated by antipyruvate carboxylase antiserum in most patients, but again the two patients with the atypical presentation showed no CRM. We propose that the different clinical presentation of human pyruvate carboxylase deficiency is a manifestation of two different mutations in the pyruvate carboxylase gene, one that results in the synthesis of a relatively inactive pyruvate carboxylase protein CRM(+ve) and one that results in the lack of expression of the gene in the form of a recognizable protein CRM(-ve).

Biotin↗
PubMed1983

[3H]biotin-labeled proteins in cultured human skin fibroblasts from patients with pyruvate carboxylase deficiency.

Biotin containing carboxylases in cultured human skin fibroblasts were radioactively labeled by addition of [8,9-3H]biotin to biotin-depleted cell cultures. Three major bands were visualized by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fibroblast proteins. These bands corresponded to pyruvate carboxylase (Mr = 125,000), the biotin-containing subunit of methyl crotonyl-CoA carboxylase (Mr = 75,000) and the biotin-containing subunit of propionyl-CoA carboxylase (Mr = 73,000) as judged by molecular weight markers, purified carboxylase protein standards, and interaction with monospecific antisera. Four out of 5 cell lines from patients with classical pyruvate carboxylase deficiency (less than 5% of normal activity) labeled with this technique displayed a normal band in the position of pyruvate carboxylase while one cell line showed complete absence of any labeled protein in this area. These results demonstrate heterogeneity in the etiology of pyruvate carboxylase deficiency.

Biotin↗
PubMed1983

Lactic acidosis, neurological deterioration and compromised cellular pyruvate oxidation due to a defect in the reoxidation of cytoplasmically generated NADH.

Two patients, one dying at 25 days and one at 20 months had 'chronic' lactic acidaemia with a high lactate to pyruvate ratio. Both showed EEG abnormalities and seizure activity and both died of respiratory failure. Investigation of cultured skin fibroblasts from these patients revealed normal pyruvate dehydrogenase and pyruvate carboxylase activities but the cells showed a decreased ability to oxidase pyruvate which was returned to normal on the addition of methylene blue. Subsequent investigations revealed that the mitochondria from the patients' cells could oxidase pyruvate normally but that the cells had an abnormal NAD to NADH ratio under standard conditions of incubation. It was concluded that both children had a redox disequilibrium in the cytoplasmic compartment due to a problem in transporting reducing equivalents from the cytoplasmic to the mitochondrial compartments.

Acidosis↗