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B H Robinson

Publications and source records attributed to B H Robinson.

At least 145 records · Page 8Linked to original sources
PubMed1990

The use of skin fibroblast cultures in the detection of respiratory chain defects in patients with lacticacidemia.

Cultured skin fibroblasts from patients with lacticacidemia were incubated with glucose for 1 h and the lactate and pyruvate production measured. Those patients with increased lactate to pyruvate ratios were further analyzed for the cause of the abnormal redox state. Two categories of patients are described. The first contains patients with either severe or partial cytochrome oxidase deficiency; this group can be broken down further into patients with Leigh's disease, Kearns-Sayre syndrome, and liver-specific cytochrome oxidase deficiency. In this group, the rise in lactate to pyruvate ratio roughly correlated with the severity of the defect. The second patient category had defects located in complex I of the mitochondrial respiratory chain. This is easily demonstrated in the most severely affected patients with the fatal infantile form of the disease. Patients with severe defects in either complex I or cytochrome oxidase had complexes that were only partially assembled. Patients with mitochondrial encephalopathy with lactic acidosis and stroke-like episodes demonstrated only minor changes in redox state and in the behavior of the mitochondrial respiratory chain.

Acidosis, Lactic↗
PubMed1990

Stimulation of porphyrinogen oxidation by mercuric ion. I. Evidence of free radical formation in the presence of thiols and hydrogen peroxide.

The etiology of mercury-induced porphyrinuria was investigated by testing the hypothesis that mercuric ions (Hg2+) promote free radical-mediated oxidation of reduced porphyrins (porphyrinogens) by compromising the antioxidant potential of endogenous thiols, particularly GSH. Studies in vitro demonstrated that porphyrinogens (uroporphyrinogen and coproporphyrinogen) readily undergo H2O2-dependent oxidization in the presence of Fe3(+)-EDTA and that this action is attenuated by GSH at biologically relevant concentrations (0.5-10 mM). At low concentrations, Hg2+ complexes with GSH in a 1:2 molar ratio to decrease the antioxidant effect of GSH. However, at Hg2+ concentrations approaching saturation-complexation with available GSH, stimulation of porphyrinogen oxidation to 2 to 3 times that mediated by the H2O2/Fe3(+)-dependent system alone is observed. Stimulation of porphyrinogen oxidation by Hg2+ plus GSH increases in a dose-related manner with the concentration of H2O2 in the reaction mixture but is independent of the presence of iron. No porphyrinogen oxidation is observed in reaction mixtures containing H2O2 and either Hg2+ or GSH alone or when Hg+ is substituted for Hg2+. Studies with reactive oxidant scavengers and ESR spectroscopy suggest the participation of free radical species in Hg:GSH-mediated porphyrinogen oxidation. A mechanism involving ligand exchange between Hg2+ and GSH, which leads to formation of GS radicals and subsequent propagation of reactive oxygen-based radical species, is proposed. These studies support the view that Hg2+ both compromises the antioxidant potential of GSH and promotes formation of reactive species via thiol complexation. These findings suggest a mechanistic basis underlying the porphyrinogenic as well as tissue-damaging properties of mercuric ions.

Animals↗
PubMed1989

Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA.

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1H NMR, CD, and thermal denaturation studies indicate that 1a (T*) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules.

Base Sequence↗
PubMed1989

Determination of [U-13C]glucose turnover into various metabolite pools for the differential diagnosis of lactic acidemias.

Stable-isotope tracer experiments performed in vitro are evaluated for their utility in differentiating between pyruvate dehydrogenase and cytochrome oxidase deficiencies, two of several enzyme defects commonly associated with the lactic acidemias. Fibroblasts of enzyme-deficient individuals and of age-matched controls are grown in medium containing [U-13C]glucose. Direct analysis of cells and conditioned culture medium provides only minor differences in the organic acid/amino acid GC-MS profiles, making differentiation of enzyme defects difficult by this method. However, differences have been found in the glucose turnover into various cell metabolites, making differentiation of these two enzyme defects possible. The cellular pool of glutamic acid experiences 13C-enrichment in both the control and cytochrome oxidase deficient lines, but not in the pyruvate dehydrogenase-deficient line. The cellular pool of an unknown, possibly an aminopentose sugar, on the other hand, experiences 13C-enrichment in the pyruvate dehydrogenase and control lines, but not in the cytochrome oxidase line. These observations, as well as other differences in the extent of enrichment into various metabolite pools, suggest that this stable-isotope approach, in vitro, is feasible and may allow these two enzyme defects to be differentiated in a definitive manner. Such stable-isotope experiments are easy to carry out with cultured cells and are inexpensive. Applications of the technique to other genetic disorders might be appropriate.

Amino Acids↗
PubMed1989

Could bovine serum albumin be the initiating antigen ultimately responsible for the development of insulin dependent diabetes mellitus?

Western blotting of either a cloned rat beta-islet tumour cell extract or isolated BB rat islets with rat anti-bovine serum albumin antiserum revealed a cross-reacting protein (Mr = 69,000). A protein of similar molecular size was observed by fluorography in proteins immunoprecipitated from islet cells labelled with (35S)-methionine using anti-bovine serum albumin antiserum. In comparing the primary structure of the beta subunits of the proteins Ia, DQ and DR a region of homology with bovine serum albumin became evident. Analysis of the amino-acid homology in relation to the DR/DQ allotypes found in the human population gave a strong correlation between the combined DR and DQ homology score with bovine serum albumin and the incidence of insulin dependent diabetes mellitus.

Adenoma, Islet Cell↗
PubMed1988

Characterization of cytochrome-c oxidase mutants in human fibroblasts.

Skin fibroblasts were selected as having cytochrome-c oxidase deficiency by activity measurements in whole cells. Each cell line was cultured in sufficient amount to isolate mitochondria for biochemical characterization. Cytochrome-c oxidase was then examined by activity measurements, by heme determination and by polypeptide analysis using antibodies specific to the enzyme subunits. The cytochrome-c oxidase activity in the different cell lines ranged from 9% to 54% of that of normal fibroblasts. Heme determinations and polypeptide analysis established that the lowered cytochrome-c oxidase activity was due to reduced amounts of the complex in the mitochondrial inner membrane. In all cases, there was defective assembly of the enzyme, with the amounts of mitochondrially coded and nuclear coded subunits being reduced proportionally. These studies show that fibroblasts can be used for prenatal diagnosis of mitochondrial diseases and are a useful system in which to study mitochondrial biogenesis.

Cell Line↗
PubMed1988

A quasi-elastic neutron-scattering study of the molecular mobility of water associated with a protein both in aqueous solution and adjacent to a charged surfactant interface.

The quasi-elastic incoherent neutron-scattering method has been used to investigate the mobility of water molecules associated with the protein, alpha-chymotrypsin, both in aqueous solution and adjacent to a charged surfactant interface. The latter was studied by solubilizing the protein as monomer in the aqueous cores of small water-in-oil microemulsion droplets (radius 3.5 nm). The droplets were stabilised by an interfacial shell of a double-chain surfactant (Aerosol-OT). The spectra of the water in both these protein-containing system contained a component corresponding to a 7-fold reduction in mobility as compared with bulk water. The integrated intensity of this 'immobilised' spectra component shows that a maximum of approx. 450 water molecules, corresponding to half complete monolayer coverage, are associated with a single protein molecule. This value of 450 may contain a contribution from exchangeable hydrogens within the protein, but this contribution is estimated to be small. The mobility of the remainder of the water is unaffected. The solvation behaviour of the protein is similar in bulk water and in the microemulsion water droplets.

Chemical Phenomena↗
PubMed1988

Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex.

A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.

Amino Acid Sequence↗
PubMed1988

In utero central nervous system damage in pyruvate dehydrogenase deficiency.

Pyruvate dehydrogenase deficiency is among the most common causes of congenital lactic acidosis. We describe siblings with congenital lactic acidosis due to a deficiency of pyruvate dehydrogenase complex. The findings of computed tomography and pathologic studies suggest that central nervous system damage had occurred in utero. These observations have implications for treatment and outcome in patients with enzymatic defects causing congenital lactic acidosis.

Brain↗
PubMed1988

3-Methylglutaconic aciduria: a phenotype in which activity of 3-methylglutaconyl-coenzyme A hydratase is normal.

3-Methylglutaconic aciduria has been found in two distinct syndromes. In one there is deficient activity of 3-methylglutaconyl coenzyme A hydratase, and the only clinical manifestation observed has been retardation of speech development. In the other, which includes a majority of the patients studied, we document that the activity of this enzyme in fibroblast extracts is normal. The phenotype of this disorder is one of profound neurological impairment with retarded psychomotor development, hypotonicity and/or spasticity, convulsions or EEG abnormalities, and sensorineural changes in the eye and ear.

Chemical Phenomena↗
PubMed1988

Cell culture studies on patients with mitochondrial diseases: molecular defects in pyruvate dehydrogenase.

There is a group of inborn errors of metabolism that result in the condition of chronic lacticacidemia of childhood. Nearly all of the defects that can be identified occur in mitochondrial proteins, and can be demonstrated in cultured skin fibroblasts from the patients concerned. One approach to the diagnosis of these defects involves a simple incubation of the fibroblast culture with glucose-containing medium followed by the measurement of accumulated lactate and pyruvate. The total amounts of lactate and pyruvate and the ratio between them is different in cells from patients with defects in the pyruvate dehydrogenase complex or the respiratory chain. Measurement of 1-14C-pyruvate oxidation to 14CO2 can also reveal defective oxidative metabolism. Localization of the defects can be achieved using individual assays for the enzymes concerned. The clinical sequelae of the different defects is discussed.

Cells, Cultured↗
PubMed1988

Gene for lipoamide dehydrogenase maps to human chromosome 7.

The gene for lipoamide dehydrogenase (LD) has been assigned to human chromosome 7 based on filter hybridization analysis of genomic DNA from rodent-human somatic cell hybrids using a cDNA probe for human LD. No indication of multiple copies of the gene was found, in accordance with previous evidence that LD in the pyruvate, alpha-ketoglutarate, and branched chain alpha-ketoacid dehydrogenase complexes is genetically as well as biochemically identical.

Chromosome Mapping↗
PubMed1987

Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases.

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.

Amino Acid Sequence↗
PubMed1987

Simulation of double-stranded branch point migration.

A structural and dynamic model has been developed for the branch point formed when two DNA double helices exchange strands during genetic recombination. This model, which generalizes most previous structural models, maintains the twofold symmetry inherent in the covalent and hydrogen bonded structure, yet has three degrees of freedom about virtual bonds, constituting a simplified junction. Using this structural model, a three-step dynamic model for branch point migration has been developed: longitudinal diffusion about the virtual bonds to achieve a structure in which the helix axes are approximately parallel; opening of the base pairs; and rotary diffusion about the helix axis to effect a migratory event. The model, which includes the possible role of electrostatic interactions, solves problems inherent in previous treatments. We find that no significant electrostatic torques arise that promote branch point migration. The absence of a kinetic mechanism to circumvent thermodynamic barriers due to mispairing suggests that an energy source is used for those situations in living systems.

Base Composition↗
PubMed1987

Variable clinical presentation in patients with defective E1 component of pyruvate dehydrogenase complex.

Clinical findings are presented for 30 patients with lactic acidemia in whom activity of the pyruvate dehydrogenase complex in fibroblasts was significantly (P = less than 0.01) below that of control cell lines. Residual activity of the activated complex ranged from 1.6% to 68.5% of control activity. Seven patients died before 6 months of age, and another five before reaching 2 years of age. Sixteen of the surviving patients and the five who died between 6 months and 2 years all had psychomotor retardation. Seventeen children had structural central nervous system damage, as determined either by computed tomography or at autopsy. The extent and location of damage varied from cerebral atrophy to the development of cystic lesions in the cerebral cortex, basal ganglia, and brain stem. Two patients had ataxic episodes only and were not developmentally delayed. This cohort of patients strongly resembles a comparable group assembled from various other reports.

Acidosis, Lactic↗