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B H Robinson

Publications and source records attributed to B H Robinson.

At least 73 records · Page 4Linked to original sources
PubMed1997

The N terminus of the Qcr7 protein of the cytochrome bc1 complex is not essential for import into mitochondria in Saccharomyces cerevisiae but is essential for assembly of the complex.

Subunit 7 of the yeast cytochrome bc1 complex is encoded by the nuclear QCR7 gene and is essential for respiration. This protein does not contain a cleavable N-terminal mitochondrial targeting sequence, and it is not understood how the Qcr7 protein is imported into mitochondria and assembled into the complex. To test the role of the N terminus of the Qcr7 protein in mitochondrial import, assembly of the complex, and proton translocation, we inactivated the endogenous QCR7 gene and expressed mutated qcr7 genes capable of synthesizing proteins truncated by 7, 10, 14, and 20 residues (Qcr7p-delta7, Qcr7p-delta10, Qcr7p-delta14, and Qcr7p-delta20, respectively) from the N terminus. In addition, we studied two mutants containing Qcr7 proteins with point mutations in addition to a delta7 truncation, Qcr7p-delta7(D13V) and Qcr7p-delta7(R10K). All the mutant proteins with the exception of Qcr7p-delta10 were present in the mitochondria at 30 degrees C, although most at lower steady-state levels than the Qcr7p from the strain overexpressing wild type QCR7. The absence of the Qcr7p-delta10 may be the result of an unstable protein or a decrease in the efficiency of mitochondrial import due to its compromised amphipathic alpha-helix and the presence of a negative charge exposed at the N terminus. Cytochrome c reductase activities and the amounts of ATP synthesized were comparable with the wild type in the strain expressing Qcr7p-delta7. The strain expressing Qcr7p-delta7(R10K) had an identical phenotype to the one containing the Qcr7p-delta7, whereas strains expressing the Qcr7p-delta10, Qcr7p-delta14, Qcr7p-delta20, and Qcr7p-delta7(D13V) were all respiration-deficient. Examination of the steady-state levels of complex III subunits showed that core protein 2, cytochrome c1, the iron-sulfur protein, and the 11-kDa subunit are reduced in respiration-deficient mutant strains. Results from deletion analyses indicate that the N-terminal 20 residues (after Met-1) of the Qcr7 protein are not essential for import into mitochondria and that the N-terminal seven residues (after Met-1) are not involved in proton translocation. The results of this work show, however, that the N terminus of the Qcr7 protein is essential for the biosynthesis of ubiquinol-cytochrome c reductase.

Amino Acid Sequence↗
PubMed1997

Excessive formation of hydroxyl radicals and aldehydic lipid peroxidation products in cultured skin fibroblasts from patients with complex I deficiency.

Previous studies suggest oxygen free radicals' involvement in the etiology of cardiomyopathy with cataracts. To investigate the role of free radicals in the pathogenesis of the cardiomyopathy with cataracts and complex I deficiency, fibroblasts from patients were assessed for hydroxyl radical formation and aldehydic lipid peroxidation products with and without redox active agents that increase free radicals. The rate of hydroxyl radical formation in patient cells was increased over 2-10-fold under basal conditions, and up to 20-fold after menadione or doxorubicin treatment compared with normal cells. We also found an overproduction of aldehydes in patient cells both under basal conditions and after treatment. Both hydroxyl radicals and toxic aldehydes such as hexanal, 4-hydroxynon-2-enal, and malondialdehyde were elevated in cells from patients with three types of complex I deficiency. In contrast, acyloins, the less toxic conjugated products of pyruvate and saturated aldehydes, were lower in the patient cells. Our data provide direct evidence for the first time that complex I deficiency is associated with excessive production of hydroxyl radicals and lipid peroxidation. The resultant damage may contribute to the early onset of cardiomyopathy and cataracts and death in early infancy in affected patients with this disease.

Aldehydes↗
PubMed1997

The role of London forces in defining noncentrosymmetric order of high dipole moment-high hyperpolarizability chromophores in electrically poled polymeric thin films.

Graphs of second harmonic generation coefficients and electro-optic coefficients (measured by ellipsometry, attenuated total reflection, and two-slit interference modulation) as a function of chromophore number density (chromophore loading) are experimentally observed to exhibit maxima for polymers containing chromophores characterized by large dipole moments and polarizabilities. Modified London theory is used to demonstrated that this behavior can be attributed to the competition of chromophore-applied electric field and chromophore-chromophore electrostatic interactions. The comparison of theoretical and experimental data explains why the promise of exceptional macroscopic second-order optical nonlinearity predicted for organic materials has not been realized and suggests routes for circumventing current limitations to large optical nonlinearity. The results also suggest extensions of measurement and theoretical methods to achieve an improved understanding of intermolecular interactions in condensed phase materials including materials prepared by sequential synthesis and block copolymer methods.

Journal Article↗
PubMed1997

Chromosomal localization of the human liver form cytochrome c oxidase subunit VIIa gene.

The chromosomal loci corresponding to human cytochrome c oxidase (COX) subunit VIIa Liver (VIIa-L) isoform genes were determined utilizing a combined approach of genomic cloning, in situ hybridization, and somatic hybrid genetics. In contrast to the proposal of E. Arnaudo et al. (Gene (Amst.), 119: 299-305, 1992) that COX VIIa-L sequences are located on chromosomes 4 and 14, we found that COX VIIa-L related sequences reside on chromosome 6, while an additional COX VIIa-L cross-reacting sequence psi-gene) was located on chromosome 4.

Animals↗
PubMed1997

Cloning, characterization, and chromosomal localization of human liver form cytochrome c oxidase subunit VIa related genes.

The chromosomal location of human cytochrome c oxidase (COX) subunit VIa Liver (VIa-L) isoform related sequences has been determined by a combination of in situ hybridization and analysis of human-hamster somatic cell hybrid panels. COX VIa-L related sequences were present on chromosomes 6 and 12. It has been verified that at least two COX VIa-L genes are on chromosome 6, one of which is a pseudogene. In total, four COX VIa-L related sequences have been cloned and their nucleotide sequences analyzed. At least three of the sequences represent pseudogenes; their relatedness to the COX VIa-L cDNA is discussed.

Animals↗
PubMed1997

Site-specific dynamics in DNA: experiments.

This chapter reviews the dynamics information obtained from experimental magnetic resonance studies of site-specifically labeled duplex DNA. A previous review (43) discusses the dynamics of duplex DNA; it develops a theory that shows how magnetic resonance experiments are used to detect those dynamics. The methods for obtaining information about dynamics as well as a summary of what is now known about the site-specific dynamics of DNA are presented. This review contains two methods sections which present results using electron paramagnetic resonance and nuclear magnetic resonance active probes.

DNA↗
PubMed1996

Assignment of the PSST subunit gene of human mitochondrial complex I to chromosome 19p13.

The cDNA for the PSST subunit of human mitochondrial nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase [complex I; NADH dehydrogenase (ubiquinone), Fe-S (20 kDa); EC 1.6.5.3] was generated by polymerase chain reaction (PCR) amplification of human cDNA. The sequence of the mature protein deduced from the cDNA codes for a protein that is closely related to the bovine protein (93% homology). Nine conservative substitutions are found in the mature protein, mainly in the N and C terminal regions. The mature human protein is missing four amino acids (PAAL) close to the N terminus that are present in the bovine protein. The N terminus of the mature protein is preceded by a presequence of 38 amino acids that, although quite different from its bovine counterpart (52% homology), has properties that are characteristic of a mitochondrial import sequence. Southern hybridization analysis predicts an estimated gene size of 3.8 kb. Northern hybridization analysis of mRNA from fibroblasts of complex I-deficient patients revealed no size or transcript level abnormalities. The cDNA of the PSST protein was used to investigate tissue-specific expression and to localize the gene for this subunit to chromosome 19p13.

Animals↗
PubMed1996

Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase.

Mitochondria were isolated from skin fibroblast cultures derived from healthy individuals (controls) and from a group patients with complex I (NADH-CoQ reductase) deficiency of the mitochondrial respiratory chain. The complex I deficient patients included those with fatal infantile lactic acidosis (FILA), cardiomyopathy with cataracts (CC), hepatopathy with tubulopathy (HT), Leigh's disease (LD), cataracts and developmental delay (CD), and lactic acidemia in the neonatal period followed by mild symptoms (MS). Production of superoxide radicals, on addition of NADH, were measured using the luminometric probe lucigenin with isolated fibroblast mitochondrial membranes. Superoxide production rates were highest with CD and decreased in the order CD >> MS > LD > control > HT > FILA = CC. The quantity of Mn-superoxide dismutase (MnSOD), as measured by ELISA techniques, however, was highest in CC and FILA and lowest in CD. Plots of MnSOD quantity versus superoxide production showed an inverse relationship for most conditions with complex I deficiency. We hypothesize that oxygen radical production is increased when complex I activity is compromised. However, the observed superoxide production rates are modulated by the variant induction of MnSOD which decreases the rates, sometimes below those seen in control fibroblast mitochondria. In turn, we show that the variant induction of MnSOD is most likely a function of the change in the redox state of the cell experienced rather than a result of the complex I defect per se.

Cardiomyopathies↗
PubMed1996

An additional mitochondrial tRNA(Ile) point mutation (A-to-G at nucleotide 4295) causing hypertrophic cardiomyopathy.

A third point mutation in the mitochondrial tRNA(Ile) gene associated with hypertrophic cardiomyopathy and respiratory chain dysfunction in heart is reported. An A-to-G transition at nucleotide position 4295 was shown to be highly evolutionarily conserved, never present in control individuals, and to segregate with the disease. A PCR-based diagnostic test and endomyocardial biopsies were used to detect both the biochemical deficiency and the level of heteroplasmy in heart. The implications of this new mitochondrial DNA point mutation are discussed.

Adenine↗
PubMed1996

Encoding of anisotropic diffusion with tetrahedral gradients: a general mathematical diffusion formalism and experimental results.

A diffusion imaging method with a tetrahedral sampling pattern has been developed for high-sensitivity diffusion analysis. The tetrahedral gradient pattern consists of four different combinations of x, y, and z gradients applied simultaneously at full strength to uniformly measure diffusion in four different directions. Signal-to-noise can be increased by up to a factor of about three using this approach, compared with diffusion measurements made using separately applied x, y, and z gradients. A mathematical formalism is presented describing six fundamental parameters: the directionally averaged diffusion coefficient D and diffusion element anisotropies eta and epsilon which are rotationally invariant, and diffusion ellipsoid orientation angles theta, phi, and omega which are rotationally variant. These six parameters contain all the information in the symmetric diffusion tensor D. Principal diffusion coefficients, reduced anisotropies, and other rotational invariants are further defined. It is shown that measurement of off-diagonal tensor elements is essential to assess anisotropy and orientation, and that the only parameter which can be measured with the orthogonal method is D. In cases of axial diffusion symmetry (e.g., fibers), the four tetrahedral diffusion measurements efficiently enable determination of D, eta, theta, and phi which contain all the diffusion information. From these four parameters, the diffusion parallel and perpendicular to the symmetry axis (D and D) and the axial anisotropy A can be determined. In more general cases, the six fundamental parameters can be determined with two additional diffusion measurements. Tetrahedral diffusion sequences were implemented on a clinical MR system. A muscle phantom demonstrates orientation independence of D, D, D, and A for large changes in orientation angles. Sample background gradients and diffusion gradient imbalances were directly measured and found to be insignificant in most cases.

Animals↗
PubMed1996

Diagnosis of complex I deficiency in patients with lactic acidemia using skin fibroblast cultures.

The requirement for a rapid and easy method of preparing mitochondrial fractions from cultured skin fibroblasts led us to compare the results obtained from such a preparation with the more traditional methods of cellular fractionation. Values for NADH-cytochrome c reductase (rotenone sensitive) were compared for a series of three controls and nine patients with complex I (NADH-coenzyme Q reductase deficiency). Values obtained for deficient cell lines varied from 19 to 64% of the control values for the long mitochondrial preparation method and from 34 to 70% of control for the rapid preparation. Mean values were statistically significantly different from the lowest control cell line (P < 0.01) in all cases. The specific activity on the basis of activity per milligram of mitochondrial protein and of activity per unit of citrate synthase activity was lower in the rapid preparation of mitochondria by some 41%, indicating a lesser degree of mitochondrial purification. However, the overall result showed that this type of rapid preparation, which uses four 9-cm petri dishes of cultured cells, can be used to diagnose mitochondrial complex I deficiency. This method will find general use in the measurement of either mitochondrial enzymes of low specific activity or mitochondrial enzymes whose measurement is made difficult by contaminating nonmitochondrial enzymes.

Cells, Cultured↗
PubMed1996

Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex.

The most common defect associated with deficiency of the pyruvate dehydrogenase (PDH) complex occurs in the E1 component, specifically due to mutations in the X-linked E1 alpha gene. Clinical sequelae of these mutations, which range from severe neonatal lactic acidosis to carbohydrate-sensitive ataxia, can be different in males and females depending on the nature of the mutation and, in the case of females, on the X-inactivation pattern in different tissues. Males have a high representation of missense mutations among the patient cohort, while females are much more likely to have DNA rearrangements, particularly toward the 3' end of the coding sequence of the gene. Missplicing mutations involving exon 6 deletion have been reported, as has a missense mutation conferring true thiamin-responsiveness of the enzyme and the patient's clinical symptoms. Pyruvate carboxylase deficiency, on the other hand, is a true autosomal recessive disease, though it has high occurrences in particular ethnic groups, especially in Algonkian-speaking Amerindians and in Arabs. In the former group the defect is a simple type in which material cross-reactive to pyruvate carboxylase antibody is present in cultured cells (CRM+ve). In the latter group, cross-reacting material is rarely present (CRM-ve). The CRM+ve patients can survive into teenage years with careful supervision, while the CRM-ve patients have complications due to hyperammonaemia and dysfunction of the urea cycle and rarely survive beyond 3 months of life.

Amino Acid Sequence↗
PubMed1996

NADH-coenzyme Q reductase (complex I) deficiency: heterogeneity in phenotype and biochemical findings.

Twelve patient cell lines with biochemically proven complex I deficiency were compared for clinical presentation and outcome, together with their sensitivity to galactose and menadione toxicity. Each patient had elevated lactate to pyruvate ratios demonstrable in fibroblast cultures. Each patient also had decreased rotenone-sensitive NADH-cytochrome c reductase (complexes I and III) with normal succinate cytochrome c reductase (complexes II and III) and cytochrome oxidase (complex IV) activity in cultured skin fibroblasts, indicating a deficient NADH-coenzyme Q reductase (complex I) activity. The patients fell into five categories: severe neonatal lactic acidosis; Leigh disease; cardiomyopathy and cataracts; hepatopathy and tubulopathy; and mild symptoms with lactic acidaemia. Cell lines from 4 out of the 12 patients were susceptible to both galactose and menadione toxicity and 3 of these also displayed low levels of ATP synthesis in digitonin-permeabilized skin fibroblasts from a number of substrates. This study highlights the heterogeneity of complex I deficiency at the clinical and biochemical level.

Acidosis, Lactic↗
PubMed1996

Localization of the human gene encoding the 13.3-kDa subunit of mitochondrial complex III (UQCRB) to 8q22 by in situ hybridization.

We have localized the human gene encoding the 13.3-kDa subunit of mitochondrial complex III (UQCRB) to chromosome 8 using both radioactive in situ hybridization and fluorescence in situ hybridization. The additional peak obtained with the former method is attributed to the higher sensitivity of this technique, which results in hybridization of the probe to the less conserved pseudogene. We therefore conclude that the functional gene is most likely located at 8q22.

Chromosome Mapping↗
PubMed1996

Familial cardiomyopathy with cataracts and lactic acidosis: a defect in complex I (NADH-dehydrogenase) of the mitochondria respiratory chain.

Four patients in one generation of a multiply consanguineous pedigree died with cardiomyopathy, cataracts, and lactic acidemia. Postmortem heart and skeletal muscle tissues from one patient were analyzed. A low (12% control) activity of NADH-CoQ reductase (complex I) in heart and normal activity in skeletal muscle mitochondria was found. Cultured skin fibroblasts obtained from two individuals in the pedigree showed elevated lactate to pyruvate ratios in the range of 2 to 3.5 times normal and decreased complex I + III activity (42 and 54% of control activities) in isolated mitochondria. Western blot analysis and enzymatic assay showed normal levels of CuZn-superoxide dismutase, but grossly elevated levels of the mitochondrial Mn-superoxide dismutase. Southern blot analysis in heart muscle cells from the patient tested revealed multiple mitochondrial DNA deletions which indicate free oxygen radical damage. We hypothesize that a nuclear-encoded defect in the respiratory chain is responsible for excessive free oxygen radical production in these infants which contributes to the prenatal onset of cardiomyopathy and cataracts.

Acidosis, Lactic↗