Biomedical subjects

Brian H Robinson

Publications and source records attributed to Brian H Robinson.

26 records · Page 2Linked to original sources

PubMed2003

Compensatory responses of protein import and transcription factor expression in mitochondrial DNA defects.

Defects in mitochondrial DNA (mtDNA) evoke distinctive responses in the nuclear genome, leading to altered mitochondrial biogenesis. We used C(2)C(12) cells depleted of mtDNA (rho(-) cells) and fibroblasts from a mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) patient to examine adaptations of the protein import machinery and transcription factors involved in mitochondrial biogenesis. In rho(-) cells, Tom20 and Tim23 protein levels were reduced by 25% and 59%, whereas mtHSP70 was induced by twofold relative to control cells. These changes were accompanied by a 21% increase in enhanced yellow fluorescent protein (EYFP) import into mitochondria in rho(-) cells (P < 0.05). In contrast, in MELAS cells mtHSP70 was elevated by 70%, whereas Tom20 and Tom34 protein levels were increased by 45% and 112% relative to control values. EYFP import was not altered in MELAS cells. In rho(-) cells, protein levels of the transcription factors nuclear respiratory factor-1 (NRF-1) and transcription factor A (Tfam) declined by 33% and 54%, whereas no change was observed for the coactivator peroxisome proliferator receptor-gamma coactivator-1alpha (PGC-1alpha). In contrast, Tfam was increased by 40% in MELAS cells. Rho(-) cells displayed reduced oxygen consumption (Vo(2)) and ATP levels, along with a twofold increase in lactate levels (P < 0.05). In electrically stimulated C(2)C(12) cells, 109%, 78%, 60%, and 67% increases were observed in mtDNA, Vo(2), cytochrome-c oxidase (COX) activity, and Tom34 levels, respectively (P < 0.05). Our findings suggest that compensatory adaptations occurred to maintain normal rates of protein import in response to mtDNA defects and support a role for contractile activity in reducing pathophysiology associated with mtDNA depletion. Because the expression of nuclear-encoded transcription factors and protein import machinery components was dependent on the type of mtDNA defect, these findings suggest involvement of distinct signaling cascades, each dependent on the type of mitochondrial defect, resulting in divergent changes in nuclear gene expression patterns.

Animals↗

PubMed2003

Macrophages as a major source of oxygen radicals in the hyperoxic newborn rat lung.

The lungs of newborn rats exposed to 60% O(2) for 14 d were found to have a greatly increased cyanide-insensitive O(2) consumption, reflecting increased reactive oxygen species (ROS) formation. Exposure of the lung to hyperoxia is known to increase the production of ROS by mitochondria. We hypothesized that macrophages may also be a major contributor to this increase. Newborn rat pups were exposed to either air or 60% O(2) for 14 d and received either intraperitoneal gadolinium chloride (GdCl(3)) to abrogate macrophage influx, or inert vehicle. Lung homogenates were equilibrated in either 21% or 100% O(2) and total and cyanide-insensitive O(2) consumption, as well as nitric oxide accumulation were measured polarographically. Citrate synthase, a marker of mitochondrial mass, and nitrotyrosine, a marker of peroxynitrite formation, were quantified by Western blot. In addition to increased macrophage numbers, the lungs of 60% O(2)-exposed animals had greatly increased cyanide-insensitive O(2) consumption (p <.05 compared to air controls) and immunoreactive nitrotyrosine (p <.05), which were all completely abrogated by treatment with GdCl(3). Exposure to 60% O(2) for 14 d had no effect on peroxynitrite-independent nitric oxide release or mitochondrial mass. We conclude that increased ROS in the lungs of newborn rats exposed to 60% O(2) for 14 d was likely to be caused, in significant part, by the presence of increased numbers of macrophages.

Administration, Inhalation↗

PubMed2003

Expression and characterization of a human pyruvate carboxylase variant by retroviral gene transfer.

Type A pyruvate carboxylase (PC) deficiency presents mainly in the Amerindian population, specifically the Ojibwa, Cree and Micmac tribes of the Algonquin-speaking peoples. The gene for PC contains a homozygous founder mutation (G1828-->A) that results in an Ala610-->Thr amino acid substitution in Ojibwa with Type A PC deficiency. The mutation is located in the highly conserved pyruvate-binding domain of PC. The present paper describes a retroviral expression system for human PC used to analyse the effects of this mutation. We show, through immunoblot analysis, PC enzyme activity assays, reverse-transcription PCR and mitochondrial-import experiments, that this mutation is disease-causing in the Ojibwa population owing to its decreased catalytic activity, decreased steady-state levels of expression and inefficient import into the mitochondria. Our data suggest that this mutation may affect the stability of the protein, resulting in decreased steady-state levels of expression, and that it may also affect the secondary structure of the protein during the import process, thereby inhibiting proper translocation into the mitochondria, where PC is active.

Amino Acid Sequence↗

PubMed2003

The effect of short-term dimethylglycine treatment on oxygen consumption in cytochrome oxidase deficiency: a double-blind randomized crossover clinical trial.

OBJECTIVE: To study the effectiveness of dimethylglycine (DMG) on oxygen consumption (VO(2)) in children with Saguenay-Lac-Saint-Jean cytochrome-c oxidase (SLSJ-COX) deficiency (OMIM 220111). STUDY DESIGN: In a crossover randomized double-blind clinical trial, 5 children with SLSJ-COX deficiency, who were stable and old enough to comply with VO(2) measurement, were treated with placebo or DMG for 3 days, and with the alternate treatment after a 2-week washout period. VO(2) was measured by indirect calorimetry before and after treatment. Dietary caloric intake was calculated for 3 days before each measurement. Mean caloric intakes per day were 1562 and 1342 kcal x m(-2) before and during placebo, 1,336 and 1,380 before and during DMG, respectively. RESULTS: DMG was well tolerated and, in all cases, resulted in markedly increased blood DMG levels (617 + 203 mmol x L(-1)), versus 0 to 2 mmol x L(-1) without treatment. Mean VO(2) was lower after administration of either DMG (-1 +/- 3 mL x min(-1) x m(-2)) or placebo (-6 +/- 4), but neither difference was statistically significant. There was no detectable effect of DMG treatment on blood levels of lactate, pyruvate, bicarbonate, or pH. VO(2) values of patients (range, 101-135 mL x min(-1) x m(-2)) were lower than published norms (150-160 mL x min(-1) x m(-2)). CONCLUSION: This study suggests that treatment with DMG does not substantially change VO(2) in children with SLSJ-COX deficiency.

Child↗

PubMed2002

Events upstream of mitochondrial protein import limit the oxidative capacity of fibroblasts in multiple mitochondrial disease.

To investigate whether protein import is defective in mitochondrial disease, we compared the rate of import and the expression of protein import machinery components in skin fibroblasts from control subjects and a patient with multiple mitochondrial disease (MMD). The patient exhibited a 35% decrease in cytochrome c oxidase activity and a 59% decrease in cellular oxygen consumption compared to control. Western blot analyses revealed that patient levels of MDH, mtHSP70, HSP60, and Tom20 protein were 57%, 20%, 75% and 100% of control cells, respectively. MDH and Tom20 mRNA levels were not different from control levels, whereas mtHSP70 mRNA were 50% greater than control. Radiolabeled MDH was imported into mitochondria with equal efficiency between patient (44% of total synthesized) and control (43%) cells, although the total MDH synthesized in patient cells was reduced by about 40%. The unaffected levels of mRNA and post-translational import into mitochondria, combined with reduced protein levels of MDH, mtHSP70, and HSP60 suggest a translational defect in this patient with MMD. This was verified by the 50% reduction in overall cellular protein synthesis in the patient compared to control. Further, the similar import rates between patient and control cells suggest an important role for Tom20, but a lesser role for mtHSP70 in regulating protein import into mitochondria.

Blotting, Southern↗

PubMed2002

Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase.

Human NADH CoQ oxidoreductase is composed of a total of 43 subunits and has been demonstrated to be a major site for the production of superoxide by mitochondria. Incubation of rat heart mitochondria with ATP resulted in the phosphorylation of two mitochondrial membrane proteins, one with a M(r) of 6 kDa consistent with the NDUFA1 (MWFE), and one at 18kDa consistent with either NDUFS4 (AQDQ) or NDUFB7 (B18). Phosphorylation of both subunits was enhanced by cAMP derivatives and protein kinase A (PKA) and was inhibited by PKA inhibitors (PKAi). When mitochondrial membranes were incubated with pyruvate dehydrogenase kinase, phosphorylation of an 18kDa protein but not a 6kDa protein was observed. NADH cytochrome c reductase activity was decreased and superoxide production rates with NADH as substrate were increased. On the other hand, with protein kinase A-driven phosphorylation, NADH cytochrome c reductase was increased and superoxide production decreased. Overall there was a 4-fold variation in electron transport rates observable at the extremes of these phosphorylation events. This suggests that electron flow through complex I and the production of oxygen free radicals can be regulated by phosphorylation events. In light of these observations we discuss a potential model for the dual regulation of complex I and the production of oxygen free radicals by both PKA and PDH kinase.

Adenosine Triphosphate↗

PubMed2002

Intron retention and frameshift mutations result in severe pyruvate carboxylase deficiency in two male siblings.

This paper describes the molecular characterization of two male siblings displaying the complex (Type B) form of pyruvate carboxylase (PC) deficiency in which severe neonatal lactic acidosis and redox abnormalities results in death within the first few weeks of life. The two male siblings were found to be compound heterozygous for a TAGG deletion at the exon15/intron15 splice site (IVS15+2-5delTAGG) and a dinucleotide deletion in exon 16 (2491-2492delGT) of the PC gene. We also demonstrate through RT-PCR and sequencing of aberrant transcripts that the IVS15+2-5delTAGG results in the retention of intron 15 during pre-mRNA splicing. In addition, both deletions are predicted to result in a frameshift to generate a premature termination codon such that the encoded mRNA could be subject to nonsense mediated decay.

Alternative Splicing↗

PubMedDate not supplied

Proteomics of bovine mitochondrial RNA-binding proteins: HES1/KNP-I is a new mitochondrial resident protein.

Proteomic analysis of bovine mitochondrial proteins with affinity to polyAdenylate or polyUridylate was performed in an effort to identify novel RNA-binding mitochondrial proteins. We have used 2D gel electrophoresis and MALDI-QqTOF mass spectrometry to identify a total of 64 proteins, of which 51 have defined mitochondrial function including 6 known RNA-binding proteins. HES1/KNP-I from the polyA-binding fraction of mitochondrial Triton extract showed exclusive mitochondrial localization when expressed in GFP-tagged form. The HES1/KNP-I gene is on human chromosome 21q22.3 and may be involved in several disorders mapped to that region. Thus, HES1/KNP-I is a proven mitochondrial resident protein with apparent tight membrane association and tentative RNA-binding properties.

Animals↗