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C Marsac

Publications and source records attributed to C Marsac.

At least 37 records · Page 2Linked to original sources
PubMed1998

Isoleucylation properties of native human mitochondrial tRNAIle and tRNAIle transcripts. Implications for cardiomyopathy-related point mutations (4269, 4317) in the tRNAIle gene.

A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNAIleand isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNAIlehas been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient isoleucylation; (ii) point mutation A4269G in the gene (A-->G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A-->G at nt 59 in the tRNA), reported in a case of fatal infantile cardiomyopathy, induces a small but significant decrease in isoleucylation. The potential implications of these findings on the understanding of the molecular mechanisms involved in the expression of pathology are discussed.

Adenine↗
PubMed1997

Biochemical and genetic studies of four patients with pyruvate dehydrogenase E1 alpha deficiency.

We report studies of four patients with pyruvate dehydrogenase complex (PDH) deficiency caused by mutations in the E1 alpha subunit. Two unrelated male patients presented with Leigh syndrome and a R263G missense mutation in exon 8. This mutation has previously been described in males with the same phenotype. The two other patients had different novel mutations: (1) an 8-bp deletion at the C-terminus (exon 11) was found in one allele of a young girl suffering from microcephaly and (2) a C88S missense mutation (exon 3) in a boy who only presented with motor neuropathy. These mutations were not found in the mothers of any of the four cases. Immunoblot analysis revealed decreased immunoreactivity for the E1 alpha and E1 beta subunits in three out of the four patients. These findings confirm that: (1) PDH deficiencies are genetically heterogeneous, (2) the R263G mutation is more frequent in male cases than are other mutations and this amino acid is a hot spot for gene mutations, (3) the last eight amino acids may be important for the conformation of the tetrameric E1-PDH enzyme, and (4) the amino acids at positions 88, 263 and 382-387 are essential for the linking of the alpha subunit with the beta subunit and for the activity of the holoenzyme.

Adolescent↗
PubMed1997

A near homoplasmic T8993G mtDNA mutation in a patient with atypic Leigh syndrome not present in the mother's tissues.

We report an interesting case of a T8993G mutation in the muscle mtDNA of a 2-year-old girl who presented with myoclonia, developmental delay, increased lactate concentration in CSF and cerebral MRI abnormalities without retinopathy, suggesting an atypical form of Leigh syndrome. Search for mutant molecules in the blood and skin fibroblasts of the patient's healthy mother as well as in the blood of the also unaffected patient's sister was unsuccessful. This "sporadic' case of 8993 mtDNA mutation may be due either to a de novo mutation arising during oogenesis (or early embryogenesis) or to a mutation pre-existing in oocytes in a few mtDNA molecules and selected through a narrow bottleneck mechanism. Whatever the mechanism involved, this observation illustrates a complete shift of the mtDNA type in only one generation: from 0 to nearly 100% of mtDNA molecules with the T8993G mutation.

Child, Preschool↗
PubMed1997

Mutations in PDX1, the human lipoyl-containing component X of the pyruvate dehydrogenase-complex gene on chromosome 11p1, in congenital lactic acidosis.

We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression.

Acidosis, Lactic↗
PubMed1996

Biochemical analysis of fibroblasts from patients with cytochrome c oxidase-associated Leigh syndrome.

Cultured skin fibroblasts from four patients with Leigh syndrome and cytochrome c oxidase deficiency were studied. Mitochondrial DNA (mtDNA) analysis excluded large-scale deletions and known point mutations associated with Leigh syndrome. The COX activities were reduced to 18-44% of healthy probands, when measured in the presence of laurylmaltoside. COX activity from patients was shown to be more temperature sensitive than COX activity from control cells. In order to determine the subunit composition of COX immunoblotting studies were performed using mono- and polyclonal antibodies to distinct subunits. A monoclonal antibody to subunit IV crossreacted with two unknown proteins of higher apparent molecular weight in mitochondria from three patients, but not in mitochondria from control and the fourth patient. Quantification of immunoreactivity revealed a decrease of subunits II/III and IV parallel to the determined enzyme activity. In contrast, a variable amount of subunit VIIa (and/or VIIb) was found in mitochondria from different patients. The results indicate a defective COX holoenzyme complex in patients with Leigh syndrome and suggest different molecular origins of the defect.

Base Sequence↗
PubMed1996

Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.

Apoptosis↗
PubMed1996

Biochemical investigations and immunoblot analyses of two unrelated patients with an isolated deficiency in complex II of the mitochondrial respiratory chain.

Mitochondrial respiratory chain defects involving complex II are comparatively rare. We report the biochemical findings in two unrelated patients who both have an isolated complex II deficiency (40-50% of control values). Western blot analysis of mitochondrial fractions showed different findings between the two patients. In one patient there was a decrease in the levels of both the Fp and Ip subunits whereas in the other patient the levels of all immunoreactive complex II subunits were normal. This is the first time that an isolated deficiency of complex II activity associated with normal levels of protein subunits, using subunit specific antisera, has been described.

Blotting, Western↗
PubMed1996

Mutation analysis of the pyruvate dehydrogenase E1 alpha gene in eight patients with a pyruvate dehydrogenase complex deficiency.

Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X-linked E1 alpha gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single-strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA. The 4 female patients were shown to carry both a normal and a mutated E1 alpha gene.

Base Sequence↗
PubMed1996

[Pharmacological modulation of alterations of endothelial cytoskeleton induced by hydrogen peroxyde and by TNF-alpha].

New selenium containing compounds which act as mimics of glutathione peroxidase (GPx) protect vascular endothelial cells (HUVEC) from the toxicity of 140 microM hydrogen peroxide. In the absence of GPx mimic, hydrogen peroxide destroys the tightness of the cellular monolayer and transforms the actin network into compact stress fibers. The pre-treatment of the cells by 4 microM of the lead-compound BXT-51072 for 1 hours inhibits the morphological modifications induced by hydrogen peroxide. This GPx mimic can also prevent the alterations of the endothelial cytoskeleton which are induced by Tumor Necrosis Factor-alpha (TNF-alpha) and which consist in a reorganization of actin filaments with the formation of stress fibers. Fluorescent labeling of polymerized actin has been performed by means of phalloidine coupled with rhodamine. The protective effect of this antioxidant catalyst against the toxicity of hydrogen peroxide and TNF-alpha includes the maintenance of a structural configuration of the cytoskeleton which is required for the function of endothelial barrier.

Antioxidants↗
PubMed1995

Impairment of the mitochondrial respiratory chain activity in diethylnitrosamine-induced rat hepatomas: possible involvement of oxygen free radicals.

Alterations in the energy metabolism of cancer cells have been reported for many years. However, the deleterious mechanisms involved in these deficiencies have not yet been clearly proved. The main goal of this study was to decipher the harmful mechanisms responsible for the respiratory chain deficiencies in the course of diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis, where mitochondrial DNA abnormalities had been previously reported. The respiratory activity of freshly isolated hepatoma mitochondria, assessed by oxygen consumption experiments and enzymatic assays, presented a severe complex I deficiency 19 months after DENA treatment, and later on, in addition, a defective complex III activity. Since respiratory complex subunits are encoded by both nuclear and mitochondrial genes, we checked whether the respiratory chain defects were due to impaired synthesis processes. The specific immunodetection of complex I failed to show any alterations in the steady-state levels of both nuclear and mitochondrial encoded subunits in the hepatomas. Moreover, in vitro protein synthesis experiments carried out on freshly isolated hepatoma mitochondria did not bring to light any modifications in the synthesis of the mitochondrial subunits of the respiratory complexes, whatever the degree of tumor progression. Finally, Southern blot analysis of mitochondrial DNA did not show any major mitochondrial DNA rearrangements in DENA-induced hepatomas. Because the synthetic processes of respiratory complexes did not seem to be implicated in the respiratory chain impairment, these deficiencies could be partly ascribed to a direct toxic impact of highly reactive molecules on these complexes, thus impairing their function. The mitochondrial respiratory chain is an important generator of noxious, reactive oxygen free radicals such as superoxide and H2O2, which are normally catabolized by powerful antioxidant scavengers. Nineteen months after DENA treatment, a general collapse of the antioxidant enzymatic system was demonstrated in the hepatomas, as recurrently observed in cancer cells. This oxidant versus antioxidant imbalance was characterized by the establishment of oxidative stress in the course of hepatocarcinogenesis, as partly shown by the important decrease of glutamine synthetase activity, an enzyme whose function is highly sensitive to oxidant reactions. This disequilibrium would result in a net increase of the steady-state concentration of superoxide generated between respiratory complexes I and III in the mitochondria. Once generated, superoxide would likely inactivate complexes I and III via oxidant reactions on their superoxide-sensitive [4Fe, 4S] clusters. The role of mitochondrial respiratory chain impairment in chemical carcinogenesis and/or the persistence of the cancerous state is further discussed.

Animals↗
PubMed1995

Clinical, biochemical, and molecular analysis of a maternally inherited case of Leigh syndrome (MILS) associated with the mtDNA T8993G point mutation.

We report a new case of Leigh disease (subacute necrotizing encephalomyelopathy) in a girl with mitochondrial DNA (mtDNA) mutation in the ATPase6 gene at nucleotide position 8993. Sequence analysis of mtDNA revealed a T-to-G transversion at nucleotide position 8993 in the ATPase6 gene. Southern blot restriction analysis of patient muscle mtDNA showed only a mutant pattern for the mutation 8993. Molecular analysis of seven subjects from the family showed that except for the father they all carried the 8993 mtDNA mutation in all studied tissues, with high percentages in the two symptomatic children and even in one asymptomatic boy.

Brain↗
PubMed1994

Chronic progressive external ophthalmoplegia is associated with a novel mutation in the mitochondrial tRNA(Asn) gene.

Chronic progressive external ophthalmoplegia (CPEO) is caused by a decreased oxidative phosphorylation (OXPHOS) activity due to large-scale deletions of the mitochondrial genome in 50% of the patients. The deletions encompass structural OXPHOS genes as well as tRNA genes, required for their expression so that the pathogenesis could be due to the deleted OXPHOS subunits or to an impaired mitochondrial translation. We have analyzed the mitochondrial genome of a patient presenting with CPEO for single base substitutions and discovered a novel heteroplasmic mutation in the tRNA(Asn) gene at position 5692 that converts a highly conserved adenine into a guanine. This mutation is unique because it is located at the transition of the anticodon loop to the anticodon stem and it leads to an additional base pair, thus reducing the number of loop-forming nucleotides from seven to five. Our findings suggest that CPEO can be caused by a single base substitution in a mitochondrial tRNA gene so that the mitochondrial protein synthesis becomes the rate limiting step in OXPHOS fidelity.

Base Sequence↗
PubMed1994

Myo-leukoencephalopathy in twins: study of 3243-myopathy, encephalopathy, lactic acidosis, and strokelike episodes mitochondrial DNA mutation.

Two dizygotic twins with myopathy and leukoencephalopathy are described. The female twin had an incomplete form of MELAS syndrome (myopathy, encephalopathy, lactic acidosis, and strokelike episodes) with severe myopathy, epileptic seizures without strokelike episodes. The male twin presented clinical features exclusively of myopathy and subclinical leukoencephalopathy. The MELAS mitochondrial DNA point mutation (MELAS-3243) was found by southern blot and polymerase chain reaction in muscle, skin fibroblasts, and blood of the female twin and was not detected in the skin fibroblasts nor in the blood of the mother, nor in any of the tissues tested in the male twin. The absence of mutation in male twin tissues raises questions about the pathogenetic significance of the mutation in this family.

Adult↗
PubMed1994

Expression of cytochrome c oxidase subunits encoded by mitochondrial or nuclear DNA in the muscle of patients with zidovudine myopathy.

The present study was carried out to determine whether a selective decrease of mitochondrial (mt) DNA-encoded cytochrome c oxidase (CCO) subunits occurs in zidovudine myopathy, as expected with a compound known to induce selective mtDNA depletion. Fourteen HIV-infected patients with zidovudine myopathy were studied. Thirteen had partial CCO deficiency assessed by histochemistry. Western blot analysis of CCO subunits (II/III, IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, and VIIc) was performed on muscle biopsy samples. We evaluated the mtDNA-encoded subunits to nuclear DNA-encoded subunits ratio with the II/III to IV ratio. Patients had either a selective decrease of mtDNA-encoded CCO subunits (3 patients), or an overall decrease affecting both mtDNA-and nuclear DNA-encoded subunits (5 patients), or a normal expression of CCO subunits (6 patients). Positive correlations could not be established between the pattern of expression of CCO subunits and total zidovudine intake, degree of inflammation, and percentages of ragged-red fibers or CCO-deficient fibers. The finding of a decrease of both mtDNA- and nuclear DNA-encoded CCO subunits suggests that a factor additional to zidovudine could be implicated in the pathogenesis of the myopathy, at least in some patients. New insights into the pathogenesis of zidovudine myopathy might come from the use of more sensitive methods, including evaluation of CCO subunits in single fibers.

Adult↗