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C T Moraes

Publications and source records attributed to C T Moraes.

At least 37 records · Page 2Linked to original sources
PubMed1999

Suppression of a mitochondrial tRNA gene mutation phenotype associated with changes in the nuclear background.

We previously have characterized a pathogenic mtDNA mutation in the tRNAAsn gene. This mutation (G5703A) was associated with a severe mitochondrial protein synthesis defect and a reduction in steady-state levels of tRNAAsn. We now show that, although transmitochondrial cybrids harboring homoplasmic levels of the mutation do not survive in galactose medium, several galactose-resistant clones could be obtained. These cell lines had restored oxidative phosphorylation function and 2-fold higher steady-state levels of tRNAAsn when compared with the parental mutant cell line. The revertant lines contained apparently homoplasmic levels of the mutation and no other detectable alteration in the tRNAAsn gene. To investigate the origin of the suppression, we transferred mtDNA from the revertants (143B/206 TK-) to a different nuclear background (143B/207 TK-, 8AGr). These new transmitochondrial cybrids became defective once again in oxidative phosphorylation and regained galactose sensitivity. However, galactose-resistant clones could also be obtained by growing the 8AGr transmitochondrial cybrids under selection. Because the original rate of reversion was higher than that expected by a classic second site nuclear mutation, and because of the aneuploid features of these cell lines, we searched for the presence of chromosomal alterations that could be associated with the revertant phenotype. These studies, however, did not reveal any gross changes. Our results suggest that modulation of the dosage or expression of unknown nuclear-coded factor(s) can compensate for a pathogenic mitochondrial tRNA gene mutation, suggesting new strategies for therapeutic intervention.

Animals↗
PubMed1999

Sequence analysis of the diabetes-protective human leukocyte antigen-DQB1*0602 allele in unaffected, islet cell antibody-positive first degree relatives and in rare patients with type 1 diabetes.

The human leukocyte antigen (HLA)-DQA1*0102/DQB1*0602/DRB1*1501 (DR2) haplotype confers strong protection from type 1 diabetes. Growing evidence suggests that such protection may be mostly encoded by the DQB1*0602 allele, and we reported that even first degree relatives with islet cell antibodies (ICA) have an extremely low diabetes risk if they carry DQB1*0602. Recently, novel variants of the DQB1*0602 and *0603 alleles were reported in four patients with type 1 diabetes originally typed as DQB1*0602 with conventional techniques. One inference from this observation is that DQB1*0602 may confer absolute protection and may never occur in type 1 diabetes. By this hypothesis, all patients typed as DQB1*0602 positive with conventional techniques should carry one of the above diabetes-permissive variants instead of the protective DQB1*0602. Such variants could also occur in ICA/DQB1*0602-positive relatives, with the implication that their diabetes risk could be significantly higher than previously estimated. We therefore sequenced the DQB1*0602 and DQA1*0102 alleles in all ICA/DQB1*0602-positive relatives (n = 8) previously described and in six rare patients with type 1 diabetes and DQB1*0602. We found that all relatives and patients carry the known DQB1*0602 and DQA1*0102 sequences, and none of them has the mtDNA A3243G mutation associated with late-onset diabetes in ICA-positive individuals. These findings suggest that diabetes-permissive DQB1*0602/3 variants may be very rare. Thus, although the protective effect associated with DQB1*0602 is extremely powerful, it is not absolute. Nonetheless, the development of diabetes in individuals with DQB1*0602 remains extremely unlikely, even in the presence of ICA, as confirmed by our further evaluation of ICA/DQB1*0602-positive relatives, none of whom has yet developed diabetes.

Adult↗
PubMed1998

Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency.

The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to introduce mtDNA from non-human apes into a human cell line lacking mtDNA (rho degrees), and succeeded in producing human-common chimpanzee, human-pigmy chimpanzee, and human-gorilla xenomitochondrial cybrids (HXC). Here, we present a comprehensive characterization of oxidative phosphorylation function in these cells. Mitochondrial complexes II, III, IV, and V had activities indistinguishable from parental human or non-human primate cells. In contrast, a complex I deficiency was observed in all HXC. Kinetic studies of complex I using decylubiquinone or NADH as limiting substrates showed that the Vmax was decreased in HXC by approximately 40%, and the Km for the NADH was significantly increased (3-fold, p < 0.001). Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone produced a mild effect in control cells (0-10% inhibition) but produced a marked inhibition of HXC respiration (50-75%). Immunoblotting analyses of three subunits of complex I (ND1, 75 and 49 kDa) showed that their relative amounts were not significantly altered in HXC cells. These results establish HXC as cellular models of complex I deficiency in humans and underscore the importance of nuclear and mitochondrial genomes co-evolution in optimizing oxidative phosphorylation function.

Animals↗
PubMed1998

Duplication and triplication with staggered breakpoints in human mitochondrial DNA.

We identified a tandem duplication and triplication of a mitochondrial DNA (mtDNA) segment in the muscle of a 57-year-old man with no evidence of a neuromuscular disorder. A large triplication of a mtDNA coding region has not been previously reported in humans. Furthermore, the rearrangements (comprising 10-12% of the muscle mtDNA pool in the propositus) were unique because the breakpoints were staggered at both ends (between mtDNA positions 3263-3272 and 16,065-16,076) and contained no identifiable direct repeats. Both sides of the breakpoint were located approximately 35 bp downstream of regions that undergo frequent strand displacement by either transcription (positions 3263-3272) or replication (positions 16,065-16,076), suggesting that topological changes generated by the movement of RNA/DNA polymerases may be associated with the genesis of a subclass of mtDNA rearrangements. The presence of low levels of these rearrangements in other normal adults also suggest that these mutations are not rare. The characterization of these rearrangements shed light on potential alternative mechanisms for the genesis of mtDNA rearrangements.

Adult↗
PubMed1998

Simultaneous transfer of mitochondrial DNA and single chromosomes in somatic cells: a novel approach for the study of defects in nuclear-mitochondrial communication.

The assembly and function of respiratory-competent mitochondria in eukaryotic cells depends on collaboration between the nuclear and mitochondrial genomes, but the molecular mechanisms underlying such cross-talk are poorly understood. Microcell-mediated chromosome transfer has been used to transfer intact chromosomes from one mammalian cell to another, helping to map loci implicated in different diseases and in the senescence process. In the present work, we show that microcells have a significant number of mitochondria which can be transferred to another cell simultaneously with a limited number of chromosomes. By fusing microcells from a colon carcinoma cell line with a mitochondrial DNA (mtDNA)-less osteosarcoma cell line, we were able to isolate transmitochondrial hybrids containing only one of three selectable chromosomes and mtDNA from the donor cell. The proportion of transmitochondrial hybrids containing one chromosomal marker with respect to the total transmitochondrial hybrids and cybrids was approximately 1% and no hybrids were isolated containing more than one nuclear marker. The genetic data correlated well with the composition and structure of the microcell preparations, which showed the presence of cytoplast-like structures and microcells containing mitochondria surrounding the micronuclei. Microcell-mediated mtDNA and chromosome transfer can be used to identify nuclear factors implicated in mtDNA maintenance and gene expression, as well as to investigate nuclear factors which modulate clinical phenotypes in mitochondrial disorders.

Bleomycin↗
PubMed1998

Mitochondrial encephalomyopathy and hypoparathyroidism associated with a duplication and a deletion of mitochondrial deoxyribonucleic acid.

Diabetes mellitus is the most frequent endocrinopathy associated with mitochondrial disorders, particularly in patients with duplications of mitochondrial DNA (mtDNA). Although hypoparathyroidism has also been described in mitochondrial diseases, there have been few molecular studies in these cases, most of which identified the presence of single mtDNA deletions in the patients' tissues. We studied muscle DNA of a 12-yr-old patient with incomplete Kearns-Sayre syndrome and hypoparathyroidism. Southern analysis showed that muscle DNA contained three populations of mtDNA: wild type (26%), deleted (65%), and duplicated (9%). To determine the sequence of the breakpoint region from deleted and duplicated mtDNA independently, we isolated the deleted and duplicated mtDNA by gel fractionation of a PstI-digested total DNA. The breakpoint was located at mtDNA positions 5788 and 15,448 for both duplicated and deleted molecules. Our study reinforces the concept that endocrinopathies other than diabetes can be associated with a duplication of mtDNA and gives additional support to the hypothesis that the duplication and deletion of mtDNA are generated from the same recombination event.

Base Sequence↗
PubMed1998

Structure of heparan sulfate: identification of variable and constant oligosaccharide domains in eight heparan sulfates of different origins.

The use of specific enzymes (heparinase and heparitinases from Flavobacterium heparinum, endoglucuronidase, alphaN-acetylglucosaminidase and beta-glucuronidase from the mollusc Anomalocardia brasiliana) and chemical methods (nitrous acid degradation, hydrazine N-deacetylation and borohydride reduction), led to the proposal of the total sequence of a heparan sulfate derived from bovine pancreas and partial sequences of heparan sulfates from different origins (bovine: lung, liver, brain; hog: liver, brain; rabbit liver; dog liver). It was shown that all the heparan sulfates contain common structural features such as: a N-acetylated and a N-sulfated domain made of glucuronic acid-containing disaccharides and a more sulfated region made of iduronic acid-containing disaccharides. Separating the two domains a peculiar tetrasaccharide made of GlcNAc-(alpha1-4)-IdoUA-(alpha1-4)-GlcNS-(alpha1-4)-IdoUA was identified in all the heparan sulfates analyzed. It was also shown that the non-reducing ends of the heparan sulfates contain the monosaccharides glucosamine N-sulfate and/or glucosamine 2,6 disulfate.

Animals↗
PubMed1997

Mitochondrial DNA defects in Brazilian patients with chronic progressive external ophthalmoplegia.

We report herein on eleven Brazilian patients with mitochondrial DNA (mtDNA) deletions, found among thirteen patients with chronic progressive external ophthalmoplegia (CPEO) and ragged-red fibers (RRF). The molecular data was correlated with the morphological and clinical findings. The muscle biopsies were studied by histochemistry, immunohistochemistry and DNA analysis. Muscle mtDNA deletions were mapped and quantitated by Southern blot analysis, polymerase chain reaction and sequencing. Of the eleven patients, ten had CPEO without multisystemic involvement and one had Kearns-Sayre syndrome. Three patients had multiple deletions, two of them with no apparent family history. Eight patients showed heteroplasmic single deletions, ranging in length from 2309 to 7566 bp; three of them had the same 'common deletion' of 4977 bp. The proportion of deleted mtDNA ranged from 14 to 89%. Immunohistochemical studies revealed decreased reactivity with the mtDNA-encoded subunit II of cytochrome c oxidase (COX) in all patients, but preserved activity with the nuclear-encoded COX subunit IV in COX-deficient fibers. Two cases presented a few COX-negative fibers with reduced COX IV immunostaining. We found a high frequency of mtDNA deletions in Brazilian patients with CPEO. There was no correlation between clinical severity, morphological findings and the size or amount of the mutated mtDNA in muscle, suggesting that there are still unknown factors influencing the disease phenotype.

Adolescent↗
PubMed1997

Oxidative phosphorylation dysfunction does not increase the rate of accumulation of age-related mtDNA deletions in skeletal muscle.

Several reports described an age-related accumulation of a particular mitochondrial DNA (mtDNA) deletion ('common deletion') in post-mitotic tissues. These findings led to the hypothesis that free radicals generated inside the mitochondria could damage mtDNA during a normal life span. The impaired electron transfer function resulting from mtDNA damage would increase the production of free radicals creating a vicious cycle. If this vicious cycle is an important player in the somatic accumulation of mtDNA deletions, patients with impaired oxidative phosphorylation (regardless of the primary defect) should have an accelerated accumulation of mtDNA deletions. We tested this hypothesis by performing three analyses: (a) comparing the amounts of the mtDNA 'common deletion' in normal controls and patients with genetically characterized mitochondrial disorders associated with pathogenic mtDNA point mutations or deletions other than the common deletion; (b) analyzing the co-segregation of the age-related mtDNA common deletion with a pathogenic mtDNA point mutation; and (c) by the detection of multiple mtDNA deletions by long PCR in controls and patients with mitochondrial disorders. We observed a positive correlation between age and common deletion levels in controls (r = 0.80) and patients (r = 0.69). The slopes of the curves were similar, suggesting that the rate of accumulation of the age-related common deletion was the same in both groups. We could not find a co-segregation of the pathogenic point mutated mtDNA molecules with the common deletion nor increased number of age-related deletions in patients. Our data do not support the hypothesis that a vicious cycle (damage to mtDNA would affect the respiratory function, leading to the generation of more free radicals, which in turn would provoke additional mtDNA damage) is an important factor in the accumulation of age-related mtDNA deletions.

Adolescent↗
PubMed1997

Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids.

The nuclear and mitochondrial genomes coevolve to optimize approximately 100 different interactions necessary for an efficient ATP-generating system. This coevolution led to a species-specific compatibility between these genomes. We introduced mitochondrial DNA (mtDNA) from different primates into mtDNA-less human cells and selected for growth of cells with a functional oxidative phosphorylation system. mtDNA from common chimpanzee, pigmy chimpanzee, and gorilla were able to restore oxidative phosphorylation in the context of a human nuclear background, whereas mtDNA from orangutan, and species representative of Old-World monkeys, New-World monkeys, and lemurs were not. Oxygen consumption, a sensitive index of respiratory function, showed that mtDNA from chimpanzee, pigmy chimpanzee, and gorilla replaced the human mtDNA and restored respiration to essentially normal levels. Mitochondrial protein synthesis was also unaltered in successful "xenomitochondrial cybrids." The abrupt failure of mtDNA from primate species that diverged from humans as recently as 8-18 million years ago to functionally replace human mtDNA suggests the presence of one or a few mutations affecting critical nuclear-mitochondrial genome interactions between these species. These cellular systems provide a demonstration of intergenus mtDNA transfer, expand more than 20-fold the number of mtDNA polymorphisms that can be analyzed in a human nuclear background, and provide a novel model for the study of nuclear-mitochondrial interactions.

Animals↗
PubMed1997

Up-regulation of D3 dopamine receptor mRNA in the nucleus accumbens of human cocaine fatalities.

The effects of chronic cocaine use on D3 receptor mRNA expression in the human nucleus accumbens was assessed by reverse transcription-polymerase chain reaction. D3 receptor/cyclophilin mRNA ratios in the nucleus accumbens were increased 6-fold in cocaine overdose victims as compared to age-matched and drug-free control subjects. This finding demonstrates that chronic cocaine exposure leads to adaptive increases in the expression of D3 receptor mRNA in a critical reward center in brain.

Autopsy↗
PubMed1997

Functional and structural features of a tandem duplication of the human mtDNA promoter region.

An approximately 260-bp tandem duplication of the human mtDNA regulatory region has been identified in patients with mitochondrial disorders and in a specific Caucasian haplogroup. The functional significance of this mtDNA duplication was difficult to assess, because it was present at very low levels in human tissues. We have isolated several transmitochondrial cybrid lines harboring this mutation, one of which (clone CA17.1) was essentially homoplasmic for the duplication. Oxidative-phosphorylation function was not impaired in clone CA17.1, suggesting that this mtDNA alteration is not pathogenic. mtDNA copy number and steady-state levels of heavy- and light-strand transcripts were unaltered in clone CA 17.1. The steady-state levels of RNAs made from the two promoters (either from the heavy-strand or from the light-strand) were also similar, indicating that oppositely oriented promoters did not interfere with each other.

Base Sequence↗
PubMed1997

A disease-associated G5703A mutation in human mitochondrial DNA causes a conformational change and a marked decrease in steady-state levels of mitochondrial tRNA(Asn).

We introduced mitochondrial DNA (mtDNA) from a patient with a mitochondrial myopathy into established mtDNA-less human osteosarcoma cells. The resulting transmitochondrial cybrid lines, containing either exclusively wild-type or mutated (G5703A transition in the tRNA[Asn] gene) mtDNA, were characterized and analyzed for oxidative phosphorylation function and steady-state levels of different RNA species. Functional studies showed that the G5703A mutation severely impairs oxidative phosphorylation function and mitochondrial protein synthesis. We detected a marked reduction in tRNA(Asn) steady-state levels which was not associated with an accumulation of intermediate transcripts containing tRNA(Asn) sequences or decreased transcription. Native polyacrylamide gel electrophoresis showed that the residual tRNA(Asn) fraction in mutant cybrids had an altered conformation, suggesting that the mutation destabilized the tRNA(Asn) secondary or tertiary structure. Our results suggest that the G5703 mutation causes a conformational change in the tRNA(Asn) which may impair aminoacylation. This alteration leads to a severe reduction in the functional tRNA(Asn) pool by increasing its in vivo degradation by mitochondrial RNases.

Base Sequence↗
PubMed1997

A novel mitochondrial G8313A mutation associated with prominent initial gastrointestinal symptoms and progressive encephaloneuropathy.

We describe a childhood mitochondrial disorder in which the clinical symptoms began and remained confined to the gastrointestinal (GI) system during the first 4 y. Seizures heralded the onset of progressive encephalopathy at age 7. Peripheral neuropathy, retinitis pigmentosa, and neural deafness developed subsequently. Laboratory investigations disclosed elevated levels of plasma lactate, and a muscle biopsy revealed ragged red fibers lacking cytochrome c oxidase activity and diminished levels of respiratory chain enzyme complexes. Molecular genetic tests failed to show any of the previously reported pathogenic mitochondrial DNA (mtDNA) mutations. We therefore screened the whole mitochondrial genome by coupling restriction digestions with single-strand conformational polymorphism (SSCP) patterns. We identified a unique SSCP in the segment that encompassed the tRNA(Lys) gene, and direct sequencing of this segment revealed a G-->A transition at an evolutionarily conserved nucleotide at mtDNA position 8313. This G8313A transition was heteroplasmic in muscle and fibroblasts of the patient, but was absent in the white blood cells and platelets from his maternal relatives. This report illustrates how GI symptoms can be the initial manifestation in a mitochondrial disorder and suggests that mitochondrial dysfunction should be considered in differentials of unexplained chronic GI symptoms, especially when lactic acidosis or other unrelated clinical signs or symptoms are present.

Adolescent↗
PubMed1996

Functional and molecular mitochondrial abnormalities associated with a C --> T transition at position 3256 of the human mitochondrial genome. The effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing.

We have previously identified a mitochondrial DNA polymorphism (a C --> T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or > 99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, overproduce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.

Blotting, Northern↗